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Sample records for human micro-vascular endothelial

  1. Bioenergetic disruption of human micro-vascular endothelial cells by antipsychotics.

    PubMed

    Elmorsy, Ekramy; Smith, Paul A

    2015-05-01

    Antipsychotics (APs) are widely used medications, however these are not without side effects such as disruption of blood brain barrier function (BBB). To investigate this further we have studied the chronic effects of the typical APs, chlorpromazine (CPZ) and haloperidol (HAL) and the atypical APs, risperidone (RIS) and clozapine (CLZ), on the bioenergetics of human micro-vascular endothelial cells (HBVECs) of the BBB. Alamar blue (AB) and ATP assays showed that these APs impair bioenergenesis in HBVECs in a concentration and time dependent manner. However since these effects were incomplete they suggest a population of cell bioenergetically heterogeneous, an idea supported by the bistable nature by which APs affected the mitochondrial transmembrane potential. CPZ, HAL and CLZ inhibited the activity of mitochondrial complexes I and III. Our data demonstrates that at therapeutic concentrations, APs can impair the bioenergetic status of HBVECs, an action that help explains the adverse side effects of these drugs when used clinically. PMID:25824037

  2. Adverse effects of antipsychotics on micro-vascular endothelial cells of the human blood-brain barrier.

    PubMed

    Elmorsy, Ekramy; Elzalabany, Laila M; Elsheikha, Hany M; Smith, Paul A

    2014-10-01

    Although the mechanisms of action of antipsychotics (APs) on neuronal function are well understood, very little is known about their effects on cells of the blood-brain barrier (BBB); one function of which is to limit the access of these amphiphilic compounds to the central nervous system. To address this question we have investigated the cytological and functional effects of four APs: chlorpromazine (CLP), haloperidol (HAL), risperidone (RIS) and clozapine (CLZ), at concentrations typical of high therapeutic dosage on a human brain microvascular endothelial cell (HBMEC) model of the BBB. At ~10 µM all four APs impaired the ability of HBMECs to reduce MTT which was followed by decreased Trypan blue exclusion and increased Lactate dehydrogenase release. These effects were associated with oxidative stress which was partly reversed by incubation in 10mM glutathione. At their EC50 concentrations for MTT reduction, all four APs disrupted cellular ultrastructure and morphology. HAL, CPZ and CLZ increased Caspase -3, -8 and -9 activity, chromatin condensation and fragmentation, data indicative of apoptosis. These events were associated with decreased transcytosis of Evans blue and increased transendothelial potential difference and electrical resistance of this BBB model. These findings suggest that at high therapeutic concentrations, CPZ and CLZ are likely to incur cytoxic effects and apoptosis of BBB endothelia with an impairment of barrier functionality. Such events may underlie the aetiology of neuroleptic associated cerebral oedema and neuroleptic malignant syndrome. PMID:25139421

  3. Force control of endothelium permeability in mechanically stressed pulmonary micro-vascular endothelial cells.

    PubMed

    Wang, Bin; Caluch, Adam; Fodil, Redouane; Féréol, Sophie; Zadigue, Patricia; Pelle, Gabriel; Louis, Bruno; Isabey, Daniel

    2012-01-01

    Mechanical factors play a key role in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS) and Ventilator-Induced Lung Injury (VILI) as contributing to alveolo-capillary barrier dysfunction. This study aims at elucidating the role of the cytoskeleton (CSK) and cell-matrix adhesion system in the stressed endothelium and more precisely in the loss of integrity of the endothelial barrier. We purposely develop a cellular model made of a monolayer of confluent Human Pulmonary Microvascular Endothelial Cells (HPMVECs) whose cytoskeleton (CSK) is directly exposed to sustained cyclic mechanical stress for 1 and 2 h. We used RGD-coated ferromagnetic beads and measured permeability before and after stress application. We find that endothelial permeability increases in the stressed endothelium, hence reflecting a loss of integrity. Structural and mechanical results suggest that this endothelial barrier alteration would be due to physically-founded discrepancies in latero-basal reinforcement of adhesion sites in response to the global increase in CSK stiffness or centripetal intracellular forces. Basal reinforcement of adhesion is presently evidenced by the marked redistribution of αvβ3 integrin with cluster formation in the stressed endothelium. PMID:22766716

  4. [Protective effect of combined administration of active ingredients of Danhong on cerebral micro-vascular endothelial cell injured by hypoxia].

    PubMed

    Zhou, Hui-fen; He, Yu; Zhang, Yu-yan; Yang, Jie-hong; Zhao, Tao; Fu, Wei; Zhou, Peng; Wan, Hai-tong

    2014-11-01

    To study the protective effect of combined administration of active ingredients of Danhong on cultured primary mice's brain microvascular endothelial cells (rBMECs) injured by hypoxia. Primary mice's brain micro-vascular endothelial cells were cultured to establish the 4 h hypoxia model. Meanwhile, active ingredients (protocatechuic aldehyde, salvianolic acid B, hydroxysafflor yellow A and tanshinol) of Danhong were administered in rBMECs. The non-toxic dosage was determined by MTT. The leakage of lactate dehydrogenase(LDH), cell superoxide dismutase (SOD) activity and MDA level were detected by the colorimetric method. The expressions of ICAM-1, MMP-9, P53 mRNA were detected by RT-PCR method. Changes in rBMECs cell cycle and early apoptosis were detected by flow cytometry. Danhong's active ingredients and prescriptions 1, 2, 3, 7, 8, 9 could be combined to significantly restrain LDH in hypoxic cells supernatant. Prescriptions 1, 2, 3, 7, 8, 9 could significantly enhance SOD activity in anoxic cells; Prescriptions 1, 2, 3, 8, 9 could significantly decrease the MDA level; Prescriptions 1, 2, 6, 7, 9 could significantly inhibit the early rB-MECs apoptosis induced by hypoxia. After hypoxia, the up-regulated P53 mRNA expression could cause retardation in G, phase and promote cell apoptosis. This proved that the regulatory function of P53 gene lay in monitoring of calibration points in G, phase. Prescriptions 1, 2, 5, 6, 7, 8, 9 could significantly down-regulate the P53 mRNA expression; Prescriptions 1, 4, 7, 8, 9 could significantly down-regulate the ICAM-1 mRNA expression; Prescriptions 1, 3, 6, 9 could significantly down-regulate the MMP-9 mRNA expression. The combined administration of Danhong's active ingredients showed a significant protective effect on primary cultured rBMECs injury induced by hypoxia Its mechanism may be related to the enhancement of cellular antioxidant capacity and the inhibition of inflammatory response and cell apoptosis. This study could

  5. Signaling hierarchy regulating human endothelial cell development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

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

    PubMed

    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 repopulation capacity of human endothelial cells derived from various tissues. Human endothelial cells were isolated from adult and fetal livers using anti-human CD31 antibody-conjugated magnetic beads. Human macrovascular endothelial cells were obtained from umbilical vein. Human microvascular endothelial cells were isolated from adipose tissue. Cells were characterized using flow cytometry. Liver engraftment and repopulation of endothelial cells was studied after intrasplenic transplantation in monocrotaline-treated immunodeficient mice. Following transplantation, human liver endothelial cells engrafted throughout the mouse liver. With immunoscanning electron microscopy, fenestrae in engrafted human liver endothelial cells were identified, a characteristic feature of liver sinusoidal endothelial cells. In contrast, CD31-negative liver cells, human macrovascular and microvascular endothelial cells were not capable of repopulating mouse liver. Characterization of human liver, macrovascular, and microvascular endothelial cells demonstrated expression of CD31, CD34, and CD146 but not CD45. Our study shows that only human liver endothelial cells, but not macro- and microvascular endothelial cells, have the unique capacity to engraft and repopulate the mouse liver. These results indicate that mature endothelial cells cannot transdifferentiate in vivo and thus do not exhibit phenotypic plasticity. Our results have set a basis for further research to the potential of human liver endothelial cells in liver-directed cell and gene therapy. PMID:23044355

  7. Human Pulmonary Endothelial Cells in Culture

    PubMed Central

    Johnson, Alice R.

    1980-01-01

    Endothelial cells were cultured from various different human vessels, including aortas, pulmonary, ovarian, and umbilical arteries, and pulmonary, ovarian, and umbilical veins. The cultured cells were identified as endothelial cells by the presence of Factor VIII antigen and antiotensin I converting enzyme (kininase II). They retained these markers for at least five passages in culture, and some cells had them for seven passages or more. Endothelial cells from the various vessels were compared with respect to their ability to metabolize angiotensins I and II and bradykinin. Cells from arteries had three to five times the angiotensin I converting enzyme activity as cells from veins. The activity of angiotensinase A (aspartyl aminopeptidase) had a similar distribution, and cells from arteries were consistently more active than cells from veins. Cultures of endothelial cells from pulmonary and umbilical vessels formed prostacyclin in response to mechanical stimulation. Media from cell monolayers that were subjected to a change of medium and gentle agitation inhibited aggregation of human platelets. This inhibitory activity was generated within 2-5 min, and it was not formed by cells that were treated with indomethacin or tranylcypromine. Addition of prostaglandin (PG)H2 to indomethacin-treated cells restored the ability to form the inhibitor, but cells treated with tranylcypromine were not responsive to PGH2. In experiments where [14C]arachidonic acid was added to the cells before stimulation, the major metabolite identified by thin-layer chromatography was 6-keto PGF1α. Thus, it appears that pulmonary endothelial cells, as well as umbilical cord cells, can form prostacyclin. In experiments comparing the ability of arterial and venous cells to form prostacyclin, arterial cells were more active than venous cells. These studies of cells from various human vessels suggest that the vascular origin of cultured endothelial cells determines how they metabolize vasoactive

  8. Proteoglycans from human umbilical vein endothelial cells.

    PubMed

    Griesmacher, A; Hennes, R; Keller, R; Greiling, H

    1987-10-01

    Human umbilical vein endothelial cells were incubated with [35S]sulphate and investigated for their proteoglycan production. By gel chromatography, ion-exchange chromatography and CsCl density-gradient centrifugation we obtained preparative amounts of the endothelial proteoheparan sulphate HSI and of proteochondroitin sulphate from the conditioned medium of mass-cultured human umbilical vein endothelial cells. Approximately 90% of the 35S-labeled material in the endothelial cell conditioned medium was proteochondroitin sulphate. This molecule, with a molecular mass of 180-200 kDa, contains four side-chains of 35-40 kDa and a core protein of 35-40 kDa. Two proteoheparan sulphate forms (HSI and HSII) from the conditioned medium were distinguished by molecular mass and transport kinetics from the cell layer to the medium in pulse-chase experiments. One major form (HSI), with an approximate molecular mass of 160-200 kDa a core protein of 55-60 kDa and three to four polysaccharide side-chains of 35 kDa each, was found enriched in the cellular membrane pellet. Another proteoheparan sulphate (HSII), with polysaccharide moieties of 20 kDa, is enriched in the subendothelial matrix (substratum). PMID:2959475

  9. Androgen receptor in human endothelial cells

    PubMed Central

    Torres-Estay, Verónica; Carreño, Daniela V; San Francisco, Ignacio F; Sotomayor, Paula; Godoy, Alejandro S; Smith, Gary J

    2015-01-01

    Androgen receptor (AR) is a ligand-inducible transcription factor, and a member of the steroid-thyroid-retinoid receptor superfamily, that mediates the biological effects of androgens in a wide range of physiological and pathological processes. AR expression was identified in vascular cells nearly 20 years ago, and recent research has shown that AR mediates a variety of actions of androgens in endothelial and vascular smooth muscle cells. In this mini-review, we review evidence indicating the importance of AR in human endothelial cell (HUVEC) homeostatic and pathogenic processes. Although a role for AR in the modulation of HUVEC biology is evident, the molecular mechanisms by which AR regulates HUVEC homeostasis and disease processes are not fully understood. Understanding these mechanisms could provide critical insights into the processes of pathogenesis of diseases ranging from cardiovascular disease to cancer that are major causes of human morbidity and mortality. PMID:25563353

  10. Phospholipid composition of cultured human endothelial cells.

    PubMed

    Murphy, E J; Joseph, L; Stephens, R; Horrocks, L A

    1992-02-01

    Detailed analyses of the phospholipid compositions of cultured human endothelial cells are reported here. No significant differences were found between the phospholipid compositions of cells from human artery, saphenous and umbilical vein. However, due to the small sample sizes, relatively large standard deviations for some of the phospholipid classes were observed. A representative composition of endothelial cells is: phosphatidylcholine 36.6%, choline plasmalogen 3.7%, phosphatidylethanolamine 10.2%, ethanolamine plasmalogen 7.6%, sphingomyelin 10.8%, phosphatidylserine 7.1%, lysophosphatidylcholine 7.5%, phosphatidylinositol 3.1%, lysophosphatidylethanolamine 3.6%, phosphatidylinositol 4,5-bisphosphate 1.8%, phosphatidic acid 1.9%, phosphatidylinositol 4-phosphate 1.5%, and cardiolipin 1.9%. The cells possess high choline plasmalogen and lysophosphatidylethanolamine contents. The other phospholipids are within the normal biological ranges expected. Phospholipids were separated by high-performance liquid chromatography and quantified by lipid phosphorus assay. PMID:1315902

  11. Replication of human endothelial cells in culture.

    PubMed

    Lewis, L J; Hoak, J C; Maca, R D; Fry, G L

    1973-08-01

    Investigative studies dealing with the properties and functions of endothelial cells have been hampered because there has been little or no success in the isolation, growth, and passage of individual cells in large numbers. We have developed a system whereby pure cultures of endothelial cells derived from umbilical veins can be subcultured for at least five serial passages. Many facets of endothelial function and interaction can be evaluated with the use of this new adaptive system of isolation and culture. PMID:4718112

  12. The effects of Δ9-Tetrahydrocannabinole treatment on gonadal micro-vascularization and affected fertility examined by SEM and 3D-morphometry

    NASA Astrophysics Data System (ADS)

    Erlbacher, K. M. T.; Minnich, B.

    2015-10-01

    The present study focuses on the effects of Δ9-tetrahydrocannabinol (THC) on the reproductive system in nude rats with special emphasis on how Δ9-THC impacts the vascularization of testes which in turn indirectly influences fertility. Basically, Δ9-tetrahydrocannabinol (THC) causes not only negative (psychoactive) effects in the human body as cannabinole administration in medical use (dose-dependent) offers multiple new treatment opportunities such as pain relief or containment of various cancers. Concerning the reproductive system it strongly influences CB-receptors along the hypothalamic-pituitary-gonadal axis resulting in reduced plasma testosterone levels. There is also altered sperm quality parameters reported such as sperm motility or sperm count. On the other hand Δ9-THC effects endothelial growth factors (VEGF, Ang-1 etc.) respectively acts on their specific receptors which in turn modify angiogenesis and vascularization of tissues and organs (e.g. tumorous tissues). This leads to new therapeutical strategies in the suppression of various cancers by inhibiting (neo-)vascularization and in turn famishment of tumorous tissues (lack of nutrition supply). Here we studied the micro-vascularization of gonads in a long-term THC-treated nude rat model by vascular corrosion casting, SEM and 3D-morphometry.

  13. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    SciTech Connect

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

    1990-11-15

    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.

  14. Selective biological response of human pulmonary microvascular endothelial cells and human pulmonary artery smooth muscle cells on cold-plasma-modified polyester vascular prostheses.

    PubMed

    Blanchemain, N; Aguilar, M R; Chai, F; Jimenez, M; Jean-Baptiste, E; El-Achari, A; Martel, B; Hildebrand, H F; Roman, J San

    2011-12-01

    The aim of this work was to improve the hemocompatibility and the selectivity according to cells of non-woven poly(ethylene terephthalate) (PET) membranes. Non-woven PET membranes were modified by a combined plasma-chemical process. The surface of these materials was pre-activated by cold-plasma treatment and poly(acrylic acid) (PAA) was grafted by the in situ free radical polymerization of acrylic acid (AA). The extent of this reaction and the number of carboxylic groups incorporated were evaluated by colorimetric titration using toluidine blue O. All samples were characterized by SEM, AFM and thermogravimetric analysis, and the mechanical properties of the PAA grafted sample were determined. A selective cell response was observed when human pulmonary artery smooth muscle cells (HPASMC) or human pulmonary micro vascular endothelial cells (HPMEC) were seeded on the modified surfaces. HPASMC proliferation decreased about 60%, while HPMEC proliferation was just reduced about 10%. PAA grafted samples did not present hemolytic activity and the platelet adhesion decreased about 28% on PAA grafted surfaces. PMID:22002636

  15. Endothelial cells derived from human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  16. Prolonged cyclic strain inhibits human endothelial cell growth.

    PubMed

    Peyton, Kelly J; Liu, Xiao-ming; Durante, William

    2016-01-01

    The vascular endothelium is continuously exposed to cyclic mechanical strain due to the periodic change in vessel diameter as a result of pulsatile blood flow. Since emerging evidence indicates the cyclic strain plays an integral role in regulating endothelial cell function, the present study determined whether application of a physiologic regimen of cyclic strain (6% at 1 hertz) influences the proliferation of human arterial endothelial cells. Prolonged exposure of human dermal microvascular or human aortic endothelial cells to cyclic strain for up to 7 days resulted in a marked decrease in cell growth. The strain-mediated anti-proliferative effect was associated with the arrest of endothelial cells in the G2/M phase of the cell cycle, did not involve cell detachment or cytotoxicity, and was due to the induction of p21. Interestingly, the inhibition in endothelial cell growth was independent of the strain regimen since prolonged application of constant or intermittent 6% strain was also able to block endothelial cell proliferation. The ability of chronic physiologic cyclic strain to inhibit endothelial cell growth represents a previously unrecognized mechanism by which hemodynamic forces maintain these cells in a quiescent, non-proliferative state. PMID:26709656

  17. Aging and vascular endothelial function in humans

    PubMed Central

    SEALS, Douglas R.; JABLONSKI, Kristen L.; DONATO, Anthony J.

    2012-01-01

    Advancing age is the major risk factor for the development of CVD (cardiovascular diseases). This is attributable, in part, to the development of vascular endothelial dysfunction, as indicated by reduced peripheral artery EDD (endothelium-dependent dilation) in response to chemical [typically ACh (acetylcholine)] or mechanical (intravascular shear) stimuli. Reduced bioavailability of the endothelium-synthesized dilating molecule NO (nitric oxide) as a result of oxidative stress is the key mechanism mediating reduced EDD with aging. Vascular oxidative stress increases with age as a consequence of greater production of reactive oxygen species (e.g. superoxide) without a compensatory increase in antioxidant defences. Sources of increased superoxide production include up-regulation of the oxidant enzyme NADPH oxidase, uncoupling of the normally NO-producing enzyme, eNOS (endothelial NO synthase) (due to reduced availability of the cofactor tetrahydrobiopterin) and increased mitochondrial synthesis during oxidative phosphorylation. Increased bioactivity of the potent endothelial-derived constricting factor ET-1 (endothelin-1), reduced endothelial production of/responsiveness to dilatory prostaglandins, the development of vascular inflammation, formation of AGEs (advanced glycation end-products), an increased rate of endothelial apoptosis and reduced expression of oestrogen receptor α (in postmenopausal females) also probably contribute to impaired EDD with aging. Several lifestyle and biological factors modulate vascular endothelial function with aging, including regular aerobic exercise, dietary factors (e.g. processed compared with non-processed foods), body weight/fatness, vitamin D status, menopause/oestrogen deficiency and a number of conventional and non-conventional risk factors for CVD. Given the number of older adults now and in the future, more information is needed on effective strategies for the prevention and treatment of vascular endothelial aging. PMID

  18. "All-laser" endothelial corneal transplant in human patients

    NASA Astrophysics Data System (ADS)

    Rossi, Francesca; Menabuoni, Luca; Malandrini, Alex; Canovetti, Annalisa; Lenzetti, Ivo; Pini, Roberto

    2012-03-01

    Femtosecond laser sculpturing of corneal tissue is commonly used for the preparation of endothelial flaps. Diode laser welding of ocular tissues is a procedure that enables minimally invasive suturing of tissues. The combination of these laser based techniques results in a new approach to minimally invasive ophthalmic surgery, such as in endothelial corneal transplant (or endothelial keratoplasty - EK). In this work we present the "all laser" EK performed in human subjects. 24 pseudophakic patients with bullous keratopathy underwent EK: the femtosecond laser was used to prepare the 100 ìm thick and 8.5 mm diameter donor Descemet endothelial flap. After staining the stromal layer of the donor flap with a liquid ICG solution, the donor flap was inserted in the recipient eye by the use of the Busin injector. Then, the endothelial layer was laser-welded to the recipient eye (10 laser spots around the periphery of the flap), in order to reduce the risk of postoperative dislocation of the transplanted flap. A transplanted flap engraftment was observed in all the treated eyes. The staining procedure used to perform laser welding also enabled to evidence the stromal side of the donor flap, so as the flap was always placed in the right side position. The endothelial cells counts in both the laserwelded flaps and in a control group were in good agreement. The proposed technique is easy to perform and enables the reduction of postoperative endothelial flap dislocations.

  19. Human cultured endothelial cells do secrete endothelin-1

    SciTech Connect

    Clozel, M.; Fischli, W. )

    1989-01-01

    Endothelin-1 (ET-1) has been identified in the conditioned medium of porcine endothelial cells. Human endothelin (ET-1) cloned from a placenta cDNA library is similar to porcine, but it is not known whether endothelin itself is secreted by human endothelial cells. To answer this question, a conditioned medium taken every 48 h from confluent cultures of umbilical vein endothelial cells was analyzed by HPLC and all fractions were tested for their ability to inhibit ({sup 125}I)ET-1 binding on human placenta membranes. Only one fraction did inhibit ({sup 125}I)ET-1 binding. When the conditioned medium was spiked with ET-1, the same single fraction inhibited ({sup 125}I)ET-1 binding showing that ET-1, itself, is present in the conditioned medium of human endothelial cells. ET-1 accumulates with time, reaching a plateau at 48 h. ET-1 secretion is not increased by a 24-h incubation of endothelial cells with phorbol myristate acetate, interleukin-1, tumor necrosis factor, thrombin or neuropeptide Y.

  20. Human endothelial cell culture plaques induced by Rickettsia rickettsii.

    PubMed Central

    Walker, D H; Firth, W T; Edgell, C J

    1982-01-01

    Primary cultures of human umbilical vein endothelial cells were inoculated with plaque-purified Rickettsia rickettsii. After adsorption of rickettsiae, monolayers were overlaid with medium containing 0.5% agarose. Small plaques appeared on day 4 postinoculation, and distinct 1- to 2-mm plaques were observed on day 5. Plaquing efficiency was less than that of primary chicken embryo cells in the same medium. Human endothelial cell monolayers were susceptible to infection by R. rickettsii and underwent necrosis as demonstrated by supravital staining. The topographic association of endothelial cell necrosis and rickettsial infection in the plaque model confirmed the direct cytopathic effect of R. rickettsii on human endothelium. Uninfected cells appeared normal by supravital staining and transmission electron microscopy. This model offers the possibility of investigating rickettsial pathogenesis and mechanisms of enhanced severity of Rocky Mountain spotted fever in specific genetically determined conditions. Images PMID:6809631

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

    NASA Astrophysics Data System (ADS)

    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.

  2. The effects of glucocorticoids on cultured human endothelial cells.

    PubMed

    Maca, R D; Fry, G L; Hoak, J C

    1978-04-01

    The effects of hydrocortisone, dexamethasone and prednisone on the morphology, replication, DNA synthesis, cell protein content and protein synthesis of cultured, human endothelial cells were evaluated. After culturing the cells with these glucocorticoids for 24-48 h, the cells covered a greater portion of the culture surface area. The mean surface area of the individual endothelial cell treated with glucocorticoids was 1.53 times greater than that of the untreated control endothelial cell. When compared with controls, the endothelial cover provided by the cells treated with glucocorticoids was more extensive and in many instances covered the entire culture surface. The change in morphology was associated with an increase in protein synthesis and protein content of the cells without an increase in DNA synthesis or cellular replication. Dexamethasone was approximately 10-fold more effective than hydrocortisone, while prednisone was the least effective. Aldosterone, DOCA, testosterone, progesterone, oestradiol and oestriol were ineffective. These studies indicate that glucocorticoids can alter the morphology and biochemistry of cultured endothelial cells and may have implications for the effects of steroids in the treatment of thrombocytopenic states and vascular disorders in man. PMID:646949

  3. Novel role of lactosylceramide in vascular endothelial growth factor-mediated angiogenesis in human endothelial cells.

    PubMed

    Rajesh, Mohanraj; Kolmakova, Antonina; Chatterjee, Subroto

    2005-10-14

    Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis associated with coronary heart disease, vascular complications in diabetes, inflammatory vascular diseases, and tumor metastasis. The mechanism of VEGF-driven angiogenesis involving glycosphingolipids such as lactosylceramide (LacCer), however, is not known. To demonstrate the involvement of LacCer in VEGF-induced angiogenesis, we used small interfering RNA (siRNA)-mediated silencing of LacCer synthase expression (GalT-V) in human umbilical vein endothelial cells. This gene silencing markedly inhibited VEGF-induced platelet endothelial cell adhesion molecule-1 (PECAM-1) expression and angiogenesis. Second, we used D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of LacCer synthase and glucosylceramide synthase, that significantly mitigated VEGF-induced PECAM-1 expression and angiogenesis. Interestingly, these phenotypic changes were reversed by LacCer but not by structurally related compounds such as glucosylceramide, digalactosylceramide, and ceramide. In a human mesothelioma cell line (REN) that lacks the endogenous expression of PECAM-1, VEGF/LacCer failed to stimulate PECAM-1 expression and tube formation/angiogenesis. In REN cells expressing human PECAM-1 gene/protein, however, both VEGF and LacCer-induced PECAM-1 protein expression and tube formation/angiogenesis. In fact, VEGF-induced but not LacCer-induced angiogenesis was mitigated by SU-1498, a VEGF receptor tyrosine kinase inhibitor. Also, VEGF/LacCer-induced PECAM-1 expression and angiogenesis was mitigated by protein kinase C and phospholipase A2 inhibitors. These results indicate that LacCer generated in VEGF-treated endothelial cells may serve as an important signaling molecule for PECAM-1 expression and in angiogenesis. This finding and the reagents developed in our report may be useful as anti-angiogenic drugs for further studies in vitro and in vivo. PMID:16151023

  4. Subtractive transcriptomics : establishing polarity drives human endothelial morphogenesis

    SciTech Connect

    Glesne, D. A.; Zhang, W.; Mandava, S.; Ursos, L.; Buell, M. E.; Makowski, L.; Rodi, D. J.; Biosciences Division

    2006-04-15

    Although investigations of mature normal and tumor-derived capillaries have resulted in characterization of these structures at the phenotypic level, less is known regarding the initial molecular cues for cellular assembly of endothelial cells into human capillaries. Here, we employ a novel combination of microenvironmental manipulation and microarray data filtration over narrowly delineated temporal data series to identify the morphogenesis component apart from the proliferation component, as pooled human microvascular-derived endothelial cells are induced to form capillary-like structures in vitro in a murine tumor-derived matrix. The 217 morphogenesis-specific genes identified using this subtractive transcriptomics approach are mostly independent of the angiogenic proteins currently used as therapeutic targets for aberrant angiogenesis. Quantitative real-time PCR was used to validate 20% of these transcripts. Immunofluorescent analysis of proliferating and tube-forming cells validates at the protein level the morphogenesis-specific expression pattern of 16 of the 217 gene products identified. The transcripts that are selectively up-regulated in tube-forming endothelial cells reveal a temporal expression pattern of genes primarily associated with intracellular trafficking, guided migration, cytoskeletal reorganization, cellular adhesion, and proliferation inhibition. These data show that a sequential upregulation of genes that establish and maintain polarity occurs during migration and morphogenesis of in vitro human endothelial cells undergoing tubulogenesis; some of which may well be effective as novel antiangiogenic drug targets.

  5. Mineralocorticoid Receptors Modulate Vascular Endothelial Function in Human Obesity

    PubMed Central

    Hwang, Moon-Hyon; Yoo, Jeung-Ki; Luttrell, Meredith; Kim, Han-Kyul; Meade, Thomas H.; English, Mark; Segal, Mark S.; Christou, Demetra D.

    2015-01-01

    Obesity increases linearly with age and is associated with impaired vascular endothelial function and increased risk for cardiovascular disease. Mineralocorticoid receptors (MR) contribute to impaired vascular endothelial function in cardiovascular disease; however, their role in uncomplicated human obesity is unknown. Because plasma aldosterone levels are elevated in obesity and adipocytes may be a source of aldosterone, we hypothesized that MR modulate vascular endothelial function in older adults in an adiposity-dependent manner. To test this hypothesis, we administered MR blockade (Eplerenone; 100 mg/day) for 1 month in a balanced, randomized, double-blind, placebo-controlled, crossover study to 22 older adults (10 men, 55–79 years) varying widely in adiposity (body mass index: 20–45 kg/m2) but who were free from overt cardiovascular disease. We evaluated vascular endothelial function (brachial artery flow-mediated dilation [FMD] via ultrasonography) and oxidative stress (plasma F2-isoprostanes and vascular endothelial cell protein expression of nitrotyrosine and NADPH oxidase p47phox) during placebo and MR blockade. In the whole group, oxidative stress (P>0.05) and FMD did not change with MR blockade (6.39±0.67 vs. 6.23±0.73 %, P=0.7, placebo vs. Eplerenone). However, individual improvements in FMD in response to Eplerenone were associated with higher total body fat (body mass index: r=0.45, P=0.02 and DXA-derived % body fat: r=0.50, P=0.009) and abdominal fat (total: r=0.61, P=0.005, visceral: r=0.67, P=0.002 and subcutaneous: r=0.48, P=0.03). In addition, greater improvements in FMD with Eplerenone were related with higher baseline fasting glucose (r=0.53, P=0.01). MR influence vascular endothelial function in an adiposity-dependent manner in healthy older adults. PMID:23786536

  6. Regulation of human endothelial progenitor cell maturation by polyurethane nanocomposites.

    PubMed

    Hung, Huey-Shan; Yang, Yi-Chun; Lin, Yu-Chun; Lin, Shinn-Zong; Kao, Wei-Chien; Hsieh, Hsien-Hsu; Chu, Mei-Yun; Fu, Ru-Huei; Hsu, Shan-hui

    2014-08-01

    The mobilization and homing of endothelial progenitor cells (EPCs) are critical to the development of an antithrombotic cardiovascular prosthesis. Polyurethane (PU) with superior elasticity may provide a mechanical environment resembling that of the natural vascular tissues. The topographical cues of PU were maximized by making nanocomposites with a small amount of gold nanoparticles (AuNPs). The nanocomposites of PU-AuNPs ("PU-Au") with a favorable response of endothelial cells were previously established. In the current study, the effect of PU and PU-Au nanocomposites on the behavior of human peripheral blood EPCs was investigated in vitro and in vivo. It was found that PU-Au promoted EPCs to become differentiated endothelial cells in vitro, confirmed by the increased expressions of CD31 and VEGF-R2 surface markers. The increased maturation of EPCs was significantly more remarkable on PU-Au, probably through the stromal derived factor 1α (SDF-1α)/CXCR4 signaling pathway. In vivo experiments showed that EPCs seeded on PU-Au coated catheters effectively reduced thrombosis by differentiation into endothelial cells. Surface endothelialization with CD31 and CD34 expression as well as intimal formation with α-SMA expression was significantly accelerated in the group receiving EPC-seeded PU-Au catheters. Moreover, the analysis of collagen deposition revealed a reduction of fibrosis in the group receiving EPC-seeded PU-Au catheters as compared to the other groups. These results suggest that EPCs engineered with a proper elastic substrate may provide unique endothelialization and antithrombogenic properties that benefit vascular tissue regeneration. PMID:24836305

  7. The expression of ADAMTS13 in human microvascular endothelial cells.

    PubMed

    Wang, Anyou; Duan, Qiaohong; Wu, Jingsheng; Liu, Xin; Sun, Zimin

    2016-06-01

    ADAMTS13, as a specific von Willebrand factor (VWF)-cleaving protease, prevents microvascular thrombosis of VWF/platelet thrombi. It has been reported that human vascular endothelial cells could also synthesize and secrete ADAMTS13, and these reports were focused in human umbilical vascular endothelial cells. Considering the particularity of its huge quantity and structure of human microvascular endothelial cells (HMECs) in the body, whether ADAMTS13 is expressed in HMECs also needs to be confirmed. To investigate whether ADAMTS13 is expressed in HMECs. Real-time PCR (RT-PCR) amplification detected ADAMTS13 mRNA in HMEC-1 cell line. The expression and distribution of ADAMTS13 protein and VWF were detected by fluorescence immunoassay and western blot. We observed the expression and distribution of ADAMTS13 in HMECs. We confirmed the expression of ADAMTS13 mRNA in HMEC-1, and found that there were some partly common distributions of ADAMTS13 protein and VWF. This study provides the evidence that HMECs also express ADAMTS13. HMECs might also be a primary source for human plasma ADAMTS13. The overlap region for the distribution of ADAMTS13 and VWF suggests that ADAMTS13 might have a potential regulation role for VWF inside cells. PMID:26366828

  8. Soluble CD40 ligand induces endothelial dysfunction in human and porcine coronary artery endothelial cells.

    PubMed

    Chen, Changyi; Chai, Hong; Wang, Xinwen; Jiang, Jun; Jamaluddin, Md Saha; Liao, Dan; Zhang, Yuqing; Wang, Hao; Bharadwaj, Uddalak; Zhang, Sheng; Li, Min; Lin, Peter; Yao, Qizhi

    2008-10-15

    The purpose of this study was to determine the effects and mechanisms of sCD40L on endothelial dysfunction in both human coronary artery endothelial cells (HCAECs) and porcine coronary artery rings. HCAECs treated with sCD40L showed significant reductions of endothelial nitric oxide synthase (eNOS) mRNA and protein levels, eNOS mRNA stability, eNOS enzyme activity, and cellular NO levels, whereas superoxide anion (O(2)(-)) production was significantly increased. sCD40L enhanced eNOS mRNA 3'UTR binding to cytoplasmic molecules and induced a unique expression pattern of 95 microRNAs. sCD40L significantly decreased mitochondrial membrane potential, and catalase and SOD activities, whereas it increased NADPH oxidase (NOX) activity. sCD40L increased phosphorylation of MAPKs p38 and ERK1/2 as well as IkappaBalpha and enhanced NF-kappaB nuclear translocation. In porcine coronary arteries, sCD40L significantly decreased endothelium-dependent vasorelaxation and eNOS mRNA levels, whereas it increased O(2)(-) levels. Antioxidant seleno-l-methionine; chemical inhibitors of p38, ERK1/2, and mitochondrial complex II; as well as dominant negative mutant forms of IkappaBalpha and NOX4 effectively blocked sCD40L-induced eNOS down-regulation in HCAECs. Thus, sCD40L reduces eNOS levels, whereas it increases oxidative stress through the unique molecular mechanisms involving eNOS mRNA stability, 3'UTR-binding molecules, microRNAs, mitochondrial function, ROS-related enzymes, p38, ERK1/2, and NF-kappaB signal pathways in endothelial cells. PMID:18658029

  9. Modeling human endothelial cell transformation in vascular neoplasias

    PubMed Central

    Wen, Victoria W.; MacKenzie, Karen L.

    2013-01-01

    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. PMID:24046386

  10. Bilirubin is an Endogenous Antioxidant in Human Vascular Endothelial Cells.

    PubMed

    Ziberna, Lovro; Martelanc, Mitja; Franko, Mladen; Passamonti, Sabina

    2016-01-01

    Bilirubin is a standard serum biomarker of liver function. Inexplicably, it is inversely correlated with cardiovascular disease risk. Given the role of endothelial dysfunction in originating cardiovascular diseases, direct analysis of bilirubin in the vascular endothelium would shed light on these relationships. Hence, we used high-performance liquid chromatography coupled with thermal lens spectrometric detection and diode array detection for the determination of endogenous cellular IXα-bilirubin. To confirm the isomer IXα-bilirubin, we used ultra-performance liquid chromatography coupled with a high-resolution mass spectrometer using an electrospray ionization source, as well as tandem mass spectrometric detection. We measured bilirubin in both arterial and venous rat endothelium (0.9-1.5 pmol mg(-1) protein). In the human endothelial Ea.hy926 cell line, we demonstrated that intracellular bilirubin (3-5 pmol mg(-1) protein) could be modulated by either extracellular bilirubin uptake, or by up-regulation of heme oxygenase-1, a cellular enzyme related to endogenous bilirubin synthesis. Moreover, we determined intracellular antioxidant activity by bilirubin, with EC50 = 11.4 ± 0.2 nM, in the range of reported values of free serum bilirubin (8.5-13.1 nM). Biliverdin showed similar antioxidant properties as bilirubin. We infer from these observations that intra-endothelial bilirubin oscillates, and may thus be a dynamic factor of the endothelial function. PMID:27381978

  11. Bilirubin is an Endogenous Antioxidant in Human Vascular Endothelial Cells

    PubMed Central

    Ziberna, Lovro; Martelanc, Mitja; Franko, Mladen; Passamonti, Sabina

    2016-01-01

    Bilirubin is a standard serum biomarker of liver function. Inexplicably, it is inversely correlated with cardiovascular disease risk. Given the role of endothelial dysfunction in originating cardiovascular diseases, direct analysis of bilirubin in the vascular endothelium would shed light on these relationships. Hence, we used high-performance liquid chromatography coupled with thermal lens spectrometric detection and diode array detection for the determination of endogenous cellular IXα-bilirubin. To confirm the isomer IXα-bilirubin, we used ultra-performance liquid chromatography coupled with a high-resolution mass spectrometer using an electrospray ionization source, as well as tandem mass spectrometric detection. We measured bilirubin in both arterial and venous rat endothelium (0.9–1.5 pmol mg−1 protein). In the human endothelial Ea.hy926 cell line, we demonstrated that intracellular bilirubin (3–5 pmol mg−1 protein) could be modulated by either extracellular bilirubin uptake, or by up-regulation of heme oxygenase-1, a cellular enzyme related to endogenous bilirubin synthesis. Moreover, we determined intracellular antioxidant activity by bilirubin, with EC50 = 11.4 ± 0.2 nM, in the range of reported values of free serum bilirubin (8.5–13.1 nM). Biliverdin showed similar antioxidant properties as bilirubin. We infer from these observations that intra-endothelial bilirubin oscillates, and may thus be a dynamic factor of the endothelial function. PMID:27381978

  12. Virulent Treponema pallidum activates human vascular endothelial cells.

    PubMed

    Riley, B S; Oppenheimer-Marks, N; Hansen, E J; Radolf, J D; Norgard, M V

    1992-03-01

    Perivascular lymphocytic infiltration, fibrin deposition, and endothelial cell abnormalities consistent with cellular activation are prominent histopathologic features of syphilis, a sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. Because activated endothelial cells play important roles in lymphocyte homing and hemostasis, the ability of virulent T. pallidum to activate cultured human umbilical vein endothelial cells (HUVEC) was investigated. T. pallidum induced the expression of intercellular adhesion molecule-1 (ICAM-1) and procoagulant activity on the surface of HUVEC. Electron microscopy of T. pallidum-stimulated HUVEC revealed extensive networks of fibrin strands not observed in cultures without treponemes. ICAM-1 expression in HUVEC also was promoted by a 47-kDa integral membrane lipoprotein purified from T. pallidum, implicating a role for spirochete membrane lipoproteins in endothelial cell activation. The combined findings are consistent with the pathology of syphilis and provide the first evidence that a pathogenic spirochetal bacterium such as T. pallidum or its constituent integral membrane lipoprotein(s) can activate directly host vascular endothelium. PMID:1347056

  13. Functional CB1 cannabinoid receptors in human vascular endothelial cells.

    PubMed Central

    Liu, J; Gao, B; Mirshahi, F; Sanyal, A J; Khanolkar, A D; Makriyannis, A; Kunos, G

    2000-01-01

    Cannabinoid CB1 receptor mRNA was detected using reverse transcription-polymerase chain reaction (RT-PCR) in endothelial cells from human aorta and hepatic artery and in the ECV304 cell line derived from human umbilical vein endothelial cells. CB1 receptor-binding sites were detected by the high-affinity antagonist radioligand [(125)I]AM-251. In ECV304 cells, both the highly potent synthetic cannabinoid agonist HU-210 and the endogenous ligand anandamide induce activation of mitogen-activated protein (MAP) kinase, and the effect of HU-210 was completely blocked, whereas the effect of anandamide was partially inhibited by SR141716A, a selective CB1 receptor antagonist. Transfection of ECV304 cells with CB1 receptor antisense, but not sense, oligonucleotides caused the same pattern of inhibition as SR141716A. This provides more definitive evidence for the involvement of CB1 receptors in MAP kinase activation and suggests that anandamide may also activate MAP kinase via an additional, CB1 receptor-independent, SR141716A-resistant mechanism. The MAP kinase activation by anandamide in ECV304 cells requires genistein-sensitive tyrosine kinases and protein kinase C (PKC), and anandamide also activates p38 kinase and c-Jun kinase. These findings indicate that CB1 receptors located in human vascular endothelium are functionally coupled to the MAP kinase cascade. Activation of protein kinase cascades by anandamide may be involved in the modulation of endothelial cell growth and proliferation. PMID:10698714

  14. Resistin increases monolayer permeability of human coronary artery endothelial cells.

    PubMed

    Jamaluddin, Md Saha; Yan, Shaoyu; Lü, Jianming; Liang, Zhengdong; Yao, Qizhi; Chen, Changyi

    2013-01-01

    Resistin has been linked to obesity, insulin resistance, atherosclerosis, and the development of cardiovascular disease. Nevertheless, the effects and the molecular mechanisms of resistin on endothelial permeability, a key event in the development of atherosclerosis, inflammation, and vascular disease, are largely unknown. In order to determine the effect of resistin on endothelial permeability, human coronary artery endothelial cells (HCAECs) were treated with clinically relevant concentrations of resistin and the endothelial permeability was measured using the Transwell system with a Texas-Red-labeled dextran tracer. The permeability of HCAEC monolayers treated with resistin (80 ng/mL) was 51% higher than the permeability of control monolayers (P<0.05). The mRNA levels of tight junction proteins zonula occludens-1 (ZO-1) and occludin in resistin-treated cells were 37% and 42% lower, respectively, than the corresponding levels in untreated cells. The protein levels of these molecules in resistin-treated cells were significantly reduced by 35% and 37%, respectively (P<0.05), as shown by flow cytometry and Western blot analysis. Superoxide dismutase (SOD) mimetic MnTBAP effectively blocked the resistin-mediated reduction of ZO-1 and occludin levels in HCAECs. In addition, superoxide anion production was increased from 21% (untreated cells) to 55% (cells treated with 40 ng/mL resistin), and 64% (resistin, 80 mg/mL) (P<0.05). The natural antioxidant Ginkgolide A effectively inhibited resistin-induced increase in permeability and the increase in superoxide anion production in HCAECs. Furthermore, resistin treatment significantly activated p38 MAPK, but not ERK1/2. Pretreatment of HCAECs with a p38 inhibitor effectively blocked resistin-induced permeability. These results provide new evidence that resistin may contribute to the vascular lesion formation via increasing endothelial permeability through the mechanism of oxidative stress and the activation of p38 MAPK. PMID

  15. Resistin Increases Monolayer Permeability of Human Coronary Artery Endothelial Cells

    PubMed Central

    Jamaluddin, Md Saha; Yan, Shaoyu; Lü, Jianming; Liang, Zhengdong; Yao, Qizhi; Chen, Changyi

    2013-01-01

    Resistin has been linked to obesity, insulin resistance, atherosclerosis, and the development of cardiovascular disease. Nevertheless, the effects and the molecular mechanisms of resistin on endothelial permeability, a key event in the development of atherosclerosis, inflammation, and vascular disease, are largely unknown. In order to determine the effect of resistin on endothelial permeability, human coronary artery endothelial cells (HCAECs) were treated with clinically relevant concentrations of resistin and the endothelial permeability was measured using the Transwell system with a Texas-Red-labeled dextran tracer. The permeability of HCAEC monolayers treated with resistin (80 ng/mL) was 51% higher than the permeability of control monolayers (P<0.05). The mRNA levels of tight junction proteins zonula occludens-1 (ZO-1) and occludin in resistin-treated cells were 37% and 42% lower, respectively, than the corresponding levels in untreated cells. The protein levels of these molecules in resistin-treated cells were significantly reduced by 35% and 37%, respectively (P<0.05), as shown by flow cytometry and Western blot analysis. Superoxide dismutase (SOD) mimetic MnTBAP effectively blocked the resistin-mediated reduction of ZO-1 and occludin levels in HCAECs. In addition, superoxide anion production was increased from 21% (untreated cells) to 55% (cells treated with 40 ng/mL resistin), and 64% (resistin, 80 mg/mL) (P<0.05). The natural antioxidant Ginkgolide A effectively inhibited resistin-induced increase in permeability and the increase in superoxide anion production in HCAECs. Furthermore, resistin treatment significantly activated p38 MAPK, but not ERK1/2. Pretreatment of HCAECs with a p38 inhibitor effectively blocked resistin-induced permeability. These results provide new evidence that resistin may contribute to the vascular lesion formation via increasing endothelial permeability through the mechanism of oxidative stress and the activation of p38 MAPK. PMID

  16. Image analysis of human corneal endothelial cells based on fractal theory

    NASA Astrophysics Data System (ADS)

    Zhang, Zhi; Luo, Qingming; Zeng, Shaoqun; Zhang, Xinyu; Huang, Dexiu; Chen, Weiguo

    1999-09-01

    A fast method is developed to quantitatively characterize the shape of human corneal endothelial cells with fractal theory and applied to analyze microscopic photographs of human corneal endothelial cells. The results show that human corneal endothelial cells possess the third characterization parameter-- fractal dimension, besides another two characterization parameter (its size and shape). Compared with tradition method, this method has many advantages, such as automatism, speediness, parallel processing and can be used to analyze large numbers of endothelial cells, the obtained values are statistically significant, it offers a new approach for clinic diagnosis of endothelial cells.

  17. Identification of endothelial antigens relevant to transplant coronary artery disease from a human endothelial cell cDNA expression library.

    PubMed

    Ationu, A

    1998-06-01

    Accelerated transplant coronary artery disease (TxCAD) results in increased expression of antiendothelial antibodies whose target antigens remain largely unidentified. One of these endothelial antigens has been identified as vimentin, a cytoskeletal protein present in cells of the blood vessel walls. In the present study, SDS-PAGE and Western blot analysis of human endothelial cell (EAHy 926) lysates probed with sera from a TxCAD patient were used to confirm immunoreactivity of antiendothelial antibodies towards several endothelial proteins. To further elucidate the identity of these putative antigens, a human endothelial cell (EAHy 926) cDNA expression library was immunoscreened with serum obtained from a TxCAD patient. Two positive cDNA clones were identified by partial nucleotide sequence analysis and GenBank/EMBL database searches for homology as the 85 kDa human CD36 antigen (a cell surface glycoprotein expressed in various cells including epithelial and endothelial cells) and a 50 kDa keratin-like protein (a member of the intermediate filament protein expressed in epithelial cells). These results are the first to demonstrate that human CD36 antigen and a keratin-like protein may be additional target proteins for the anti-endothelial antibodies associated with TxCAD. PMID:9852639

  18. [Isolation of endothelial cells from human umbilical cords and development of low-cost culture medium].

    PubMed

    Karasaki, Y

    1996-12-01

    To study the role of endothelial cells in important biological phenomena such as thrombosis and atherosclerosis, it is necessary to have a sufficient amount of endothelial cells. This report shows modified methods to isolate endothelial cells from the human umbilical vein and the components of potent and low-cost culture medium for the cells. PMID:8981651

  19. Modulation of Human Vascular Endothelial Cell Behaviors by Nanotopographic Cues

    PubMed Central

    Liliensiek, S.J.; Wood, J.A.; Yong, J.; Auerbach, R.; Nealey, P.F.; Murphy, C.J.

    2010-01-01

    Basement membranes possess a complex three dimensional topography in the nanoscale and submicron range which have been shown to profoundly modulate a large menu of fundamental cell behaviors. Using the topographic features found in native vascular endothelial basement membranes as a guide, polyurethane substrates were fabricated containing anisotropically ordered ridge and groove structures and isotropically ordered pores from 200 nm to 2000 nm in size. We investigated the impact of biomimetic length-scale topographic cues on orientation/elongation, proliferation and migration on four human vascular endothelial cell-types from large and small diameter vessels. We found that all cell-types exhibited orientation and alignment with the most pronounced response on anisotropically ordered ridges ≥ 800 nm. HUVEC cells were the only cell-type examined to demonstrate a decrease in proliferation in response to the smallest topographic features regardless of surface order. On anisotropically ordered surfaces all cell types migrated preferentially parallel to the long axis of the ridges, with the greatest increase in cell migration being observed on the 1200 nm pitch. In contrast, cells did not exhibit any preference in direction or increase in migration speed on isotropically ordered surfaces. Overall, our data demonstrate that surface topographic features impact vascular endothelial cell behavior and that the impact of features varies with the cell behavior being considered, topographic feature scale, surface order, and the anatomic origin of the cell being investigated. PMID:20400175

  20. [Isolation, culture and identification of human umbilical vein endothelial cells].

    PubMed

    Chen, Xiaocui; Chen, Bangdang; Yang, Yining; Zhou, Yun; Liu, Fen; Gai, Mintao; Chen, Qingjie; Ma, Yitong

    2016-03-01

    Objective To establish a simple, reliable and efficient isolation and culture method of human umbilical vein endothelial cells (HUVECs) in vitro. Methods Type 2 collagenase was used to digest umbilical cord and separate HUVECs. The cells were cultured in the endothelial cell culture medium (ECM). The cell morphology was observed under an inverted phase-contrast microscope. Immunofluorescence technique was applied to detect the expression of von Willebrand factor (vWF). Cell purity was determined by detecting CD31 level on cell surface with flow cytometry. Tube formation assay was used to test the function of the endothelial cells after cryopreservation in vitro. Results HUVECs successfully isolated were proved with high purity and good activity. HUVECs of primary generation could merge into a single layer one week after isolation. The cells showed a typical cobblestone-like arrangement. Immunofluorescence technique validated that the cells could widely express vWF and the expression frequency of CD31 was 93.1%. The cells were still capable of forming the lumen structure after cryopreservation, indicating that the standardized cryopreservation method could well maintain the cell function. Conclusion This is a simple, reliable and efficient method of isolating and culturing HUVECs in vitro. PMID:26927551

  1. Modulation of human vascular endothelial cell behaviors by nanotopographic cues.

    PubMed

    Liliensiek, Sara J; Wood, Joshua A; Yong, Jiang; Auerbach, Robert; Nealey, Paul F; Murphy, Christopher J

    2010-07-01

    Basement membranes possess a complex three-dimensional topography in the nanoscale and submicron range which have been shown to profoundly modulate a large menu of fundamental cell behaviors. Using the topographic features found in native vascular endothelial basement membranes as a guide, polyurethane substrates were fabricated containing anisotropically ordered ridge and groove structures and isotropically ordered pores from 200 nm to 2000 nm in size. We investigated the impact of biomimetic length-scale topographic cues on orientation/elongation, proliferation and migration on four human vascular endothelial cell-types from large and small diameter vessels. We found that all cell-types exhibited orientation and alignment with the most pronounced response on anisotropically ordered ridges > or =800 nm. HUVEC cells were the only cell-type examined to demonstrate a decrease in proliferation in response to the smallest topographic features regardless of surface order. On anisotropically ordered surfaces all cell-types migrated preferentially parallel to the long axis of the ridges, with the greatest increase in cell migration being observed on the 1200 nm pitch. In contrast, cells did not exhibit any preference in direction or increase in migration speed on isotropically ordered surfaces. Overall, our data demonstrate that surface topographic features impact vascular endothelial cell behavior and that the impact of features varies with the cell behavior being considered, topographic feature scale, surface order, and the anatomic origin of the cell being investigated. PMID:20400175

  2. Syndecan-2 downregulation impairs angiogenesis in human microvascular endothelial cells

    SciTech Connect

    Noguer, Oriol Villena, Joan; Lorita, Jordi; Vilaro, Senen; Reina, Manuel

    2009-03-10

    The formation of new blood vessels, or angiogenesis, is a necessary process during development but also for tumour growth and other pathologies. It is promoted by different growth factors that stimulate endothelial cells to proliferate, migrate, and generate new tubular structures. Syndecans, transmembrane heparan sulphate proteoglycans, bind such growth factors through their glycosaminoglycan chains and could transduce the signal to the cytoskeleton, thus regulating cell behaviour. We demonstrated that syndecan-2, the major syndecan expressed by human microvascular endothelial cells, is regulated by growth factors and extracellular matrix proteins, in both bidimensional and tridimensional culture conditions. The role of syndecan-2 in 'in vitro' tumour angiogenesis was also examined by inhibiting its core protein expression with antisense phosphorothioate oligonucleotides. Downregulation of syndecan-2 reduces spreading and adhesion of endothelial cells, enhances their migration, but also impairs the formation of capillary-like structures. These results suggest that syndecan-2 has an important function in some of the necessary steps that make up the angiogenic process. We therefore propose a pivotal role of this heparan sulphate proteoglycan in the formation of new blood vessels.

  3. Glycoproteomic Analysis of the Secretome of Human Endothelial Cells*

    PubMed Central

    Yin, Xiaoke; Bern, Marshall; Xing, Qiuru; Ho, Jenny; Viner, Rosa; Mayr, Manuel

    2013-01-01

    Previous proteomics studies have partially unraveled the complexity of endothelial protein secretion but have not investigated glycosylation, a key modification of secreted and membrane proteins for cell communication. In this study, human umbilical vein endothelial cells were kept in serum-free medium before activation by phorbol-12-myristate-13 acetate, a commonly used secretagogue that induces exocytosis of endothelial vesicles. In addition to 123 secreted proteins, the secretome was particularly rich in membrane proteins. Glycopeptides were enriched by zwitterionic hydrophilic interaction liquid chromatography resins and were either treated with PNGase F and H218O or directly analyzed using a recently developed workflow combining higher-energy C-trap dissociation (HCD) with electron-transfer dissociation (ETD) for a hybrid linear ion trap–orbitrap mass spectrometer. After deglycosylation with PNGase F in the presence of H218O, 123 unique peptides displayed 18O-deamidation of asparagine, corresponding to 86 proteins with a total of 121 glycosylation sites. Direct glycopeptide analysis via HCD-ETD identified 131 glycopeptides from 59 proteins and 118 glycosylation sites, of which 41 were known, 51 were predicted, and 26 were novel. Two methods were compared: alternating HCD-ETD and HCD-product-dependent ETD. The former detected predominantly high-intensity, multiply charged glycopeptides, whereas the latter preferentially selected precursors with complex/hybrid glycans for fragmentation. Validation was performed by means of glycoprotein enrichment and analysis of the input, the flow-through, and the bound fraction. This study represents the most comprehensive characterization of endothelial protein secretion to date and demonstrates the potential of new HCD-ETD workflows for determining the glycosylation status of complex biological samples. PMID:23345538

  4. Glycated serum albumin stimulates expression of endothelial cell specific molecule-1 in human umbilical vein endothelial cells: Implication in diabetes mediated endothelial dysfunction.

    PubMed

    Nirala, Bikesh K; Perumal, Vivekanandan; Gohil, Nivedita K

    2015-07-01

    Pro-inflammatory conditions induced by products of protein glycation in diabetes substantially enhance the risk of endothelial dysfunction and related vascular complications. Endothelial cell specific molecule-1 (ESM-1) or endocan has been demonstrated as a potential biomarker in cancer and sepsis. Its role in diabetes-induced pathologies remains unknown. The expression of ESM-1 gene is under cytokine regulation, indicating its role in endothelium-dependent pathological disorders. In this study, we investigated the effect of advanced glycated human serum albumin (AGE-HSA) on the production of ESM-1. We show that AGE-HSA exerts a modulating role on the expression of ESM-1 in human umbilical vein endothelial cells. It up-regulates expression of ESM-1 protein in a dose-dependent manner which correlates with its messenger RNA (mRNA) transcription. RAGE and galectin-3, both AGE receptors, show antagonistic action on its expression. While gene silencing of RAGE has down-regulatory effect, that of galectin-3 has up-regulatory effect on AGE-induced expression of ESM-1. Inhibition of MAPKKK and JNK pathways did not alter the expression. In contrast, phosphatidylinositol 3 kinase (PI3K) inhibition significantly up-regulated ESM-1 expression. In conclusion, these results suggest that AGE-induced activation of human umbilical vein endothelial cells promotes formation of endocan which is an endothelial dysfunction marker and may be related to vascular disease in diabetes. PMID:25963575

  5. Antiproliferative effect of elevated glucose in human microvascular endothelial cells

    NASA Technical Reports Server (NTRS)

    Kamal, K.; Du, W.; Mills, I.; Sumpio, B. E.

    1998-01-01

    Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P < 0.05) HMEC-1 proliferation after 7, 10, and 14 days. This effect was not mimicked by 20 mM mannitol. The antiproliferative effect was more pronounced with longer exposure (1-14 days) to elevated glucose and was irreversible 4 days after a 10-day exposure. The antiproliferative effect was partially reversed in the presence of a PKA inhibitor, Rp-cAMP (10-50 microM), and/or a PKC inhibitor, Calphostin C (10 nM). HMEC-1 exposed to elevated glucose (20 mM) for 14 days caused an increase in cyclic AMP accumulation, PKA, and PKC activity but was not associated with the activation of downstream events such as CRE and AP-1 binding activity. These data support the hypothesis that HMEC-1 is a suitable model to study the deleterious effects of elevated glucose on microvascular endothelial cells. Continued studies with HMEC-1 may prove advantageous in delineation of the molecular

  6. Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin.

    PubMed

    Yu, Young; Wise, Steven G; Michael, Praveesuda L; Bax, Daniel V; Yuen, Gloria S C; Hiob, Matti A; Yeo, Giselle C; Filipe, Elysse C; Dunn, Louise L; Chan, Kim H; Hajian, Hamid; Celermajer, David S; Weiss, Anthony S; Ng, Martin K C

    2015-01-01

    The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs) are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE) and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVβ3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants. PMID:26115013

  7. Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin

    PubMed Central

    Yu, Young; Wise, Steven G.; Michael, Praveesuda L.; Bax, Daniel V.; Yuen, Gloria S. C.; Hiob, Matti A.; Yeo, Giselle C.; Filipe, Elysse C.; Dunn, Louise L.; Chan, Kim H.; Hajian, Hamid; Celermajer, David S.; Weiss, Anthony S.; Ng, Martin K. C.

    2015-01-01

    The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs) are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE) and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVβ3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants. PMID:26115013

  8. 3D map of the human corneal endothelial cell

    PubMed Central

    He, Zhiguo; Forest, Fabien; Gain, Philippe; Rageade, Damien; Bernard, Aurélien; Acquart, Sophie; Peoc’h, Michel; Defoe, Dennis M.; Thuret, Gilles

    2016-01-01

    Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin, braced like a drum. Lateral membranes, which support enzymatic pumps, presented complex expansions resembling interdigitated foot processes at the basal surface. Using computer-aided design and drafting software, we obtained a first simplified 3D model of CECs. By comparing their expression with those in epithelial, stromal and trabecular corneal cells, we selected 9 structural or functional proteins for which 3D patterns were specific to CECs. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions. PMID:27381832

  9. 3D map of the human corneal endothelial cell.

    PubMed

    He, Zhiguo; Forest, Fabien; Gain, Philippe; Rageade, Damien; Bernard, Aurélien; Acquart, Sophie; Peoc'h, Michel; Defoe, Dennis M; Thuret, Gilles

    2016-01-01

    Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin, braced like a drum. Lateral membranes, which support enzymatic pumps, presented complex expansions resembling interdigitated foot processes at the basal surface. Using computer-aided design and drafting software, we obtained a first simplified 3D model of CECs. By comparing their expression with those in epithelial, stromal and trabecular corneal cells, we selected 9 structural or functional proteins for which 3D patterns were specific to CECs. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions. PMID:27381832

  10. Volume changes of human endothelial cells induced by photodynamic treatment

    NASA Astrophysics Data System (ADS)

    Leunig, Andreas; Staub, Frank; Plesnila, Nick; Peters, Jurgen; Feyh, Jens; Gutmann, Ralph; Goetz, Alwin E.

    1996-01-01

    Photodynamic therapy (PDT) has shown promising results in treatment of malignant tumors. However, the mechanisms leading to tumor destruction during PDT are still not completely understood. In addition to effects on the microcirculation, damage to cellular structures has been observed following exposure of cells to PDT. A phenomenon preceding these events might possibly be cell swelling. We therefore studied the influence of treatment with Photofrin (PF) and laser light on volume changes and cell viability of endothelial cells. Endothelial cells were obtained from human umbilical cord veins (HUVEC) by an adaption of the method of Maruyama (1963). After subcultivation the cells were harvested and transferred as a cell suspension into a specially designed incubation chamber. Cells received either PF in concentrations of 1.5 or 3.0 (mu) g/ml and laser illumination (630 nm; 40 mW/cm2, 4 Joule), PF alone, or laser treatment only. Following start of PF incubation and after phototreatment cell samples were taken for volume measurements using flow cytometry and for studies of cellular morphology using scanning electron microscopy. Simultaneously, cell viability was monitored by the trypan blue exclusion test and colorimetric MTT assay. (abstract truncated)

  11. Three electrophysiological phenotypes of cultured human umbilical vein endothelial cells.

    PubMed

    Yu, K; Ruan, D Y; Ge, S Y

    2002-09-01

    The conventional whole cell patch-clamp technique was used to measure the resting membrane conductance and membrane currents of nonstimulated cultured human umbilical vein endothelial cells (HUVECs) in different ionic conditions. Three electrophysiological phenotypes of cultured HUVECs (n = 122) were determined: first, 20% of cells as type I mainly displaying the inwardly rectifying potassium current (IKi); second, 38% of cells as type II in which IKi was super-posed on a TEA-sensitive, delayed rectifying current; third, 27% of cells as type III predominantly displaying the outwardly rectifying current which was sensitive to TEA and slightly inhibited by a chloride channel blocker niflumic acid (N.A.). In cells of type I, the mean zero-current potential (V0) was dependent on extracellular K+ ([K+]o) but not on Cl-, indicating major permeability to K+. Whereas V0 of type II was also affected by extracellular Cl- ([Cl-]o), indicating the contribution of an outward Cl- current in setting V0. The cells of type III were not sensitive to decrease of [Cl-]o and the outward current was activated in a relative stable voltage range. This varying phenotypic expression and multipotential behavior of HUVECs suggests that the electrical features of HUVEC may be primarily determined by embryonic origin and local effect of the microenvironment. This research provided the detailed electrophysiological knowledge of the endothelial cells. PMID:12537354

  12. Endothelial human dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling

    PubMed Central

    Whitsett, Jennifer; Filho, Artur Rangel; Sethumadhavan, Savitha; Celinska, Joanna; Widlansky, Michael; Vásquez-Vivar, Jeannette

    2013-01-01

    Tetrahydrobiopterin (BH4) is required for NO synthesis and inhibition of superoxide release from eNOS. Clinical trials using BH4 to treat endothelial dysfunction have produced mixed results. Poor outcomes may be explained by the rapid systemic and cellular oxidation of BH4. One of the oxidation products of BH4, 7,8-dihydrobiopterin (7,8-BH2), is recycled back to BH4 by dihydrofolate reductase (DHFR). This enzyme is ubiquitously distributed and shows a wide range of activity depending on species-specific factors and cell type. Information about the kinetics and efficiency of BH4 recycling in human endothelial cells receiving BH4 treatment is lacking. To characterize this reaction, we applied a novel multi-electrode coulometric HPLC method that enabled the direct quantification of 7,8-BH2 and BH4 which is not possible with fluorescent-based methodologies. We found that basal untreated BH4 and 7,8-BH2 concentrations in human ECs is lower than bovine and murine endothelioma cells. Treatment of human ECs with BH4 transiently increased intracellular BH4 while accumulating the more stable 7,8-BH2. This was different from bovine or murine ECs that resulted in preferential BH4 increase. Using BH4 diastereomers, 6S-BH4 and 6R-BH4, the narrow contribution of enzymatic DHFR recycling to total intracellular BH4 was demonstrated. Reduction of 7,8-BH2 to BH4 occurs at very slow rates in cells and needs supra-physiological levels of 7,8-BH2, indicating this reaction is kinetically limited. Activity assays verified that hDHFR has very low affinity for 7,8-BH2 (DHF7,8-BH2) and folic acid inhibits 7,8-BH2 recycling. We conclude that low activity of endothelial DHFR is an important factor limiting the benefits of BH4 therapies which may be further aggravated by folate supplements. PMID:23707606

  13. Perfluorooctane sulfonate (PFOS) induces reactive oxygen species (ROS) production in human microvascular endothelial cells: role in endothelial permeability

    PubMed Central

    Qian, Yong; Ducatman, Alan; Ward, Rebecca; Leonard, Steve; Bukowski, Valerie; Guo, Nancy Lan; Shi, Xianglin; Vallyathan, Val; Castranova, Vincent

    2011-01-01

    Perfluorooctane sulfonate (PFOS) is a member of perfluoroalkyl acids (PFAA) containing an 8-carbon backbone. PFOS is a man-made chemical with carbon-fluorine bonds that are one of the strongest in organic chemistry and widely used in industry. Human occupational and environmental exposure to PFOS occurs globally. PFOS is non-biodegradable and persistent in the human body and environment. In this study, data demonstrated that exposure of human microvascular endothelial cells (HMVEC) to PFOS induced the production of reactive oxygen species (ROS) at both high and low concentrations. Morphologically, it was found that exposure to PFOS induced actin filament remodeling and endothelial permeability changes in HMVEC. Furthermore, data demonstrated the production of ROS plays a regulatory role in PFOS-induced actin filament remodeling and the increase in endothelial permeability. Our results indicate that the generation of ROS may play a role in PFOS-induced aberrations of the endothelial permeability barrier. The results generated from this study may provide a new insight into the potential adverse effects of PFOS exposure on humans at the cellular level. PMID:20391123

  14. Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin.

    PubMed

    Gospodarowicz, D; Brown, K D; Birdwell, C R; Zetter, B R

    1978-06-01

    Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of

  15. Directed Endothelial Progenitor Differentiation from Human Pluripotent Stem Cells Via Wnt Activation Under Defined Conditions.

    PubMed

    Bao, Xiaoping; Lian, Xiaojun; Palecek, Sean P

    2016-01-01

    Efficient derivation of endothelial cells and their progenitors from human pluripotent stem cells (hPSCs) can facilitate studies of human vascular development, disease modeling, drug discovery, and cell-based therapy. Here we provide a detailed protocol for directing hPSCs to functional endothelial cells and their progenitors in a completely defined, growth factor- and serum-free system by temporal modulation of Wnt/β-catenin signaling via small molecules. We demonstrate a 10-day, two-stage process that recapitulates endothelial cell development, in which hPSCs first differentiate to endothelial progenitors that then generate functional endothelial cells and smooth muscle cells. Methods to characterize endothelial cell identity and function are also described. PMID:27590162

  16. Expression and endocytosis of VEGF and its receptors in human colonic vascular endothelial cells.

    PubMed

    Wang, Dongfang; Lehman, Richard E; Donner, David B; Matli, Mary R; Warren, Robert S; Welton, Mark L

    2002-06-01

    Normal human colonic microvascular endothelial cells (HUCMEC) have been isolated from surgical specimens by their adherence to Ulex europaeus agglutinin bound to magnetic dynabeads that bind alpha-L-fucosyl residues on the endothelial cell membrane. Immunocytochemistry demonstrated the presence of a range of endothelial-specific markers on HUCMEC, including the von Willebrand factor, Ulex europaeus agglutinin, and platelet endothelial cell adhesion molecule-1. The growing cells form monolayers with the characteristic cobblestone morphology of endothelial cells and eventually form tube-like structures. HUCMEC produce vascular endothelial growth factor (VEGF) and express the receptors, kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase, through which VEGF mediates its actions in the endothelium. VEGF induces the tyrosine phosphorylation of KDR and a proliferative response from HUCMEC comparable to that elicited from human umbilical vein endothelial cells (HUVEC). On binding to HUCMEC or HUVEC, (125)I-labeled VEGF internalizes or dissociates to the medium. Once internalized, (125)I-labeled VEGF is degraded and no evidence of ligand recycling was observed. However, significantly less VEGF is internalized, and more is released to the medium from HUCMEC than HUVEC. Angiogenesis results from the proliferation and migration of microvascular, not large-vessel, endothelial cells. The demonstration that microvascular endothelial cells degrade less and release more VEGF to the medium than large-vessel endothelial cells identifies a mechanism permissive of the role of microvascular cells in angiogenesis. PMID:12016135

  17. An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

    1998-04-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

  18. Nanoliposomes protect against human arteriole endothelial dysfunction induced by β-amyloid peptide.

    PubMed

    Truran, Seth; Weissig, Volkmar; Madine, Jillian; Davies, Hannah A; Guzman-Villanueva, Diana; Franco, Daniel A; Karamanova, Nina; Burciu, Camelia; Serrano, Geidy; Beach, Thomas G; Migrino, Raymond Q

    2016-02-01

    We tested whether nanoliposomes containing phosphatidylcholine, cholesterol and phosphatidic acid (NLPA) prevent β-amyloid 1-42 (Aβ42) fibrillation and Aβ42-induced human arteriole endothelial dysfunction. NLPA abolished Aβ42 fibril formation (thioflavin-T fluorescence/electron microscopy). In ex-vivo human adipose and leptomeningeal arterioles, Aβ42 impaired dilator response to acetylcholine that was reversed by NLPA; this protection was abolished by L-NG-nitroarginine methyl ester. Aβ42 reduced human umbilical vein endothelial cell NO production that was restored by NLPA. Nanoliposomes prevented Aβ42 amyloid formation, reversed Aβ42-induced human microvascular endothelial dysfunction and may be useful in Alzheimer's disease. PMID:26661197

  19. Transcript Analysis Reveals a Specific HOX Signature Associated with Positional Identity of Human Endothelial Cells

    PubMed Central

    Toshner, Mark; Dunmore, Benjamin J.; McKinney, Eoin F.; Southwood, Mark; Caruso, Paola; Upton, Paul D.; Waters, John P.; Ormiston, Mark L.; Skepper, Jeremy N.; Nash, Gerard; Rana, Amer A.; Morrell, Nicholas W.

    2014-01-01

    The endothelial cell has a remarkable ability for sub-specialisation, adapted to the needs of a variety of vascular beds. The role of developmental programming versus the tissue contextual environment for this specialization is not well understood. Here we describe a hierarchy of expression of HOX genes associated with endothelial cell origin and location. In initial microarray studies, differential gene expression was examined in two endothelial cell lines: blood derived outgrowth endothelial cells (BOECs) and pulmonary artery endothelial cells. This suggested shared and differential patterns of HOX gene expression between the two endothelial lines. For example, this included a cluster on chromosome 2 of HOXD1, HOXD3, HOXD4, HOXD8 and HOXD9 that was expressed at a higher level in BOECs. Quantative PCR confirmed the higher expression of these HOXs in BOECs, a pattern that was shared by a variety of microvascular endothelial cell lines. Subsequently, we analysed publically available microarrays from a variety of adult cell and tissue types using the whole “HOX transcriptome” of all 39 HOX genes. Using hierarchical clustering analysis the HOX transcriptome was able to discriminate endothelial cells from 61 diverse human cell lines of various origins. In a separate publically available microarray dataset of 53 human endothelial cell lines, the HOX transcriptome additionally organized endothelial cells related to their organ or tissue of origin. Human tissue staining for HOXD8 and HOXD9 confirmed endothelial expression and also supported increased microvascular expression of these HOXs. Together these observations suggest a significant involvement of HOX genes in endothelial cell positional identity. PMID:24651450

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

    PubMed Central

    Muid, Suhaila; Froemming, Gabriele R. Anisah; Rahman, Thuhairah; Ali, A. Manaf; Nawawi, Hapizah M.

    2016-01-01

    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, there is a

  1. Virulent Treponema pallidum promotes adhesion of leukocytes to human vascular endothelial cells.

    PubMed Central

    Riley, B S; Oppenheimer-Marks, N; Radolf, J D; Norgard, M V

    1994-01-01

    Perivasculitis and endothelial cell abnormalities are characteristic histopathologic features of syphilis, a sexually transmitted disease caused by Treponema pallidum. To extend earlier studies demonstrating that T. pallidum activates endothelial cells, we now show that virulent T. pallidum, but not heat-killed T. pallidum or nonpathogenic Treponema phagedenis, promotes increased adherence of lymphocytes and monocytes to human umbilical vein endothelial cells. Lymphocytes and monocytes are the two cell types prominent in the histopathology of syphilis. Recognition that T. pallidum can stimulate endothelial cells to bind leukocytes provides important insights into the early mechanisms of syphilis immunopathogenesis. Images PMID:7927729

  2. Ultrasound fails to induce proliferation of human brain and mouse endothelial cell lines

    NASA Astrophysics Data System (ADS)

    Rodemer, Claus; Jenne, Jürgen; Fatar, Marc; Hennerici, Michael G.; Meairs, Stephen

    2012-11-01

    Both in vitro and in vivo studies suggest that ultrasound (US) is capable of inducing angiogenesis. There is no information, however, on whether ultrasound can induce proliferation of brain endothelial cells. We therefore explored the angiogenic potential of ultrasound on a novel immortalised human brain endothelial cell line (hCMEC/D3) and on mouse brain microvascular endothelial cells (bEND3). Ultrasound failed to enhance cell proliferation in both cell lines at all acoustic pressures studied. Endothelial cell damage occurred at 0.24 MPa with significantly slower proliferation. Cells growing in Opticell{trade mark, serif} dishes did not show damage or reduced proliferation at these pressures.

  3. SIRT3 Deficiency Induces Endothelial Insulin Resistance and Blunts Endothelial-Dependent Vasorelaxation in Mice and Human with Obesity

    PubMed Central

    Yang, Lu; Zhang, Julei; Xing, Wenjuan; Zhang, Xing; Xu, Jie; Zhang, Haifeng; Chen, Li; Ning, Xiaona; Ji, Gang; Li, Jia; Zhao, Qingchuan; Gao, Feng

    2016-01-01

    Recent evidence implicates the critical role of Sirtuin 3 (SIRT3) in the development of many metabolic diseases, but the contribution of SIRT3 to vascular homeostasis remains largely unknown. The aim of this study was to investigate the role of SIRT3 in endothelial insulin resistance and vascular dysfunction in obesity. We found an impaired insulin-induced mesenteric vasorelaxation and concomitant reduced vascular SIRT3 expression in morbid obese human subjects compared with the non-obese subjects. Downregulation of SIRT3 in cultured human endothelial cells increased mitochondrial reactive oxygen species (mtROS) and impaired insulin signaling as evidenced by decreased phosphorylation of Akt and endothelial nitric oxide synthase and subsequent reduced nitric oxide (NO) release. In addition, obese mice induced by 24-week high-fat diet (HFD) displayed an impaired endothelium-dependent vasorelaxation to both insulin and acetylcholine, which was further exacerbated by the gene deletion of Sirt3. Scavenging of mtROS not only restored insulin-stimulated NO production in SIRT3 knockdown cells, but also improved insulin-induced vasorelaxation in SIRT3 knockout mice fed with HFD. Taken together, our findings suggest that SIRT3 positively regulates endothelial insulin sensitivity and show that SIRT3 deficiency and resultant increased mtROS contribute to vascular dysfunction in obesity. PMID:27000941

  4. Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue.

    PubMed

    Ugele, B; Lange, F

    2001-01-01

    Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary. PMID:11573814

  5. SIRT3 Deficiency Induces Endothelial Insulin Resistance and Blunts Endothelial-Dependent Vasorelaxation in Mice and Human with Obesity.

    PubMed

    Yang, Lu; Zhang, Julei; Xing, Wenjuan; Zhang, Xing; Xu, Jie; Zhang, Haifeng; Chen, Li; Ning, Xiaona; Ji, Gang; Li, Jia; Zhao, Qingchuan; Gao, Feng

    2016-01-01

    Recent evidence implicates the critical role of Sirtuin 3 (SIRT3) in the development of many metabolic diseases, but the contribution of SIRT3 to vascular homeostasis remains largely unknown. The aim of this study was to investigate the role of SIRT3 in endothelial insulin resistance and vascular dysfunction in obesity. We found an impaired insulin-induced mesenteric vasorelaxation and concomitant reduced vascular SIRT3 expression in morbid obese human subjects compared with the non-obese subjects. Downregulation of SIRT3 in cultured human endothelial cells increased mitochondrial reactive oxygen species (mtROS) and impaired insulin signaling as evidenced by decreased phosphorylation of Akt and endothelial nitric oxide synthase and subsequent reduced nitric oxide (NO) release. In addition, obese mice induced by 24-week high-fat diet (HFD) displayed an impaired endothelium-dependent vasorelaxation to both insulin and acetylcholine, which was further exacerbated by the gene deletion of Sirt3. Scavenging of mtROS not only restored insulin-stimulated NO production in SIRT3 knockdown cells, but also improved insulin-induced vasorelaxation in SIRT3 knockout mice fed with HFD. Taken together, our findings suggest that SIRT3 positively regulates endothelial insulin sensitivity and show that SIRT3 deficiency and resultant increased mtROS contribute to vascular dysfunction in obesity. PMID:27000941

  6. Human hepatocytes and endothelial cells in organotypic membrane systems.

    PubMed

    Salerno, Simona; Campana, Carla; Morelli, Sabrina; Drioli, Enrico; De Bartolo, Loredana

    2011-12-01

    The realization of organotypic liver model that exhibits stable phenotype is a major challenge in the field of liver tissue engineering. In this study we developed liver organotypic co-culture systems by using synthetic and biodegradable membranes with primary human hepatocytes and human umbilical vein endothelial cells (HUVEC). Synthetic membranes prepared by a polymeric blend constituted of modified polyetheretherketone (PEEK-WC) and polyurethane (PU) and biodegradable chitosan membranes were developed by phase inversion technique and used in homotypic and organotypic culture systems. The morphological and functional characteristics of cells in the organotypic co-culture membrane systems were evaluated in comparison with homotypic cultures and traditional systems. Hepatocytes in the organotypic co-culture systems exhibit compact polyhedral cells with round nuclei and well demarcated cell-cell borders like in vivo, as a result of heterotypic interaction with HUVECs. In addition HUVECs formed tube-like structures directly through the interactions with the membranes and hepatocytes and indirectly through the secretion of ECM proteins which secretion improved in the organotypic co-culture membrane systems. The heterotypic cell-cell contacts have beneficial effect on the hepatocyte albumin production, urea synthesis and drug biotransformation. The developed organotypic co-culture membrane systems elicit liver specific functions in vitro and could be applied for the realization of engineered liver tissues to be used in tissue engineering, drug metabolism studies and bioartificial liver devices. PMID:21871658

  7. Focally regulated endothelial proliferation and cell death in human synovium.

    PubMed Central

    Walsh, D. A.; Wade, M.; Mapp, P. I.; Blake, D. R.

    1998-01-01

    Angiogenesis and vascular insufficiency each may support the chronic synovial inflammation of rheumatoid arthritis. We have shown by quantitative immunohistochemistry and terminal uridyl deoxynucleotide nick end labeling that endothelial proliferation and cell death indices were each increased in synovia from patients with rheumatoid arthritis compared with osteoarthritic and noninflamed controls, whereas endothelial fractional areas did not differ significantly among disease groups. Markers of proliferation were associated with foci immunoreactive for vascular endothelial growth factor and integrin alpha(v)beta3, whereas cell death was observed in foci in which immunoreactivities for these factors were weak or absent. No association was found with thrombospondin immunoreactivity. The balance between angiogenesis and vascular regression in rheumatoid synovitis may be determined by the focal expression of angiogenic and endothelial survival factors. Increased endothelial cell turnover may contribute to microvascular dysfunction and thereby facilitate persistent synovitis. Images Figure 1 Figure 3 Figure 4 PMID:9502411

  8. Plastic Compressed Collagen as a Novel Carrier for Expanded Human Corneal Endothelial Cells for Transplantation

    PubMed Central

    Levis, Hannah J.; Peh, Gary S. L.; Toh, Kah-Peng; Poh, Rebekah; Shortt, Alex J.; Drake, Rosemary A. L.; Mehta, Jodhbir S.; Daniels, Julie T.

    2012-01-01

    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. PMID:23226443

  9. Transdifferentiation of human endothelial progenitors into smooth muscle cells.

    PubMed

    Ji, HaYeun; Atchison, Leigh; Chen, Zaozao; Chakraborty, Syandan; Jung, Youngmee; Truskey, George A; Christoforou, Nicolas; Leong, Kam W

    2016-04-01

    Access to smooth muscle cells (SMC) would create opportunities for tissue engineering, drug testing, and disease modeling. Herein we report the direct conversion of human endothelial progenitor cells (EPC) to induced smooth muscle cells (iSMC) by induced expression of MYOCD. The EPC undergo a cytoskeletal rearrangement resembling that of mesenchymal cells within 3 days post initiation of MYOCD expression. By day 7, the reprogrammed cells show upregulation of smooth muscle markers ACTA2, MYH11, and TAGLN by qRT-PCR and ACTA2 and MYH11 expression by immunofluorescence. By two weeks, they resemble umbilical artery SMC in microarray gene expression analysis. The iSMC, in contrast to EPC control, show calcium transients in response to phenylephrine stimulation and a contractility an order of magnitude higher than that of EPC as determined by traction force microscopy. Tissue-engineered blood vessels constructed using iSMC show functionality with respect to flow- and drug-mediated vasodilation and vasoconstriction. PMID:26874281

  10. Adhesion Molecule Expression in Human Endothelial Cells under Simulated Microgravity

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

    Voglauer, Regina; Chang, Martina Wei-Fen; Dampier, Brigitta; Wieser, Matthias; Baumann, Kristin; Sterovsky, Thomas; Schreiber, Martin; Katinger, Hermann; Grillari, Johannes . E-mail: j.grillari@iam.boku.ac.at

    2006-04-01

    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.

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

    PubMed Central

    McCabe, Kathryn L.; Kunzevitzky, Noelia J.; Chiswell, Brian P.; Xia, Xin; Goldberg, Jeffrey L.; Lanza, Robert

    2015-01-01

    Aim To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. Materials and Methods 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. Results 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. Conclusion 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. PMID:26689688

  13. Innate Immunity in Human Embryonic Stem Cells: Comparison with Adult Human Endothelial Cells

    PubMed Central

    Badiger, Rekha; Paul-Clark, Mark; Moreno, Laura; Lendvai, Zsuzsanna; Wright, Jamie S.; Ali, Nadire N.; Harding, Sian E.; Mitchell, Jane A.

    2010-01-01

    Treatment of human disease with human embryonic stem cell (hESC)-derived cells is now close to reality, but little is known of their responses to physiological and pathological insult. The ability of cells to respond via activation of Toll like receptors (TLR) is critical in innate immune sensing in most tissues, but also extends to more general danger sensing, e.g. of oxidative stress, in cardiomyocytes. We used biomarker release and gene-array analysis to compare responses in hESC before and after differentiation, and to those in primary human endothelial cells. The presence of cardiomyocytes and endothelial cells was confirmed in differentiated cultures by immunostaining, FACS-sorting and, for cardiomyocytes, beating activity. Undifferentiated hESC did not respond with CXCL8 release to Gram positive or Gram negative bacteria, or a range of PAMPs (pathogen associated molecular patterns) for TLRs 1-9 (apart from flagellin, an activator of TLR5). Surprisingly, lack of TLR-dependent responses was maintained over 4 months of differentiation of hESC, in cultures which included cardiomyocytes and endothelial cells. In contrast, primary cultures of human aortic endothelial cells (HAEC) demonstrated responses to a broad range of PAMPs. Expression of downstream TLR signalling pathways was demonstrated in hESC, and IL-1β, TNFα and INFγ, which bypass the TLRs, stimulated CXCL8 release. NFκB pathway expression was also present in hESC and NFκB was able to translocate to the nucleus. Low expression levels of TLRs were detected in hESC, especially TLRs 1 and 4, explaining the lack of response of hESC to the main TLR signals. TLR5 levels were similar between differentiated hESC and HAEC, and siRNA knockdown of TLR5 abolished the response to flagellin. These findings have potential implications for survival and function of grafted hESC-derived cells. PMID:20463927

  14. Effects of Parietaria judaica pollen extract on human microvascular endothelial cells.

    PubMed

    Taverna, Simona; Flugy, Anna; Colomba, Paolo; Barranca, Marilisa; De Leo, Giacomo; Alessandro, Riccardo

    2008-08-01

    Pollinosis from Parietaria judaica is one of the main causes of allergy in the Mediterranean area. The present study is designed to assess if P. judaica pollens contain bioactive compounds able to elicit a functional response in endothelial cells. We have demonstrated that addition of pollen extract to human lung microvascular endothelial cells (HMVEC-L) induces a modification of cell morphology, actin cytoskeletal rearrangements and an increase in endothelial cell permeability. We further showed that the treatment of endothelial cells with pollen extract causes an increase of E-selectin and VCAM-1 protein levels as well as an increase of IL-8 production. The stimulation of cell-cell adhesion molecules was paralleled by a dose-dependent increase of adhesion of polymorphonuclear cells (PMNs) to HMVEC-L monolayer. Our results suggest for the first time that pollen affect directly endothelial cells (EC) modulating critical functions related to the inflammatory response. PMID:18515075

  15. Speckle-correlation monitoring of the internal micro-vascular flow

    NASA Astrophysics Data System (ADS)

    Zimnyakov, D. A.; Khmara, M. B.; Vilensky, M. A.; Kozlov, V. V.; Gorfinkel, I. V.; Zdrajevsky, R. A.

    2009-10-01

    The results of experimental study of possibility to monitor the micro-vascular blood flow in superficial tissues of various organs with the use of endoscope-based full-field speckle correlometer are presented. The blood microcirculation monitoring was carried out in the course of the laparotomy of abdominal cavity of laboratory animals (rats). Transfer of laser light to the area of interest and scattered radiation from the probed zone to the detector (CMOS camera) was carried out via fiber-optic bundles of endoscopic system. Microscopic hemodynamics was analyzed for small intestine, liver, spleen, kidney, and pancreas under different conditions (normal state, provocated peritonitis and ischemia, administration of vasodilative agents such as papaverine, lidocaine). The prospects and problems of internal monitoring of microvascular flow in laboratory and clinical conditions are discussed.

  16. Micro-vascular shape-memory polymer actuators with complex geometries obtained by laser stereolithography

    NASA Astrophysics Data System (ADS)

    Díaz Lantada, Andrés; de Blas Romero, Adrián; Chacón Tanarro, Enrique

    2016-06-01

    In our work we present the complete development process of geometrically complex micro-vascular shape-memory polymer actuators. The complex geometries and three-dimensional networks are designed by means of computer aided design resources. Manufacture is accomplished, in a single step, by means of laser stereolithography, directly from the computer-aided design files with the three dimensional geometries of the different actuators under development. To our knowledge, laser stereolithography is applied here for the first time to the development of shape memory polymer devices with complex geometries and inner micro-vasculatures for their activation using a thermal fluid. Final testing of the developed actuators helps to validate the approach and to put forward some present challenges.

  17. A Methodology for Concomitant Isolation of Intimal and Adventitial Endothelial Cells from the Human Thoracic Aorta

    PubMed Central

    Leclercq, Anne; Veillat, Véronique; Loriot, Sandrine; Spuul, Pirjo; Madonna, Francesco; Roques, Xavier; Génot, Elisabeth

    2015-01-01

    Background Aortic diseases are diverse and involve a multiplicity of biological systems in the vascular wall. Aortic dissection, which is usually preceded by aortic aneurysm, is a leading cause of morbidity and mortality in modern societies. Although the endothelium is now known to play an important role in vascular diseases, its contribution to aneurysmal aortic lesions remains largely unknown. The aim of this study was to define a reliable methodology for the isolation of aortic intimal and adventitial endothelial cells in order to throw light on issues relevant to endothelial cell biology in aneurysmal diseases. Methodology/Principal Findings We set up protocols to isolate endothelial cells from both the intima and the adventitia of human aneurysmal aortic vessel segments. Throughout the procedure, analysis of cell morphology and endothelial markers allowed us to select an endothelial fraction which after two rounds of expansion yielded a population of >90% pure endothelial cells. These cells have the features and functionalities of freshly isolated cells and can be used for biochemical studies. The technique was successfully used for aortic vessel segments of 20 patients and 3 healthy donors. Conclusions/Significance This simple and highly reproducible method allows the simultaneous preparation of reasonably pure primary cultures of intimal and adventitial human endothelial cells, thus providing a reliable source for investigating their biology and involvement in both thoracic aneurysms and other aortic diseases. PMID:26599408

  18. Substrates for Expansion of Corneal Endothelial Cells towards Bioengineering of Human Corneal Endothelium

    PubMed Central

    Navaratnam, Jesintha; Utheim, Tor P.; Rajasekhar, Vinagolu K.; Shahdadfar, Aboulghassem

    2015-01-01

    Corneal endothelium is a single layer of specialized cells that lines the posterior surface of cornea and maintains corneal hydration and corneal transparency essential for vision. Currently, transplantation is the only therapeutic option for diseases affecting the corneal endothelium. Transplantation of corneal endothelium, called endothelial keratoplasty, is widely used for corneal endothelial diseases. However, corneal transplantation is limited by global donor shortage. Therefore, there is a need to overcome the deficiency of sufficient donor corneal tissue. New approaches are being explored to engineer corneal tissues such that sufficient amount of corneal endothelium becomes available to offset the present shortage of functional cornea. Although human corneal endothelial cells have limited proliferative capacity in vivo, several laboratories have been successful in in vitro expansion of human corneal endothelial cells. Here we provide a comprehensive analysis of different substrates employed for in vitro cultivation of human corneal endothelial cells. Advances and emerging challenges with ex vivo cultured corneal endothelial layer for the ultimate goal of therapeutic replacement of dysfunctional corneal endothelium in humans with functional corneal endothelium are also presented. PMID:26378588

  19. Photodynamic effect of hypericin in primary cultures of human umbilical endothelial cells and glioma cell lines.

    PubMed

    Stupáková, Viktória; Varinská, Lenka; Mirossay, Andrej; Sarisský, Marek; Mojzis, Ján; Dankovcík, Róbert; Urdzík, Peter; Ostró, Alexander; Mirossay, Ladislav

    2009-06-01

    Hypericin is the most powerful naturally occurring photosensitizer and as such there is renaissant interest in the potentials of this compound for anticancer photodynamic therapy (PDT). The purpose of this study was to investigate the hypericin-mediated photodynamic therapy effects on normal human umbilical endothelial cells (HUVECs) in comparison with cancer human glioma cell lines U-87 MG and U-373 MG, in in vitro conditions. The data suggest that endothelial cells as well as glioma cell lines are sensitive only to photoactivated hypericin. The inhibitory effects of photoactivated hypericin did not differ in endothelial compared with tumor cells in cytotoxicity MTT and DNA fragmentation assays. However, an important difference in sensitivity was found between the above mentioned cell types in migration and metalloproteinases inhibition assays performed as cell function tests. The findings in both function tests were supported by the high sensitivity of endothelial cells in an additional angiogenesis test of tubular formation in vitro. PMID:19173218

  20. Phyllanthus emblica L. Enhances Human Umbilical Vein Endothelial Wound Healing and Sprouting

    PubMed Central

    Chularojmontri, Linda; Suwatronnakorn, Maneewan; Wattanapitayakul, Suvara K.

    2013-01-01

    Endothelial dysfunction is the hallmark of impaired wound healing and increased risk of cardiovascular disease. Antioxidants from natural sources decrease oxidative stress and protect against cellular damage caused by reactive oxygen species (ROS). In this study, we examined the antioxidant constituents and capacity of Phyllanthus emblica L. (PE) fruit in freeze-dried power form. The pharmacological properties of PE were investigated using human umbilical vein endothelial cells (HUVECs) in the aspects of endothelial cell proliferation, nitric oxide (NO) production, wound healing, cell migration, in vitro angiogenesis, and VEGF gene expression. The ASC content of PE was 1.574% + 0.046% (w/w) as determined by HPLC and the total phenolic content was 36.1% ± 0.7% gallic acid equivalent when measured by Folin-Ciocalteu assay. The FRAP assay revealed a relatively high antioxidant capacity at 3,643 + 192.5 µmole/mg. PE at 0.1 to 10 µg/mL did not significantly influence endothelial cell proliferation, but at higher concentrations PE decreased cell survival to 62%. PE significantly promoted NO production, endothelial wound closure, endothelial sprouting, and VEGF mRNA expression. Therefore, PE is a candidate for antioxidant supplement that promotes endothelial function and restores wound healing competency. PMID:23606890

  1. Effects of glucocorticoids on the interaction of lymphoblastoid cells with human endothelial cells in vitro.

    PubMed

    Maca, R D; Fry, G L; Hakes, A D

    1978-08-01

    The adhesive characteristics of cultured acute lymphocytic leukemia cells (CCRF-CEM), lymphoma cells (Raji), and freshly isolated acute lymphocytic leukemia cells to human cultured endothelial cells were studied. An assay system was used whereby these neoplastic cells were allowed to interact with endothelial cells while being continuously agitated on a rocking platform. All cell lines adhered significantly to the endothelium monolayers. This process appeared not to be dependent upon intact microtubular or microfilament function. Likewise, removing surface sialic acid from either cell type did not alter this process. In contrast incubating the endothelial cells for 24 or 48 hr with dexamethasone decreased adhesiveness of either CCRF-CEM or Raji cells to the endothelial cells by approximately 40%. Incubating these cells with hydrocortisone instead of dexamethasone for 48 hr was equally as effective in altering the endothelial cell adhesiveness. The decreased adhesiveness could be blocked by cycloheximide, indicating that this altered adhesiveness of the endothelial cells involves protein synthesis, presumably of a surface protein. We suggest that this assay system may provide a means to evaluate other agents that can alter the surface characteristics of endothelial cells, which may have important implications in various disease states such as inflammation, thrombogenesis, and metastatic disease. PMID:276420

  2. A simple and biosafe method for isolation of human umbilical vein endothelial cells.

    PubMed

    Lei, Jinghui; Peng, Sha; Samuel, Sonia B; Zhang, Suli; Wu, Ye; Wang, Pengli; Li, Ya-Feng; Liu, Huirong

    2016-09-01

    Human umbilical vein endothelial cells (HUVECS) are used as an irreplaceable tool for the study of vascular diseases. However, the technicians who isolate HUVECs are largely exposed to potential infectious threats. Here we report the development of a specialized instrument to protect researchers from known or unknown infectious agents when they operate on human umbilical cords. This instrument can be assembled by common laboratory supplies and adapted to accommodate umbilical cords of different lengths. When the cord is enclosed within the instrument, the risk of sample contamination and operator infection is greatly reduced. Using our instrument, endothelial cells were successfully isolated from human umbilical veins without contamination. The cells were verified by their cobblestone-like morphology and by immunofluorescence staining (Factor VIII and CD31 positivity and α-SMA negativity). Our instrument simplifies and optimizes the cell extraction process, and most importantly elevates the biosafety to a higher level during the isolation of human umbilical vein endothelial cells. PMID:27335213

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

    SciTech Connect

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

    1987-05-01

    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 /sup 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 /sup 51/Cr release from radiolabeled monolayers.

  4. Differential regulation of angiopoietin 1 and angiopoietin 2 during dengue virus infection of human umbilical vein endothelial cells: implications for endothelial hyperpermeability.

    PubMed

    Ong, Siew Pei; Ng, Mah Lee; Chu, Justin Jang Hann

    2013-12-01

    Infection with dengue virus (DV) can result in dengue hemorrhagic fever and dengue shock syndrome, where patients suffer from bleeding and plasma leakage involving endothelial cells. Angiopoietins (Ang) 1 and 2 are important angiogenic factors that affect endothelial barrier integrity. In this study, DV was observed to induce endothelial leakage at multiplicity of infection of 10 in primary human umbilical vein endothelial cells (HUVEC) with interendothelial gap formation. Immunostaining of vascular endothelial cadherin (VE-cadherin) and zona occludin 1 (ZO-1) showed the absence of these endothelial junctional proteins at the cell-cell contact zones between adjacent cells. In addition, Ang1 that is required for protecting against endothelial hyperpermeability was found to be down-regulated during DV infection. Treatment with increasing concentrations of recombinant Ang1 was shown to prevent DV-induced endothelial hyperpermeability in a dose-dependent manner by preventing the down-regulation of VE-cadherin and ZO-1 at cell membrane. In contrast, the expression of Ang2, the natural antagonist of Ang1, was observed to be up-regulated during DV infection. Recombinant Ang2 added to HUVEC at non-toxic concentrations showed decreased in transendothelial electrical resistance reading and the down-regulation of VE-cadherin and ZO-1. These findings suggest that DV reduces the expression of Ang1 and enhances the expression of Ang2 in endothelial cells and that this imbalance of Ang 1 and Ang 2 may play a contributing role to the increased permeability of human primary endothelial cells during DV infection. PMID:23989887

  5. CD40-TRAF Signaling Upregulates CX3CL1 and TNF-α in Human Aortic Endothelial Cells but Not in Retinal Endothelial Cells

    PubMed Central

    Greene, Jennifer A.; Portillo, Jose-Andres C.; Lopez Corcino, Yalitza; Subauste, Carlos S.

    2015-01-01

    CD40, CX3CL1 and TNF-α promote atheroma and neointima formation. CD40 and TNF-α are also central to the development of diabetic retinopathy while CX3CL1 may play a role in the pathogenesis of this retinopathy. The purpose of this study was to examine whether CD40 ligation increases CX3CL1 and TNF-α protein expression in human endothelial cells from the aorta and retina. CD154 (CD40 ligand) upregulated membrane-bound and soluble CX3CL1 in human aortic endothelial cells. CD154 triggered TNF-α production by human aortic endothelial cells. TNF Receptor Associated Factors (TRAF) are key mediators of CD40 signaling. Compared to human aortic endothelial cells that express wt CD40, cells that express CD40 with a mutation that prevents TRAF2,3 recruitment, or CD40 with a mutation that prevents TRAF6 recruitment exhibited a profound inhibition of CD154-driven upregulation of membrane bound and soluble CX3CL1 as well as of TNF-α secretion. While both CD154 and TNF-α upregulated CX3CL1 in human aortic endothelial cells, these stimuli could act independently of each other. In contrast to human aortic endothelial cells, human retinal endothelial cells did not increase membrane bound or soluble CX3CL1 expression or secrete TNF-α in response to CD154 even though CD40 ligation upregulated ICAM-1 and CCL2 in these cells. Moreover, TNF-α did not upregulate CX3CL1 in retinal endothelial cells. In conclusion, CD40 ligation increases CX3CL1 protein levels and induces TNF-α production in endothelial cells. However, endothelial cells are heterogeneous in regards to these responses. Human aortic but not retinal endothelial cells upregulated CX3CL1 and TNF-α in response to CD40 ligation, as well as upregulated CX3CL1 in response to TNF-α. These dissimilarities may contribute to differences in regulation of inflammation in large vessels versus the retina. PMID:26710229

  6. Expression of the vascular endothelial growth factor receptor, KDR, in human placenta.

    PubMed Central

    Vuckovic, M; Ponting, J; Terman, B I; Niketic, V; Seif, M W; Kumar, S

    1996-01-01

    Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor known to act directly on vascular endothelial cells by promoting cell proliferation and permeability. To date, 3 structurally related cell surface receptors for VEGF, Flt-1, Flt-4 and KDR, have been identified and shown to be human type III receptor tyrosine kinases. The establishment of a vascular network is crucial to the development of the placenta and occurs through both angiogenesis and vasculogenesis. The signals controlling these processes are unclear. Immunohistochemical and in situ hybridisation techniques have localised VEGF in the trophoblast layers and VEGF binding to placental vascular endothelial cells and haemangioblasts has been shown, suggesting a role for VEGF and its receptors in development of the vascular network. In this study we have used specific antibodies to localise KDR and endothelial cells in 1st and 3rd trimester human placenta. The staining showed a colocalisation of KDR with endothelial cells and haemangioblasts. No staining of trophoblast cells was observed, but strong staining of the endothelial cells was seen in the villous stroma adjacent to areas of trophoblast proliferation. Images Fig. 1 Fig. 2 PMID:8621335

  7. CD133 positive progenitor endothelial cell lines from human cord blood.

    PubMed

    Paprocka, Maria; Krawczenko, Agnieszka; Dus, Danuta; Kantor, Aneta; Carreau, Aude; Grillon, Catherine; Kieda, Claudine

    2011-08-01

    Endothelial progenitor cells (EPCs) modulate postnatal vascularization and contribute to vessel regeneration in adults. Stem cells and progenitor cells were found in umbilical cord blood, bone marrow, and mobilized peripheral blood cells, from where they were isolated and cultured. However, the yield of progenitor cells is usually not sufficient for clinical application and the quality of progenitor cells varies. The aim of the study was the immortalization of early progenitor cells with high proliferative potential, capable to differentiate to EPCs and, further, toward endothelial cells. Two cell lines, namely HEPC-CB.1 and HEPC-CB.2 (human endothelial progenitor cells-cord blood) were isolated. As assessed by specific antibody labeling and flow cytometric analysis, they express a panel of stem cell markers: CD133, CD13, CD271, CD90 and also endothelial cell markers: CD202b, CD309 (VEGFR2), CD146, CD105, and CD143 but they do not present markers of finally differentiated endothelial cells: CD31, vWf, nor CD45 which is a specific hematopoietic cell marker. Using the multiplex Cytometric Bead Assay, the simultaneous production of proangiogenic cytokines IL8, angiogenin, and VEGF was demonstrated in normoxia and was shown to be increased by hypoxia. Both cell lines, similarly as mature endothelial cells, underwent in vitro pre-angiogenic process, formed pseudovessel structures and present an accelerated angiogenesis in hypoxic conditions. To date, these are the first CD133 positive established cell lines from human cord blood cells. PMID:21710642

  8. Impairment of endothelial progenitor cell function and vascularization capacity by aldosterone in mice and humans

    PubMed Central

    Thum, Thomas; Schmitter, Kerstin; Fleissner, Felix; Wiebking, Volker; Dietrich, Bernd; Widder, Julian D.; Jazbutyte, Virginija; Hahner, Stefanie; Ertl, Georg; Bauersachs, Johann

    2011-01-01

    Aims Hyperaldosteronism is associated with vascular injury and increased cardiovascular events. Bone marrow-derived endothelial progenitor cells (EPCs) play an important role in endothelial repair and vascular homeostasis. We hypothesized that hyperaldosteronism impairs EPC function and vascularization capacity in mice and humans. Methods and results We characterized the effects of aldosterone and mineralocorticoid receptor (MR) blockade on EPC number and function as well as vascularization capacity and endothelial function. Treatment of human EPC with aldosterone induced translocation of the MR and impaired multiple cellular functions of EPC, such as differentiation, migration, and proliferation in vitro. Impaired EPC function was rescued by pharmacological blockade or genetic ablation of the MR. Aldosterone protein kinase A (PKA) dependently increased reactive oxygen species formation in EPC. Aldosterone infusion in mice impaired EPC function, EPC homing to vascular structures and vascularization capacity in a MR-dependent but blood pressure-independent manner. Endothelial progenitor cells from patients with primary hyperaldosteronism compared with controls of similar age displayed reduced migratory potential. Impaired EPC function was associated with endothelial dysfunction. MR blockade in patients with hyperaldosteronism improved EPC function and arterial stiffness. Conclusion Endothelial progenitor cells express a MR that mediates functional impairment by PKA-dependent increase of reactive oxygen species. Normalization of EPC function may represent a novel mechanism contributing to the beneficial effects of MR blockade in cardiovascular disease prevention and treatment. PMID:20926363

  9. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo

    NASA Astrophysics Data System (ADS)

    Plate, Karl H.; Breier, Georg; Weich, Herbert A.; Risau, Werner

    1992-10-01

    CLINICAL and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth1,2. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo3,4. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular prolifer-ations5'6. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.

  10. Isolation and expansion of human and mouse brain microvascular endothelial cells.

    PubMed

    Navone, Stefania E; Marfia, Giovanni; Invernici, Gloria; Cristini, Silvia; Nava, Sara; Balbi, Sergio; Sangiorgi, Simone; Ciusani, Emilio; Bosutti, Alessandra; Alessandri, Giulio; Slevin, Mark; Parati, Eugenio A

    2013-09-01

    Brain microvascular endothelial cells (BMVECs) have an important role in the constitution of the blood-brain barrier (BBB). The BBB is involved in the disease processes of a number of neurological disorders in which its permeability increases. Isolation of BMVECs could elucidate the mechanism involved in these processes. This protocol describes how to isolate and expand human and mouse BMVECs. The procedure covers brain-tissue dissociation, digestion and cell selection. Cells are selected on the basis of time-responsive differential adhesiveness to a collagen type I-precoated surface. The protocol also describes immunophenotypic characterization, cord formation and functional assays to confirm that these cells in endothelial proliferation medium (EndoPM) have an endothelial origin. The entire technique requires ∼7 h of active time. Endothelial cell clusters are readily visible after 48 h, and expansion of BMVECs occurs over the course of ∼60 d. PMID:23928501

  11. Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemoattractant, MCP-1/JE.

    PubMed Central

    Rollins, B. J.; Yoshimura, T.; Leonard, E. J.; Pober, J. S.

    1990-01-01

    We have demonstrated inducible expression of the mRNA encoding the monocyte chemoattractant MCP-1, the human homolog of the JE gene, in endothelial cells within 3 hours of treatment with IL-1 beta and tumor necrosis factor. IFN-gamma also induced expression of this mRNA after 24 hours, but to a lesser extent. MCP-1/JE protein steadily accumulated in the medium of endothelial cells during a 48-hour exposure to IL-1 beta. Medium conditioned by IL-1 beta-treated endothelial cells contained monocyte chemoattractant activity that was immunoadsorbed by anti-MCP-1 antibodies. These results suggest that endothelial cells secrete a monocyte chemoattractant, MCP-1/JE, in response to inflammatory mediators, and thus may contribute to the accumulation of monocytes at sites of inflammation. Images Figure 1 Figure 2 PMID:2113354

  12. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells

    PubMed Central

    Patsch, Christoph; Challet-Meylan, Ludivine; Thoma, Eva C.; Urich, Eduard; Heckel, Tobias; O’Sullivan, John F; Grainger, Stephanie J; Kapp, Friedrich G.; Sun, Lin; Christensen, Klaus; Xia, Yulei; Florido, Mary H. C.; He, Wei; Pan, Wei; Prummer, Michael; Warren, Curtis R.; Jakob-Roetne, Roland; Certa, Ulrich; Jagasia, Ravi; Freskgård, Per-Ola; Adatto, Isaac; Kling, Dorothee; Huang, Paul; Zon, Leonard I; Chaikof, Elliot L.; Gerszten, Robert E.; Graf, Martin; Iacone, Roberto; Cowan, Chad A.

    2015-01-01

    The use of human pluripotent stem cells for in vitro disease modeling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies over 80% within six days. Upon purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease. PMID:26214132

  13. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells.

    PubMed

    Patsch, Christoph; Challet-Meylan, Ludivine; Thoma, Eva C; Urich, Eduard; Heckel, Tobias; O'Sullivan, John F; Grainger, Stephanie J; Kapp, Friedrich G; Sun, Lin; Christensen, Klaus; Xia, Yulei; Florido, Mary H C; He, Wei; Pan, Wei; Prummer, Michael; Warren, Curtis R; Jakob-Roetne, Roland; Certa, Ulrich; Jagasia, Ravi; Freskgård, Per-Ola; Adatto, Isaac; Kling, Dorothee; Huang, Paul; Zon, Leonard I; Chaikof, Elliot L; Gerszten, Robert E; Graf, Martin; Iacone, Roberto; Cowan, Chad A

    2015-08-01

    The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease. PMID:26214132

  14. Oxidized-LDL induce the expression of heat shock protein 70 in human endothelial cells.

    PubMed

    Zhu, W; Roma, P; Pellegatta, F; Catapano, A L

    1994-04-15

    Heat shock proteins are detectable in human atherosclerotic plaques, especially in endothelial cells. In this report we show by immunofluorescence that incubation "in vitro" with OxLDL is a stress capable of inducing the expression of heat shock protein 70 in both the EAhy-926 cell line and human umbilical vein endothelial cells (HUVEC). This induction was parallel to the cytotoxicity of oxidized LDL as determined by [3H]adenine release. When cells were confluent, however, both effects were greatly reduced. We speculate that induction of hsp70 is related to the cytotoxicity of oxidized LDL and that the detection of heat shock proteins in human atherosclerotic plaques is a further indication for the presence "in vivo" of oxidized LDL. These observations may be relevant to the understanding of endothelial response to injury in proatherosclerotic events. PMID:8166710

  15. Sacrificial component fabrication for optimised production of micro-vascular polymer composite

    NASA Astrophysics Data System (ADS)

    Dalton, B.; Dixon, D.; McIlhagger, A.; Archer, E.

    2015-02-01

    Smart functional materials are a viable future goal for advanced applications in aerospace, space and medical applications. In this work micro-vascular polymer composite systems have been developed using sacrificial fibres produced from catalyst loaded Poly(lactic acid). The sacrificial fibres have been produced via a published technique which treated PLA in a solvent catalyst mixture of 60% Trifluoroethanol, 40% H2O dispersed with 10 wt% tin (II) oxalate catalyst. A second process of polymer extrusion of PLA using graded fill contents of tin (II) oxalate has also been developed for the up scaled production of fibres as an alternative to solution treatment. Thermal analysis (TGA) was used to compare sacrificial fibre specimens. PLA fibres produced via the polymer extrusion method outperformed solution treated fibres displaying a lower degradation onset temperature (average 25°C lower), higher degradation rates (observed through a derivative curve comparison) and lower residual catalyst content (0.67% solvent treated fibre against 0.16% extruded fibre). The continuous extrusion process is solvent free and is suitable for high volume production. This work has been carried out to fully understand the fabrication issues with sacrificial components.

  16. Small Spacecraft Active Thermal Control: Micro-Vascular Composites Enable Small Satellite Cooling

    NASA Technical Reports Server (NTRS)

    Ghosh, Alexander

    2016-01-01

    The Small Spacecraft Integrated Power System with Active Thermal Control project endeavors to achieve active thermal control for small spacecraft in a practical and lightweight structure by circulating a coolant through embedded micro-vascular channels in deployable composite panels. Typically, small spacecraft rely on small body mounted passive radiators to discard heat. This limits cooling capacity and leads to the necessity to design for limited mission operations. These restrictions severely limit the ability of the system to dissipate large amounts of heat from radios, propulsion systems, etc. An actively pumped cooling system combined with a large deployable radiator brings two key advantages over the state of the art for small spacecraft: capacity and flexibility. The use of a large deployable radiator increases the surface area of the spacecraft and allows the radiation surface to be pointed in a direction allowing the most cooling, drastically increasing cooling capacity. With active coolant circulation, throttling of the coolant flow can enable high heat transfer rates during periods of increased heat load, or isolate the radiator during periods of low heat dissipation.

  17. Engineering of Surface Functionality onto Polystyrene Microcarriers for the Attachment and Growth of Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Xiong, Gordon M.; Foord, John S.; Griffiths, Jon-Paul; Parker, Emily M.; Moloney, Mark G.; Choong, Cleo

    2014-08-01

    This work reports the effects of introducing diverse chemical functionalities onto the surface of polystyrene microcarrier beads on their ability to function as injectable cell carriers. Cellular adhesion and proliferation, as well as cellular outgrowths from microcarrier surfaces, using human umbilical vein endothelial cells (HUVECs), were examined in detail. It was observed that initial cell adhesion appeared to be most significantly decreased by hydrophobicity, whilst cell proliferation appeared to be improved in most chemical functional groups over unmodified polystyrene. Overall, our study highlights the importance of surface chemistry in directing the growth and function of human endothelial cells.

  18. Human microvascular endothelial cells express receptors for platelet-derived growth factor

    SciTech Connect

    Beitz, J.G.; Kim, Insoon; Calabresi, P.; Frackelton, A.R. Jr. )

    1991-03-01

    Endothelial cells have been widely thought to be unresponsive to platelet-derived growth factor (PDGF, a major growth factor released from stimulated platelets at the sites of vascular insults) and devoid of PDGF receptors. Nevertheless, in examining the growth-factor responses of microvascular endothelial cells isolated from human omental adipose tissue, the authors were surprised to detect PDGF-induced tyrosine phosphorylation of a 180-kDa glycoprotein, subsequently identified as the cellular receptor for PDGF by specific immunoprecipitation. Scatchard analysis of {sup 125}I-labeled PDGF binding to human microvascular endothelial cells revealed 30,000 PDGF receptors per cell with a K{sub d} of 0.14 nM. Normal cellular consequences of receptor activation were also observed, including tyrosine phosphorylation of a 42-kDa protein and serine phosphorylation of ribosomal protein S6. Furthermore, PDGF was mitogenic for these cells. Microvascular endothelial cells play a central role in neovascularization required for wound healing and solid tumor growth. Thus, the discovery of functional PFDG receptors on human microvascular endothelial cells suggests a direct role for PDGF in this process.

  19. Sonic hedgehog derived from human pancreatic cancer cells augments angiogenic function of endothelial progenitor cells.

    PubMed

    Yamazaki, Madoka; Nakamura, Kazumasa; Mizukami, Yusuke; Ii, Masaaki; Sasajima, Junpei; Sugiyama, Yoshiaki; Nishikawa, Tomoya; Nakano, Yasuhiro; Yanagawa, Nobuyuki; Sato, Kazuya; Maemoto, Atsuo; Tanno, Satoshi; Okumura, Toshikatsu; Karasaki, Hidenori; Kono, Toru; Fujiya, Mikihiro; Ashida, Toshifumi; Chung, Daniel C; Kohgo, Yutaka

    2008-06-01

    Hedgehog signaling is important in the pathogenesis of pancreatic cancer. Several recent observations suggest the involvement of sonic hedgehog (SHH) in postnatal neovascularization. We identified a novel role for SHH in tumor-associated angiogenesis in pancreatic cancer. Immunohistochemical analysis revealed that patched homolog 1 (PTCH1), both a receptor for and transcriptional target of hedgehog signaling, was expressed in a small fraction of endothelial cells within pancreatic cancer, but not in normal pancreatic tissue. When endothelial progenitor cells (EPC) isolated from human peripheral blood were cultured with supernatant from SHH-transfected 293 cells or pancreatic cancer cells, mRNA levels of vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 and angiopoietin-1 were significantly increased, whereas no such induction was observed in human umbilical vein endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMVEC). HUVEC tube formation was stimulated when cocultured with EPC, and preconditioning EPC with supernatant from KP-1 N pancreatic cancer cells highly expressing SHH significantly enhanced the effect. The effect was partially attenuated by specific inhibition of SHH with cyclopamine or a neutralizing antibody. These findings suggest that tumor-derived SHH can induce angiogenesis, and this is mediated by its effects on EPC specifically. Targeting SHH would be a novel therapeutic approach that can inhibit not only proliferation of cancer cells but also EPC-mediated angiogenesis. PMID:18422746

  20. Ionizing radiation increases adhesiveness of human aortic endothelial cells via a chemokine-dependent mechanism.

    PubMed

    Khaled, Saman; Gupta, Kiran B; Kucik, Dennis F

    2012-05-01

    Exposure to radiation from a variety of sources is associated with increased risk of heart disease and stroke. Since radiation also induces inflammation, a possible mechanism is a change in the adhesiveness of vascular endothelial cells, triggering pro-atherogenic accumulation of leukocytes. To investigate this mechanism at the cellular level, the effect of X rays on adhesiveness of cultured human aortic endothelial cells (HAECs) was determined. HAECs were grown as monolayers and exposed to 0 to 30 Gy X rays, followed by measurement of adhesiveness under physiological shear stress using a flow chamber adhesion assay. Twenty-four hours after irradiation, HAEC adhesiveness was increased, with a peak effect at 15 Gy. Radiation had no significant effect on surface expression of the endothelial adhesion molecules ICAM-1 and VCAM-1. Antibody blockade of the leukocyte integrin receptors for ICAM-1 and VCAM-1, however, abolished the radiation-induced adhesiveness. Since these leukocyte integrins can be activated by chemokines presented on the endothelial cell surface, the effect of pertussis toxin (PTX), an inhibitor of chemokine-mediated integrin activation, was tested. PTX specifically inhibited radiation-induced adhesiveness, with no significant effect on nonirradiated cells. Therefore, radiation induces increased adhesiveness of aortic endothelial cells through chemokine-dependent signaling from endothelial cells to leukocytes, even in the absence of increased expression of the adhesion molecules involved. PMID:22087741

  1. Size and composition effects of household particles on inflammation and endothelial dysfunction of human coronary artery endothelial cells

    NASA Astrophysics Data System (ADS)

    Lin, Lian-Yu; Liu, I.-Jung; Chuang, Hsiao-Chi; Lin, Hui-Yi; Chuang, Kai-Jen

    2013-10-01

    People spend generally 90 percent of their time indoors, yet toxicity of household particles has not been thoroughly investigated before. The objective of this study is to examine particle size and components effects of household particles on human coronary artery endothelial cells (HCAEC). We used two micro-orifice uniform deposit impactors to collect 60 sets of indoor particulate matters (PM) from 30 houses in Taipei, Taiwan. Polycyclic aromatic hydrocarbons (PAHs) effects of household particles were determined by high-resolution gas chromatograph/high-resolution mass spectrometer, respectively. HCAEC were exposed to household particles extracts in three size ranges: PM0.1 (diameters less than 0.1 μm), PM1.0-0.1 (diameters between 1.0 and 0.1 μm), and PM10-1.0 (diameters between 10 and 1.0 μm) at 50 μg mL-1 for 4 h, and interleukin-6 (IL-6), endothelin-1 (ET-1), and nitric oxide (NO) concentrations in the medium were measured. We found that household PM1.0-0.1 was associated with increased IL-6 and ET-1 production and decreased NO synthesis. Naphthalene of PM1.0-0.1 was highly correlated with IL-6 and ET-1 production and NO reduction. We concluded that size and compositions of household particles were both important factors on inflammation and endothelial dysfunction in HCAEC.

  2. Definitive proof of endothelialization of a Dacron arterial prosthesis in a human being.

    PubMed

    Wu, M H; Shi, Q; Wechezak, A R; Clowes, A W; Gordon, I L; Sauvage, L R

    1995-05-01

    A 10 mm woven Dacron axillofemoral bypass graft was removed from a 65-year-old patient during redo surgery after an implant period of 26 months, because of a large seroma that surrounded the entire length of the graft. Tissue blocks were taken from representative areas along the entire length of the graft surface and evaluated by light microscopy with hematoxylin and eosin and Masson trichrome staining, scanning electron microscopy, transmission electron microscopy, and immunocytochemical staining. Paraffin-embedded sections were stained with smooth muscle cell alpha-actin, which demonstrated smooth muscle cells in the pseudointima, and Ham 56 stain to identify macrophages. Endothelial factor VIII/von Willebrand factor and Ulex europaeus agglutinin identified human endothelial cells on the flow surface, in areas far removed from the anastomoses to the native vessels. This is the first definitive proof in a human of endothelialization of a synthetic arterial graft beyond the pannus ingrowth zone. PMID:7769746

  3. Alloproliferation of purified CD4+ T cells to adult human heart endothelial cells, and study of second-signal requirements.

    PubMed Central

    McDouall, R M; Page, C S; Hafizi, S; Yacoub, M H; Rose, M L

    1996-01-01

    Human endothelial cells have been shown to be capable of causing direct allostimulation of T cells. However, the majority of immunological studies of human endothelial cells have been performed on cells of fetal origin. Here we use endothelial cells isolated from the adult human heart, both large vessel (coronary artery, pulmonary artery and aorta) and also microvascular. We have examined the ability of all these endothelial cells to cause direct allostimulation of T cells, and show that purified CD4+ T cells can proliferate in response to adult human heart endothelial cells, the response being dependent on pretreatment of the endothelial cells with interferon-gamma (IFN-gamma) and inhibited by anti-HLA-DR monoclonal antibody. The proliferative responses of CD8+ T cells to adult but not fetal endothelial cells was inconsistent and weak. Proliferative responses were not blocked by CTLA4-Ig, which inhibits T-cell responses to "classical' antigen-presenting cells (APC), but > 50% inhibition was achieved with monoclonal antibody to lymphocyte function-associated antigen-3 (LFA-3). These results show that adult human cardiovascular endothelial cells are capable of causing allostimulation of resting CD4+ T cells, using a different second signal to classical APC. In view of these findings endothelial cells should be considered as APC following solid organ transplantation. PMID:8943718

  4. Human vascular tissue models formed from human induced pluripotent stem cell derived endothelial cells

    PubMed Central

    Belair, David G.; Whisler, Jordan A.; Valdez, Jorge; Velazquez, Jeremy; Molenda, James A.; Vickerman, Vernella; Lewis, Rachel; Daigh, Christine; Hansen, Tyler D.; Mann, David A.; Thomson, James A.; Griffith, Linda G.; Kamm, Roger D.; Schwartz, Michael P.; Murphy, William L.

    2015-01-01

    Here we describe a strategy to model blood vessel development using a well-defined iPSC-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats. PMID:25190668

  5. ETS transcription factor ETV2 directly converts human fibroblasts into functional endothelial cells.

    PubMed

    Morita, Rimpei; Suzuki, Mayu; Kasahara, Hidenori; Shimizu, Nana; Shichita, Takashi; Sekiya, Takashi; Kimura, Akihiro; Sasaki, Ken-ichiro; Yasukawa, Hideo; Yoshimura, Akihiko

    2015-01-01

    Transplantation of endothelial cells (ECs) is a promising therapeutic approach for ischemic disorders. In addition, the generation of ECs has become increasingly important for providing vascular plexus to regenerated organs, such as the liver. Although many attempts have been made to generate ECs from pluripotent stem cells and nonvascular cells, the minimum number of transcription factors that specialize in directly inducing vascular ECs remains undefined. Here, by screening 18 transcription factors that are important for both endothelial and hematopoietic development, we demonstrate that ets variant 2 (ETV2) alone directly converts primary human adult skin fibroblasts into functional vascular endothelial cells (ETVECs). In coordination with endogenous FOXC2 in fibroblasts, transduced ETV2 elicits expression of multiple key endothelial development factors, including FLI1, ERG, and TAL1, and induces expression of endothelial functional molecules, including EGFL7 and von Willebrand factor. Consequently, ETVECs exhibits EC characteristics in vitro and forms mature functional vasculature in Matrigel plugs transplanted in NOD SCID mice. Furthermore, ETVECs significantly improve blood flow recovery in a hind limb ischemic model using BALB/c-nu mice. Our study indicates that the creation of ETVECs provides further understanding of human EC development induced by ETV2. PMID:25540418

  6. Tailoring Material Properties of Cardiac Matrix Hydrogels to Induce Endothelial Differentiation of Human Mesenchymal Stem Cells

    PubMed Central

    Jeffords, Megan E.; Wu, Jinglei; Shah, Mickey; Hong, Yi; Zhang, Ge

    2015-01-01

    Cardiac matrix hydrogel has shown great promise as an injectable biomaterial due to the possession of cardiac-specific extracellular matrix composition. A cardiac matrix hydrogel facilitating neovascularization will further improve its therapeutic outcomes in cardiac repair. In this study, we explored the feasibility of tailoring material properties of cardiac matrix hydrogels using a natural compound, genipin, to promote endothelial differentiation of stem cells. Our results demonstrated that the genipin crosslinking could increase the mechanical properties of the cardiac matrix hydrogel to a stiffness range promoting endothelial differentiation of human mesenchymal stem cells (hMSCs). It also decreased the swelling ratio and prolonged degradation without altering gelation time. Human mesenchymal stem cells cultured on the genipin crosslinked cardiac matrix hydrogels showed great viability. After 1-day culture, hMSCs demonstrated down-regulation of early endothelial marker expression and up-regulation of mature endothelial marker expression. Especially for 1 mM genipin crosslinked cardiac matrix hydrogels, hMSCs showed particularly significant expression of mature endothelial cell marker vWF. These attractive results indicate the potential of using genipin crosslinked cardiac matrix hydrogels to promote rapid vascularization for cardiac infarction treatment through minimally invasive therapy. PMID:25946697

  7. Inactivation of the Human Cytomegalovirus US20 Gene Hampers Productive Viral Replication in Endothelial Cells

    PubMed Central

    Cavaletto, Noemi; Luganini, Anna

    2015-01-01

    ABSTRACT The human cytomegalovirus (HCMV) US12 gene family includes a group of 10 contiguous genes (US12 to US21) encoding predicted seven-transmembrane-domain (7TMD) proteins that are nonessential for replication within cultured fibroblasts. Nevertheless, inactivation of some US12 family members affects virus replication in other cell types; e.g., deletion of US16 or US18 abrogates virus growth in endothelial and epithelial cells or in human gingival tissue, respectively, suggesting a role for some US12 proteins in HCMV cell tropism. Here, we provide evidence that another member, US20, impacts the ability of a clinical strain of HCMV to replicate in endothelial cells. Through the use of recombinant HCMV encoding tagged versions of the US20 protein, we investigated the expression pattern, localization, and topology of the US20-encoded protein (pUS20). We show that pUS20 is expressed as a partially glycosylated 7TMD protein which accumulates late in infection in endoplasmic reticulum-derived peripheral structures localized outside the cytoplasmic virus assembly compartment (cVAC). US20-deficient mutants generated in the TR clinical strain of HCMV exhibited major growth defects in different types of endothelial cells, whereas they replicated normally in fibroblasts and epithelial cells. While the attachment and entry phases in endothelial cells were not significantly affected by the absence of US20 protein, US20-null viruses failed to replicate viral DNA and express representative E and L mRNAs and proteins. Taken together, these results indicate that US20 sustains the HCMV replication cycle at a stage subsequent to entry but prior to E gene expression and viral DNA synthesis in endothelial cells. IMPORTANCE Human cytomegalovirus (HCMV) is a major pathogen in newborns and immunocompromised individuals. A hallmark of HCMV pathogenesis is its ability to productively replicate in an exceptionally broad range of target cells, including endothelial cells, which represent

  8. Neutrophil killing of human umbilical vein endothelial cells is oxygen radical-mediated and enhanced by TNF-. alpha

    SciTech Connect

    Dame, M.K.; Varani, J.; Weinberg, J.M.; Ward, P.A. )

    1991-03-11

    Human umbilical vein endothelial cells are sensitive to killing by activated human neutrophils. Killing is inhibited in the presence of catalase and deferoxamine mesylate but not soybean trypsin inhibitor. Reagent hydrogen peroxide can substitute for activated neutrophils in producing endothelial cell injury. These data suggest that lethal injury is due to the production of oxygen radicals by activated neutrophils. In these respects, the human umbilical vein endothelial cells are similar to rat pulmonary artery endothelial cells in that pretreatment with TNF-{alpha} increases sensitivity to injury by activated neutrophils. In part, the increased endothelial cell sensitivity to killing by neutrophils may be due to up-regulation of surface adhesion molecules. However, it was observed that cells passaged more than two times in culture did not demonstrate increased killing after treatment with TNF-{alpha} while up-regulation of neutrophil adhesion could be detected through several additional passages. Although the human umbilical vein endothelial cells are qualitatively similar to rat pulmonary artery endothelial cells in their sensitivity to killing, they are quantitatively much more resistant. What accounts for the relative resistance of the human umbilical vein endothelial cells is not fully understood. In the rat pulmonary artery endothelial cells, killing is known to be dependent on an intraendothelial source of iron. Pre-treatment of the human umbilical vein endothelial cells with 8-hydroxyquinoline-bound iron increased their sensitivity to oxidant injury. These data suggest that the availability of iron within the human umbilical vein endothelial cells may be a limiting factor in sensitivity to oxygen radical-mediated injury.

  9. Isolation and chromosomal localization of the human endothelial nitric oxide synthase (NOS3) gene

    SciTech Connect

    Robinson, L.J.; Michel, T.; Weremowicz, S.; Morton, C.C. )

    1994-01-15

    Endothelial NOS activity is a major determinant of vascular tone and blood pressure, and in several important (and sometimes hereditary) disease states, such as hypertension, diabetes, and atherosclerosis, the endothelial NO signaling system appears to be abnormal. To explore the relationship of the endothelial NOS activity, the authors isolated the human gene encoding the endothelial NOS. Genomic clones containing the 5[prime] end of this gene were identified in a human genomic library by applying a polymerase chain reaction (PCR)-based approach. Identification of the human gene for endothelial NOS (NOS3) was confirmed by nucleotide sequence analysis of the first coding exon, which was found to be identical to its cognate cDNA. The NOS3 gene spans at least 20 kb and appears to contain multiple introns. The transcription start site and promoter region of the NOS3 gene were identified by primer extension and ribonuclease protection assays. Sequencing of the putative promoter revealed consensus sequences for the shear stress-response element, as well as cytokine-responsive cis regulatory sequences, both possible important to the roles played by NOS3 in the normal and the diseased cardiovascular system. The authors also mapped the chromosomal location of the NOS3 gene. First, a chromosomal panel of human-rodent somatic cell hybrids was screened using PCR with oligonucleotide primers derived from the NOS3 genomic clone. The specificity of the amplified PCR product was confirmed by human and hamster genomic DNA controls, as well as by Southern blot analysis, using the NOS3 cDNA as probe. Definitive chromosomal assignment of the NOS3 gene to human chromosome 7 was based upon 0% discordancy; fluorescence in situ hybridization sublocalized the NOS3 gene to 7q36. The identification and characterization of the NOS3 gene may lead to further insights into heritable disease states associated with this gene product. 41 refs., 3 figs., 1 tab.

  10. Endothelial Interleukin-6 defines the tumorigenic potential of primary human cancer stem cells

    PubMed Central

    Krishnamurthy, Sudha; Warner, Kristy A.; Dong, Zhihong; Imai, Atsushi; Nör, Carolina; Ward, Brent B.; Helman, Joseph I.; Taichman, Russell S.; Bellile, Emily L.; McCauley, Laurie K.; Polverini, Peter J.; Prince, Mark E.; Wicha, Max S.; Nör, Jacques E.

    2014-01-01

    Head and neck squamous cell carcinomas (HNSCC) contain a small sub-population of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of IL-6, we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling. PMID:25078284

  11. Endothelial interleukin-6 defines the tumorigenic potential of primary human cancer stem cells.

    PubMed

    Krishnamurthy, Sudha; Warner, Kristy A; Dong, Zhihong; Imai, Atsushi; Nör, Carolina; Ward, Brent B; Helman, Joseph I; Taichman, Russell S; Bellile, Emily L; McCauley, Laurie K; Polverini, Peter J; Prince, Mark E; Wicha, Max S; Nör, Jacques E

    2014-11-01

    Head and neck squamous cell carcinomas (HNSCC) contain a small subpopulation of stem cells endowed with unique capacity to generate tumors. These cancer stem cells (CSC) are localized in perivascular niches and rely on crosstalk with endothelial cells for survival and self-renewal, but the mechanisms involved are unknown. Here, we report that stromal interleukin (IL)-6 defines the tumorigenic capacity of CSC sorted from primary human HNSCC and transplanted into mice. In search for the cellular source of Interleukin-6 (IL-6), we observed a direct correlation between IL-6 levels in tumor-associated endothelial cells and the tumorigenicity of CSC. In vitro, endothelial cell-IL-6 enhanced orosphere formation, p-STAT3 activation, survival, and self-renewal of human CSC. Notably, a humanized anti-IL-6R antibody (tocilizumab) inhibited primary human CSC-mediated tumor initiation. Collectively, these data demonstrate that endothelial cell-secreted IL-6 defines the tumorigenic potential of CSC, and suggest that HNSCC patients might benefit from therapeutic inhibition of IL-6/IL-6R signaling. PMID:25078284

  12. Early gene response of human brain endothelial cells to Listeria monocytogenes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene expression of human brain microvascular endothelial cells (HBMEC) to Listeria monocytogenes at 4 hour infection was analyzed. Four hours after infection, the expression of 456 genes of HBMEC had changed (p<0.05). We noted that many active genes were involved in the formyl-methionylleucylph...

  13. Preserved endothelial function in human obesity in the absence of insulin resistance

    PubMed Central

    2013-01-01

    Background Insulin resistance (IR) is frequently associated with endothelial dysfunction and has been proposed to play a major role in cardiovascular disease (CVD). On the other hand, obesity has long been related to IR and increased CVD. However it is not known if IR is a necessary condition for endothelial dysfunction in human obesity, allowing for preserved endothelial function in obese people when absent. Therefore, the purpose of the study was to assess the relationship between IR and endothelial dysfunction in human obesity and the mechanisms involved. Methods Twenty non-insulin resistant morbid obese (NIR-MO), 32 insulin resistant morbid obese (IR-MO), and 12 healthy subjects were included. Serum concentrations of glucose, insulin, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), resistin and adiponectin were determined. IR was evaluated by HOMA-index. Endothelium-dependent relaxation to bradykinin (BK) in mesenteric microvessels was assessed in wire myograph. Results Serum IL-6, and TNF-α levels were elevated only in IR-MO patients while resistin was elevated and adiponectin reduced in all MO individuals. Mesenteric arteries from IR-MO, but not from NIR-MO subjects displayed blunted relaxation to BK. Vasodilatation was improved in IR-MO arteries by the superoxide scavenger, superoxide dismutase (SOD) or the mitochondrial-targeted SOD mimetic, mito-TEMPO. NADPH oxidase inhibitors (apocynin and VAS2870) and the nitric oxide synthase (NOS) cofactor, tetrahydrobiopterin failed to modify BK-induced vasodilatations. Superoxide generation was higher in vessels from IR-MO subjects and reduced by mito-TEMPO. Blockade of TNF-α with infliximab, but not inhibition of inducible NOS or cyclooxygenase, improved endothelial relaxation and decreased superoxide formation. Conclusions Endothelial dysfunction is observed in human morbid obesity only when insulin resistance is present. Mechanisms involved include augmented mitochondrial superoxide generation, and

  14. Utilization of Microgravity Bioreactor for Differentiation and Growth of Human Vascular Endothelial Cells

    NASA Technical Reports Server (NTRS)

    Chen, Chu-Huang; Pellis, Neal R.

    1997-01-01

    The goal was to delineate mechanisms of genetic responses to angiogenic stimulation of human coronary arterial and dermal microvascular endothelial cells during exposure to microgravity. The NASA-designed rotating-wall vessel was used to create a three-dimensional culture environment with low shear-stress and microgravity simulating that in space. The primary specific aim was to determine whether simulated microgravity enhances endothelial cell growth and whether the growth enhancement is associated by augmented expression of Basic Fibroblast Growth Factor (BFGF) and c-fos, an immediate early gene and component of the transcription factor AP-1.

  15. Impact of Corneal Endothelial Dysfunctions on Intraocular Oxygen Levels in Human Eyes

    PubMed Central

    Huang, Andrew J. W.; Shui, Ying-Bo; Han, Yu-Ping; Bai, Fang; Siegfried, Carla J.; Beebe, David C.

    2015-01-01

    Purpose We studied the implications of corneal endothelial dysfunctions on oxidative stress in the anterior segment via in vivo measurements of oxygen partial pressure (pO2) in the anterior chamber (AC) of human eyes. Methods We recruited 51 patients undergoing cataract surgery and/or endothelial keratoplasty (EK). Endothelial cell density (ECD; n = 33) and central corneal thickness (CCT; n = 41) were measured on patients with relatively clear corneas. Before surgery, an oxygen sensor was introduced into the AC via a peripheral corneal paracentesis. In all patients, seven measurements of pO2 were obtained by positioning the flexible tip near the endothelium at the central cornea, at four cardinal subendothelial locations near the midperipheral cornea, and in the mid-AC and AC angle. In patients with pseudophakia or eyes undergoing cataract surgery, pO2 also was measured near the lens surface and in the posterior chamber. Results Consistent with our previous reports, a steep oxygen gradient was noted in the anterior segment of normal controls (n = 24). In patients with endothelial dysfunctions (n = 27), there was a significant increase of pO2 at all five subendothelial locations without a significant increase of pO2 in the AC angle. By regression analyses, subendothelial pO2 correlated inversely with ECD and positively with CCT in patients with endothelial dysfunctions. Conclusions This study demonstrates an even steeper intraocular oxygen gradient in eyes with corneal endothelial dysfunctions. It suggests that the reduced oxygen consumption in corneal endothelial cells may increase oxidative stress in the AC and the existence of an alternative aqueous inflow pathway that maintains a relatively low and constant pO2 at the AC angle. PMID:26447982

  16. Non-nucleoside reverse transcriptase inhibitor efavirenz increases monolayer permeability of human coronary artery endothelial cells.

    PubMed

    Jamaluddin, Md Saha; Lin, Peter H; Yao, Qizhi; Chen, Changyi

    2010-01-01

    Highly active antiretroviral therapy (HAART) is often associated with endothelial dysfunction and cardiovascular complications. In this study, we determined whether HIV non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) could increase endothelial permeability. Human coronary artery endothelial cells (HCAECs) were treated with EFV (1, 5 and 10 microg/ml) and endothelial permeability was determined by a transwell system with a fluorescence-labeled dextran tracer. HCAECs treated with EFV showed a significant increase of endothelial permeability in a concentration-dependent manner. With real time PCR analysis, EFV significantly reduced the mRNA levels of tight junction proteins claudin-1, occludin, zonula occluden-1 and junctional adhesion molecule-1 compared with controls (P<0.05). Protein levels of these tight junction molecules were also reduced substantially in the EFV-treated cells by western blot and flow cytometry analyses. In addition, EFV also increased superoxide anion production with dihydroethidium and cellular glutathione assays, while it decreased mitochondrial membrane potential with JC-staining. Antioxidants (ginkgolide B and MnTBAP) effectively blocked EFV-induced endothelial permeability and mitochondrial dysfunction. Furthermore, EFV increased the phosphorylation of MAPK JNK and IkappaBalpha, thereby increasing NFkappaB translocation to the nucleus. Chemical JNK inhibitor and dominant negative mutant JNK and IkappaBalpha adenoviruses effectively blocked the effects of EFV on HCAECs. Thus, EFV increases endothelial permeability which may be due to the decrease of tight junction proteins and the increase of superoxide anion. JNK and NFkappaB activation may be directly involved in the signal transduction pathway of EFV action in HCAECs. PMID:19674747

  17. Different forms of human vascular adhesion protein-1 (VAP-1) in blood vessels in vivo and in cultured endothelial cells: implications for lymphocyte-endothelial cell adhesion models.

    PubMed

    Salmi, M; Jalkanen, S

    1995-10-01

    Vascular endothelium plays a pivotal role in controlling leukocyte extravasation from the blood into the tissues. Vascular adhesion protein-1 (VAP-1) is a novel endothelial cell molecule which mediates lymphocyte binding to the vascular lining (Salmi, M., and Jalkanen, S., Science 1992. 257:1407). In this study, we analyzed endothelial cell type-specific differences of VAP-1. In vivo, VAP-1 is a 90/170-kDa molecule which is mainly expressed on the lumenal surface and in cytoplasmic granules of peripheral lymph node-type postcapillary venules (high endothelial venules, HEV). In tonsil HEV, VAP-1 is modified with abundant sialic acids. VAP-1 is also detectable in the cytoplasm of human umbilical vein endothelial cells (HUVEC) and in an endothelial cell hybrid EaHy-926, although both cell types lack detectable surface VAP-1. Cultured endothelial cells do not express MECA-79-defined peripheral lymph node addressins either. VAP-1 was not translocated onto the endothelial cell surface after stimulation with multiple cytokines, mitogens or secretagogues which induced expression of other known endothelial adhesion molecules. Biochemical analyses revealed that VAP-1 is a approximately 180-kDa protein in these endothelial cell types. Digestions with neuraminidase, O-glycanase and N-glycanase, as well as treatment of cells with tunicamycin and benzyl-N-acetylgalactosaminide, did not alter the molecular mass of VAP-1 in EaHy-926. Pulse-chase experiments showed that VAP-1 is directly synthesized as a 180-kDa molecule without any detectable precursors. Thus, in cultured endothelial cells, VAP-1 is a 180-kDa protein which is devoid of post-translational modifications, and in particular, lacks the sialic acids crucial for the function of VAP-1 in tonsil vessels. Notably, the endothelial cell types commonly used as a model in studying lymphocyte-endothelial cell interactions lack surface expression of VAP-1 and peripheral node addressins, and hence are inherently of limited use in

  18. Transfer of ultrasmall iron oxide nanoparticles from human brain-derived endothelial cells to human glioblastoma cells.

    PubMed

    Halamoda Kenzaoui, Blanka; Angeloni, Silvia; Overstolz, Thomas; Niedermann, Philippe; Chapuis Bernasconi, Catherine; Liley, Martha; Juillerat-Jeanneret, Lucienne

    2013-05-01

    Nanoparticles (NPs) are being used or explored for the development of biomedical applications in diagnosis and therapy, including imaging and drug delivery. Therefore, reliable tools are needed to study the behavior of NPs in biological environment, in particular the transport of NPs across biological barriers, including the blood-brain tumor barrier (BBTB), a challenging question. Previous studies have addressed the translocation of NPs of various compositions across cell layers, mostly using only one type of cells. Using a coculture model of the human BBTB, consisting in human cerebral endothelial cells preloaded with ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) and unloaded human glioblastoma cells grown on each side of newly developed ultrathin permeable silicon nitride supports as a model of the human BBTB, we demonstrate for the first time the transfer of USPIO NPs from human brain-derived endothelial cells to glioblastoma cells. The reduced thickness of the permeable mechanical support compares better than commercially available polymeric supports to the thickness of the basement membrane of the cerebral vascular system. These results are the first report supporting the possibility that USPIO NPs could be directly transferred from endothelial cells to glioblastoma cells across a BBTB. Thus, the use of such ultrathin porous supports provides a new in vitro approach to study the delivery of nanotherapeutics to brain cancers. Our results also suggest a novel possibility for nanoparticles to deliver therapeutics to the brain using endothelial to neural cells transfer. PMID:23578059

  19. Aescin protection of human vascular endothelial cells exposed to cobalt chloride mimicked hypoxia and inflammatory stimuli.

    PubMed

    Montopoli, Monica; Froldi, Guglielmina; Comelli, Maria Cristina; Prosdocimi, Marco; Caparrotta, Laura

    2007-03-01

    Human vascular endothelial cells (HUVECs) were exposed to CoCl2 as an in vitro model of hypoxia. Expression of VCAM-1 (vascular cell adhesion molecule), reduction of PECAM-1 (platelet endothelial cell adhesion molecule) and cytoskeletal changes without alterations in cell viability were observed. HUVECs were also exposed to Escherichia coli lipopolysaccaride (LPS) as an in vitro model of inflammation: significant IL-6 release was measured. Pre-treatment of HUVECs with aescin prevented, in a concentration-dependent fashion (0.1-1 microM), the action of CoCl2 on VCAM-1 and PECAM-1, also preserving endothelial cell morphology. Furthermore, aescin pre-treatment reduced IL-6 release from LPS-activated vascular endothelium. PMID:17310430

  20. Kaempferia parviflora ethanolic extract promoted nitric oxide production in human umbilical vein endothelial cells.

    PubMed

    Wattanapitayakul, Suvara K; Suwatronnakorn, Maneewan; Chularojmontri, Linda; Herunsalee, Angkana; Niumsakul, Somchit; Charuchongkolwongse, Suphan; Chansuvanich, Nuchattra

    2007-04-01

    The rhizomes of Kaempferia parviflora (KP) (Zingiberaceae) have been used in Thai traditional medicine for health promotion and for the treatment of digestive disorders and gastric ulcer. This study investigated effect of KP on endothelial function. Studies in human umbilical vein endothelial cells (HUVEC) showed that KP dose-dependently increased nitrite concentrations in culture media after 48 h incubation. eNOS mRNA and protein expression were also enhanced. The induction of eNOS mRNA was detected at 4 h and plateau at 48 h while iNOS expression was not observed. These data demonstrate that KP has a great potential for a supplemental use in vascular endothelial health promotion. PMID:17113256

  1. Lysophosphatidic acid stimulates thrombomodulin lectin-like domain shedding in human endothelial cells

    SciTech Connect

    Wu Hualin; Lin ChiIou; Huang Yuanli; Chen, Pin-Shern; Kuo, Cheng-Hsiang; Chen, Mei-Shing; Wu, G.C.-C.; Shi, G.-Y.; Yang, H.-Y.; Lee Hsinyu

    2008-02-29

    Thrombomodulin (TM) is an anticoagulant glycoprotein highly expressed on endothelial cell surfaces. Increased levels of soluble TM in circulation have been widely accepted as an indicator of endothelial damage or dysfunction. Previous studies indicated that various proinflammatory factors stimulate TM shedding in various cell types such as smooth muscle cells and epithelial cells. Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in biological fluids during endothelial damage or injury. In the present study, we first observed that LPA triggered TM shedding in human umbilical vein endothelial cells (HUVECs). By Cyflow analysis, we showed that the LPA-induced accessibility of antibodies to the endothelial growth factor (EGF)-like domain of TM is independent of matrix metalloproteinases (MMPs), while LPA-induced TM lectin-like domain shedding is MMP-dependent. Furthermore, a stable cell line expressing TM without its lectin-like domain exhibited a higher cell proliferation rate than a stable cell line expressing full-length TM. These results imply that LPA induces TM lectin-like domain shedding, which might contribute to the exposure of its EGF-like domain for EGF receptor (EGFR) binding, thereby stimulating subsequent cell proliferation. Based on our findings, we propose a novel mechanism for the exposure of TM EGF-like domain, which possibly mediates LPA-induced EGFR transactivation.

  2. Cocaine inhibits human endothelial cell IL-8 production: the role of transforming growth factor-beta.

    PubMed

    Mao, J T; Zhu, L X; Sharma, S; Chen, K; Huang, M; Santiago, S J; Gulsurd, J; Tashkin, D P; Dubinett, S M

    1997-10-10

    Cocaine use is associated with modulation of a broad range of biological functions including the capacity to influence cytokine production in murine and human immunoeffector cells. Little is known, however, regarding the effects of cocaine on endothelial cell cytokine production. Because the vascular endothelium actively participates in acute and chronic inflammatory responses and interleukin-8 (IL-8) is one of the key cytokines involved in the inflammatory process, modification of the production of IL-8 by vascular endothelial cells may interfere with the host response to infection or tissue injury. We investigated the effect of cocaine on endothelial cell IL-8 production. Conditioned supernatant from EA.hy 926 cells were evaluated by ELISA following in vitro cocaine exposure. Cocaine decreased IL-8 production in a dose-responsive manner, and this reduction correlated with down-regulation of IL-8 mRNA expression. Cocaine also increased the production of TGF-beta by EA.hy 926 cells and anti-TGF-beta abrogated the cocaine-mediated decrement of IL-8 production, indicating that cocaine down-regulates endothelial IL-8 production by increasing TGF-beta. Our findings suggest that the immunomodulatory effects of cocaine may be mediated, in part, by modification of endothelial-derived cytokine production. PMID:9344494

  3. Expression of endothelial cell-specific receptor tyrosine kinases and growth factors in human brain tumors.

    PubMed Central

    Hatva, E.; Kaipainen, A.; Mentula, P.; Jääskeläinen, J.; Paetau, A.; Haltia, M.; Alitalo, K.

    1995-01-01

    Key growth factor-receptor interactions involved in angiogenesis are possible targets for therapy of CNS tumors. Vascular endothelial growth factor (VEGF) is a highly specific endothelial cell mitogen that has been shown to stimulate angiogenesis, a requirement for solid tumor growth. The expression of VEGF, the closely related placental growth factor (PIGF), the newly cloned endothelial high affinity VEGF receptors KDR and FLT1, and the endothelial orphan receptors FLT4 and Tie were analyzed by in situ hybridization in normal human brain tissue and in the following CNS tumors: gliomas, grades II, III, IV; meningiomas, grades I and II; and melanoma metastases to the cerebrum. VEGF mRNA was up-regulated in the majority of low grade tumors studied and was highly expressed in cells of malignant gliomas. Significantly elevated levels of Tie, KDR, and FLT1 mRNAs, but not FLT4 mRNA, were observed in malignant tumor endothelia, as well as in endothelia of tissues directly adjacent to the tumor margin. In comparison, there was little or no receptor expression in normal brain vasculature. Our results are consistent with the hypothesis that these endothelial receptors are induced during tumor progression and may play a role in tumor angiogenesis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7856749

  4. Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoposis

    SciTech Connect

    Zhou Yijun . E-mail: zhou-yijun@hotmail.com; Wang Jiahe; Zhang Jin

    2006-06-02

    Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-{kappa}B, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-{kappa}B, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease.

  5. Study of apoptosis induced by cytostatics and vegetal extracts on human endothelial cell line.

    PubMed

    Bârzu, Simona Natalia; Bădulescu, Maria Mihaela; Lupu, Andreea Roxana; Cremer, Lidia; Szegli, G; Kerek, F; Călugăru, Ana

    2008-01-01

    Angiogenesis, the biological process by which new capillaries are formed from pre-existing vessels, is a tightly controlled and complex process involving several factors with both stimulating and inhibiting steps. In solid tumor growth, a specific clinical turning point is the transition to the vascular phase. Once it develops an intrinsic vascular network, a tumor grows indefinitely. Tumor angiogenesis depends mainly on the release by neoplasic cells of growth factors specific for endothelial cells (ECs), able to stimulate growth of the host blood vessels. The aim of this study was to analyze the apoptotic effect of some cytostatics, Vinblastine, Rapamycin and Doxorubicin, and vegetal extracts (called VOB) isolated and purified from Vitis sp., on human EA.hy926 endothelial cell line. In a proliferation assay using Crystal Violet, we demonstrated that Vinblastine and Rapamycin cytostatics have synergistic effect on endothelial cell line EA.hy926 growth inhibition. The inhibitory effects of Vinblastine and Doxorubicin were enhanced by VOB vegetal extracts. A combined treatment of cytostatics and VOB vegetal extracts resulted in a stronger antiproliferative effect of EA.hy926 endothelial cells. Results obtained regarding the apoptosis induced on EA.hy926 endothelial cells showed that each compound alone was able to induce a significant percent of apoptotic cells in a dose-dependent manner. PMID:19284159

  6. Cytokine factors present in dengue patient sera induces alterations of junctional proteins in human endothelial cells.

    PubMed

    Appanna, Ramapraba; Wang, Seok Mui; Ponnampalavanar, Sasheela A; Lum, Lucy Chai See; Sekaran, Shamala Devi

    2012-11-01

    Plasma leakage in severe dengue has been postulated to be associated with skewed cytokine immune responses. In this study, the association of cytokines with vascular permeability in dengue patients was investigated. Human serum samples collected from 48 persons (13 with dengue fever, 29 with dengue hemorrhagic fever, and 6 healthy) were subjected to cytokines analysis by using Luminex Multiplex Technology. Selected serum samples from patients with dengue hemorrhagic fever sera and recombinant human cytokines were then tested for roles on inducing vascular permeability by treatment of human umbilical vein endothelial cells. Confocal immunofluorescence staining indicated morphologic alteration of human umbilical vein endothelial cells treated with serum samples from patients with dengue hemorrhagic fever compared with serum samples from healthy persons. The findings suggest that cytokines produced during dengue hemorrhagic infections could induce alterations in the vascular endothelium, which may play a fundamental role in the pathophysiology of dengue. PMID:22987650

  7. Direct cytotoxic action of Shiga toxin on human vascular endothelial cells.

    PubMed Central

    Obrig, T G; Del Vecchio, P J; Brown, J E; Moran, T P; Rowland, B M; Judge, T K; Rothman, S W

    1988-01-01

    To help explain a role of the Shiga toxin family in hemorrhagic colitis and hemolytic-uremic syndrome in humans, it has been hypothesized that these toxins cause direct damage to the vascular endothelium. We now report that Shiga toxin purified from Shigella dysenteriae 1 does indeed have a direct cytotoxic effect on vascular endothelial cells in cultures. Human umbilical vein endothelial cells (HUVEC) in confluent monolayers were reduced 50% by 10(-8) M Shiga toxin after a lag period of 48 to 96 h. In comparison, nonconfluent HUVEC were reduced 50% by 10(-10) M Shiga toxin within a 24-h period. These data suggest that dividing endothelial cells are more sensitive to Shiga toxin than are quiescent cells in confluent monolayers. Both confluent and nonconfluent HUVEC specifically bound 125I-Shiga toxin. However, in response to the toxin, rates of incorporation of [3H]leucine into protein were more severely reduced in nonconfluent cells than in confluent cells. Toxin inhibition of protein synthesis preceded detachment of cells from the substratum. The specific binding of 125I-Shiga toxin to human endothelial cells and the cytotoxic response were both toxin dose dependent and neutralized by anti-Shiga toxin antibody. Heat-denatured Shiga toxin was without the cytotoxic effect. In addition, the complete culture system contained less than 0.1 ng of bacterial endotoxin per ml, as measured by the Limulus amoebocyte lysate test. PMID:3044997

  8. Nuclear p120 catenin unlocks mitotic block of contact-inhibited human corneal endothelial monolayers without disrupting adherent junctions.

    PubMed

    Zhu, Ying-Ting; Chen, Hung-Chi; Chen, Szu-Yu; Tseng, Scheffer C G

    2012-08-01

    Contact inhibition ubiquitously exists in non-transformed cells that are in contact with neighboring cells. This phenomenon explains the poor regenerative capacity of in vivo human corneal endothelial cells during aging, injury and surgery. This study demonstrated that the conventional approach of expanding human corneal endothelial cells by disrupting contact inhibition with EDTA followed by bFGF activated canonical Wnt signaling and lost the normal phenotype to endothelial-mesenchymal transition, especially if TGFβ1 was added. By contrast, siRNA against p120 catenin (CTNND1) also uniquely promoted proliferation of the endothelial cells by activating trafficking of p120 catenin to the nucleus, thus relieving repression by nuclear Kaiso. This nuclear p120-catenin-Kaiso signaling is associated with activation of RhoA-ROCK signaling, destabilization of microtubules and inhibition of Hippo signaling, but not with activation of Wnt-β-catenin signaling. Consequently, proliferating human corneal endothelial cells maintained a hexagonal shape, with junctional expression of N-cadherin, ZO-1 and Na(+)/K(+)-ATPase. Further expansion of human corneal endothelial monolayers with a normal phenotype and a higher density was possible by prolonging treatment with p120 catenin siRNA followed by its withdrawal. This new strategy of perturbing contact inhibition by selective activation of p120-catenin-Kaiso signaling without disrupting adherent junction could be used to engineer surgical grafts containing normal human corneal endothelial cells to meet a global corneal shortage and for endothelial keratoplasties. PMID:22505615

  9. Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase.

    PubMed

    Bevan, Heather S; Slater, Sadie C; Clarke, Hayley; Cahill, Paul A; Mathieson, Peter W; Welsh, Gavin I; Satchell, Simon C

    2011-10-01

    Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10-20 dyn/cm(2)) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm(2) had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-N(G)-monomethyl arginine (l-NMMA) and l-N(G)-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall. PMID:21775480

  10. Concise Review: An Update on the Culture of Human Corneal Endothelial Cells for Transplantation.

    PubMed

    Parekh, Mohit; Ferrari, Stefano; Sheridan, Carl; Kaye, Stephen; Ahmad, Sajjad

    2016-02-01

    The cornea forms the front window of the eye, enabling the transmission of light to the retina through a crystalline lens. Many disorders of the cornea lead to partial or total blindness, and therefore corneal transplantation becomes mandatory. Recently, selective corneal layer (as opposed to full thickness) transplantation has become popular because this leads to earlier rehabilitation and visual outcomes. Corneal endothelial disorders are a common cause of corneal disease and transplantation. Corneal endothelial transplantation is successful but limited worldwide because of lower donor corneal supply. Alternatives to corneal tissue for endothelial transplantation therefore require immediate attention. The field of human corneal endothelial culture for transplantation is rapidly emerging as a possible viable option. This manuscript provides an update regarding these developments. Significance: The cornea is the front clear window of the eye. It needs to be kept transparent for normal vision. It is formed of various layers of which the posterior layer (the endothelium) is responsible for the transparency of the cornea because it allows the transport of ions and solutes to and from the other layers of the cornea. Corneal blindness that results from the corneal endothelial dysfunction can be treated using healthy donor tissues. There is a huge demand for human donor corneas but limited supply, and therefore there is a need to identify alternatives that would reduce this demand. Research is underway to understand the isolation techniques for corneal endothelial cells, culturing these cells in the laboratory, and finding possible options to transplant these cells in the patients. This review article is an update on the recent developments in this field. PMID:26702128

  11. Pterostilbene, an Active Constituent of Blueberries, Stimulates Nitric Oxide Production via Activation of Endothelial Nitric Oxide Synthase in Human Umbilical Vein Endothelial Cells.

    PubMed

    Park, Seong Hoon; Jeong, Sun-Oh; Chung, Hun-Teag; Pae, Hyun-Ock

    2015-09-01

    Endothelial dysfunction, a key process in development of cardiovascular diseases, is largely due to reduced nitric oxide (NO) derived from endothelial NO synthase (eNOS). Resveratrol has been reported to stimulate NO production via estrogen receptor α (ERα) activation in endothelial cells. Here, we investigated whether two natural methylated analogs of resveratrol, pterostilbene (Pts) and trans-3,5,4'-trimethoxystilbene (TMS), similarly to resveratrol, could influence endothelial NO release in human umbilical vein endothelial cells (HUVECs). In HUVECs exposed to Pts or TMS, NO production and phosphorylation of eNOS, protein kinase B (Akt), and ERα were measured by using a fluorimetric NO assay kit and Western blot analysis, respectively. Dimethylated Pts, but not trimethylated TMS, stimulated dose-dependent NO production via eNOS phosphorylation. Pts also stimulated dose-dependent phosphorylation of Akt, but not of ERα. NO production and eNOS phosphorylation in response to Pts were significantly abolished by the phosphoinositide 3-kinase (PI3K)/Akt inhibitor LY294002, but not by the ERα antagonist ICI182780. Our results suggest that Pts, but not TMS, is capable of inducing eNOS phosphorylation and the subsequent NO release, presumably, by activating PI3K/Akt pathway. The potential efficacy of Pts, an active constituent of blueberries, may aid in the prevention of cardiovascular diseases characterized by endothelial dysfunction. PMID:26008990

  12. Intrinsic FGF2 and FGF5 promotes angiogenesis of human aortic endothelial cells in 3D microfluidic angiogenesis system

    PubMed Central

    Seo, Ha-Rim; Jeong, Hyo Eun; Joo, Hyung Joon; Choi, Seung-Cheol; Park, Chi-Yeon; Kim, Jong-Ho; Choi, Ji-Hyun; Cui, Long-Hui; Hong, Soon Jun; Chung, Seok; Lim, Do-Sun

    2016-01-01

    The human body contains different endothelial cell types and differences in their angiogenic potential are poorly understood. We compared the functional angiogenic ability of human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using a three-dimensional (3D) microfluidic cell culture system. HAECs and HUVECs exhibited similar cellular characteristics in a 2D culture system; however, in the 3D microfluidic angiogenesis system, HAECs exhibited stronger angiogenic potential than HUVECs. Interestingly, the expression level of fibroblast growth factor (FGF)2 and FGF5 under vascular endothelial growth factor (VEGF)-A stimulation was significantly higher in HAECs than in HUVECs. Moreover, small interfering RNA-mediated knockdown of FGF2 and FGF5 more significantly attenuated vascular sprouting induced from HAECs than HUVECs. Our results suggest that HAECs have greater angiogenic potential through FGF2 and FGF5 upregulation and could be a compatible endothelial cell type to achieve robust angiogenesis. PMID:27357248

  13. Intrinsic FGF2 and FGF5 promotes angiogenesis of human aortic endothelial cells in 3D microfluidic angiogenesis system.

    PubMed

    Seo, Ha-Rim; Jeong, Hyo Eun; Joo, Hyung Joon; Choi, Seung-Cheol; Park, Chi-Yeon; Kim, Jong-Ho; Choi, Ji-Hyun; Cui, Long-Hui; Hong, Soon Jun; Chung, Seok; Lim, Do-Sun

    2016-01-01

    The human body contains different endothelial cell types and differences in their angiogenic potential are poorly understood. We compared the functional angiogenic ability of human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using a three-dimensional (3D) microfluidic cell culture system. HAECs and HUVECs exhibited similar cellular characteristics in a 2D culture system; however, in the 3D microfluidic angiogenesis system, HAECs exhibited stronger angiogenic potential than HUVECs. Interestingly, the expression level of fibroblast growth factor (FGF)2 and FGF5 under vascular endothelial growth factor (VEGF)-A stimulation was significantly higher in HAECs than in HUVECs. Moreover, small interfering RNA-mediated knockdown of FGF2 and FGF5 more significantly attenuated vascular sprouting induced from HAECs than HUVECs. Our results suggest that HAECs have greater angiogenic potential through FGF2 and FGF5 upregulation and could be a compatible endothelial cell type to achieve robust angiogenesis. PMID:27357248

  14. Bortezomib induces autophagic death in proliferating human endothelial cells

    SciTech Connect

    Belloni, Daniela; Veschini, Lorenzo; Foglieni, Chiara; Dell'Antonio, Giacomo; Caligaris-Cappio, Federico; Ferrarini, Marina; Ferrero, Elisabetta

    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.

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

    SciTech Connect

    Cheng Chingwen Yeh Juching; Fan Taiping; Smith, Stephen K.; Charnock-Jones, D. Stephen

    2008-01-11

    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.

  16. Magnetic field-guided cell delivery with nanoparticle-loaded human corneal endothelial cells.

    PubMed

    Moysidis, Stavros N; Alvarez-Delfin, Karen; Peschansky, Veronica J; Salero, Enrique; Weisman, Alejandra D; Bartakova, Alena; Raffa, Gabriella A; Merkhofer, Richard M; Kador, Karl E; Kunzevitzky, Noelia J; Goldberg, Jeffrey L

    2015-04-01

    To improve the delivery and integration of cell therapy using magnetic cell guidance for replacement of corneal endothelium, here we assess magnetic nanoparticles' (MNPs') effects on human corneal endothelial cells (HCECs) in vitro. Biocompatible, 50 nm superparamagnetic nanoparticles endocytosed by cultured HCECs induced no short- or long-term change in viability or identity. Assessment of guidance of the magnetic HCECs in the presence of different magnet shapes and field strengths showed a 2.4-fold increase in delivered cell density compared to gravity alone. After cell delivery, HCECs formed a functional monolayer, with no difference in tight junction formation between MNP-loaded and control HCECs. These data suggest that nanoparticle-mediated magnetic cell delivery may increase the efficiency of cell delivery without compromising HCEC survival, identity or function. Future studies may assess the safety and efficacy of this therapeutic modality in vivo. From the clinical editor: The authors show in this article that magnetic force facilitates the delivery of human corneal endothelial cells loaded by superparamagnetic nanoparticles to cornea, without changing their morphology, identity or functional properties. This novel idea can potentially have vast impact in the treatment of corneal endothelial dystrophies by providing self-endothelial cells after ex-vivo expansion. PMID:25596075

  17. Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells

    PubMed Central

    Reinhard, Nathalie R.; van Helden, Suzanne F.; Anthony, Eloise C.; Yin, Taofei; Wu, Yi I.; Goedhart, Joachim; Gadella, Theodorus W. J.; Hordijk, Peter L.

    2016-01-01

    Endothelial cells line the vasculature and are important for the regulation of blood pressure, vascular permeability, clotting and transendothelial migration of leukocytes and tumor cells. A group of proteins that that control the endothelial barrier function are the RhoGTPases. This study focuses on three homologous (>88%) RhoGTPases: RhoA, RhoB, RhoC of which RhoB and RhoC have been poorly characterized. Using a RhoGTPase mRNA expression analysis we identified RhoC as the highest expressed in primary human endothelial cells. Based on an existing RhoA FRET sensor we developed new RhoB/C FRET sensors to characterize their spatiotemporal activation properties. We found all these RhoGTPase sensors to respond to physiologically relevant agonists (e.g. Thrombin), reaching transient, localized FRET ratio changes up to 200%. These RhoA/B/C FRET sensors show localized GEF and GAP activity and reveal spatial activation differences between RhoA/C and RhoB. Finally, we used these sensors to monitor GEF-specific differential activation of RhoA/B/C. In summary, this study adds high-contrast RhoB/C FRET sensors to the currently available FRET sensor toolkit and uncover new insights in endothelial and RhoGTPase cell biology. This allows us to study activation and signaling by these closely related RhoGTPases with high spatiotemporal resolution in primary human cells. PMID:27147504

  18. Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells.

    PubMed

    Reinhard, Nathalie R; van Helden, Suzanne F; Anthony, Eloise C; Yin, Taofei; Wu, Yi I; Goedhart, Joachim; Gadella, Theodorus W J; Hordijk, Peter L

    2016-01-01

    Endothelial cells line the vasculature and are important for the regulation of blood pressure, vascular permeability, clotting and transendothelial migration of leukocytes and tumor cells. A group of proteins that that control the endothelial barrier function are the RhoGTPases. This study focuses on three homologous (>88%) RhoGTPases: RhoA, RhoB, RhoC of which RhoB and RhoC have been poorly characterized. Using a RhoGTPase mRNA expression analysis we identified RhoC as the highest expressed in primary human endothelial cells. Based on an existing RhoA FRET sensor we developed new RhoB/C FRET sensors to characterize their spatiotemporal activation properties. We found all these RhoGTPase sensors to respond to physiologically relevant agonists (e.g. Thrombin), reaching transient, localized FRET ratio changes up to 200%. These RhoA/B/C FRET sensors show localized GEF and GAP activity and reveal spatial activation differences between RhoA/C and RhoB. Finally, we used these sensors to monitor GEF-specific differential activation of RhoA/B/C. In summary, this study adds high-contrast RhoB/C FRET sensors to the currently available FRET sensor toolkit and uncover new insights in endothelial and RhoGTPase cell biology. This allows us to study activation and signaling by these closely related RhoGTPases with high spatiotemporal resolution in primary human cells. PMID:27147504

  19. Ghrelin stimulates angiogenesis in human microvascular endothelial cells: Implications beyond GH release

    SciTech Connect

    Li Aihua; Cheng Guangli; Zhu Genghui; Tarnawski, Andrzej S. . E-mail: atarnawski@yahoo.com

    2007-02-09

    Ghrelin, a peptide hormone isolated from the stomach, releases growth hormone and stimulates appetite. Ghrelin is also expressed in pancreas, kidneys, cardiovascular system and in endothelial cells. The precise role of ghrelin in endothelial cell functions remains unknown. We examined the expression of ghrelin and its receptor (GHSR1) mRNAs and proteins in human microvascular endothelial cells (HMVEC) and determined whether ghrelin affects in these cells proliferation, migration and in vitro angiogenesis; and whether MAPK/ERK2 signaling is important for the latter action. We found that ghrelin and GHSR1 are constitutively expressed in HMVEC. Treatment of HMVEC with exogenous ghrelin significantly increased in these cells proliferation, migration, in vitro angiogenesis and ERK2 phosphorylation. MEK/ERK2 inhibitor, PD 98059 abolished ghrelin-induced in vitro angiogenesis. This is First demonstration that ghrelin and its receptor are expressed in human microvascular endothelial cells and that ghrelin stimulates HMVEC proliferation, migration, and angiogenesis through activation of ERK2 signaling.

  20. Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells

    SciTech Connect

    Bikfalvi, A.; Dupuy, E.; Inyang, A.L.; Tobelem, G. ); Fayein, N.; Courtois, Y. ); Leseche, G. )

    1989-03-01

    The binding, internalization, and degradation of basic fibroblast growth factor (bFGF) in human omental microvascular endothelial cells (HOME cells) were investigated. Binding studies of bFGF in human endothelial cells have not yet been reported. Basic FGF bound to HOME cells. The number of low-affinity binding sites was found to be variable. Washing the cells with 2 M phosphate-buffered saline removed completely {sup 125}I-bFGF bound to low-affinity binding sites but decreased also the high-affinity binding. The majority of the surface-bound {sup 125}I-bFGF was removed by washing the cells with acetic acid buffer at pH 3. At this temperature, degradation of the internalized ligand was followed after 1 hour by the appearance of three major bands of 15,000 10,000, and 8,000 Da and was inhibited by chloroquine. These results demonstrated two classes of binding sites for bFGF in HOME cells; the number of high-affinity binding sites being larger than the number reported for bovine capillary endothelial cells. The intracellular processing of bFGF in HOME cells seems to be different from that of heparin binding growth factor-1 in murine lung capillary endothelial cells and of eye-derived growth factor-1 in Chinese hamster fibroblasts.

  1. Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells

    PubMed Central

    D’Alessio, Alessio; Esposito, Bianca; Giampietri, Claudia; Ziparo, Elio; Pober, Jordan S; Filippini, Antonio

    2012-01-01

    Abstract Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine. PMID:21645239

  2. Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

    PubMed Central

    Burns, Jorge S.; Kristiansen, Malthe; Kristensen, Lars P.; Larsen, Kenneth H.; Nielsen, Maria O.; Christiansen, Helle; Nehlin, Jan; Andersen, Jens S.; Kassem, Moustapha

    2011-01-01

    Background Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. Methodology/Principal Findings Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. Conclusions Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was

  3. Effects of Fe particle irradiation on human endothelial barrier structure and function

    NASA Astrophysics Data System (ADS)

    Sharma, Preety; Guida, Peter; Grabham, Peter

    2014-07-01

    Space travel involves exposure to biologically effective heavy ion radiation and there is consequently a concern for possible degenerative disorders in humans. A significant target for radiation effects is the microvascular system, which is crucial to healthy functioning of the tissues. Its pathology is linked to disrupted endothelial barrier function and is not only a primary event in a range of degenerative diseases but also an important influencing factor in many others. Thus, an assessment of the effects of heavy ion radiation on endothelial barrier function would be useful for estimating the risks of space travel. This study was aimed at understanding the effects of high LET Fe particles (1 GeV/n) and is the first investigation of the effects of charged particles on the function of the human endothelial barrier. We used a set of established and novel endpoints to assess barrier function after exposure. These include, trans-endothelial electrical resistance (TEER), morphological effects, localization of adhesion and cell junction proteins (in 2D monolayers and in 3D tissue models), and permeability of molecules through the endothelial barrier. A dose of 0.50 Gy was sufficient to cause a progressive reduction in TEER measurements that were significant 48 hours after exposure. Concurrently, there were morphological changes and a 14% loss of cells from monolayers. Gaps also appeared in the normally continuous cell-border localization of the tight junction protein - ZO-1 but not the Platelet endothelial cell adhesion molecule (PECAM-1) in both monolayers and in 3D vessel models. Disruption of barrier function was confirmed by increased permeability to 3 kDa and 10 kDa dextran molecules. A dose of 0.25 Gy caused no detectible change in cell number, morphology, or TEER, but did cause barrier disruption since there were gaps in the cell border localization of ZO-1 and an increased permeability to 3 kDa dextran. These results indicate that Fe particles potently have

  4. Induction of connective tissue growth factor (CTGF) in human endothelial cells by lysophosphatidic acid, sphingosine-1-phosphate, and platelets.

    PubMed

    Muehlich, Susanne; Schneider, Nadine; Hinkmann, Fabian; Garlichs, Christoph D; Goppelt-Struebe, Margarete

    2004-08-01

    Endothelial dysfunction is characterized by multiple interactions between endothelial cells and components of the blood. This study focussed on the induction of the pro-atherogenic connective tissue growth factor (CTGF) in endothelial cells by bioactive lipids and platelets. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) led to a time- and concentration-dependent increase in CTGF mRNA and protein expression in the human endothelial cell line EAHY 926 and in primary cultures of human umbilical vein endothelial cells (HUVEC). As both cell types expressed various receptors for LPA and S1P, signaling pathways were further characterized by pharmacological means: induction of CTGF was pertussis toxin-insensitive and inhibition of activation of p42/44 MAP kinases only partially reduced CTGF expression. On the contrary, interference with the RhoA signaling pathway by simvastatin, an inhibitor of geranylgeranyltransferases, or the Rho-kinase inhibitor Y27632 prevented induction of CTGF. Co-incubation of endothelial cells with freshly isolated human platelets significantly increased the expression of CTGF mRNA in endothelial cells, which was also sensitive to simvastatin. Up-regulation of CTGF in endothelial cells, induced by LPA, S1P, or platelets, may contribute to the initiation and progression of atherosclerosis. Interference of simvastatin with the synthesis of this pro-atherogenic factor further supports the anti-atherogenic role of statins. PMID:15262182

  5. Interleukin-33 stimulates GM-CSF and M-CSF production by human endothelial cells.

    PubMed

    Montanari, Eliana; Stojkovic, Stefan; Kaun, Christoph; Lemberger, Christof E; de Martin, Rainer; Rauscher, Sabine; Gröger, Marion; Maurer, Gerald; Neumayer, Christoph; Huk, Ihor; Huber, Kurt; Demyanets, Svitlana; Wojta, Johann

    2016-08-01

    Interleukin (IL)-33, a member of the IL-1 family of cytokines, is involved in various inflammatory conditions targeting amongst other cells the endothelium. Besides regulating the maturation and functions of myeloid cells, granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) have been shown to play a role in such pathologies too. It was the aim of our study to investigate a possible influence of IL-33 on GM-CSF and M-CSF production by human endothelial cells. IL-33, but not IL-18 or IL-37, stimulated GM-CSF and M-CSF mRNA expression and protein production by human umbilical vein endothelial cells (HUVECs) and human coronary artery ECs (HCAECs) through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in an IL-1-independent way. This effect was inhibited by the soluble form of ST2 (sST2), which is known to act as a decoy receptor for IL-33. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor fluvastatin could also be shown to moderately reduce the IL-33-mediated effect on M-CSF, but not on GM-CSF expression. In addition, IL-33, IL-1β, GM-CSF and M-CSF were detected in endothelial cells of human carotid atherosclerotic plaques using immunofluorescence. Upregulation of GM-CSF and M-CSF production by human endothelial cells, an effect that appears to be mediated by NF-κB and to be independent of IL-1, may be an additional mechanism through which IL-33 contributes to inflammatory activation of the vessel wall. PMID:27173404

  6. Tetrahydrobiopterin Role in human umbilical vein endothelial dysfunction in maternal supraphysiological hypercholesterolemia.

    PubMed

    Leiva, Andrea; Fuenzalida, Bárbara; Salsoso, Rocío; Barros, Eric; Toledo, Fernando; Gutiérrez, Jaime; Pardo, Fabián; Sobrevia, Luis

    2016-04-01

    Maternal physiological hypercholesterolemia (MPH) allows a proper foetal development; however, maternal supraphysiological hypercholesterolemia (MSPH) associates with foetal endothelial dysfunction and early development of atherosclerosis. MSPH courses with reduced endothelium-dependent dilation of the human umbilical vein due to reduced endothelial nitric oxide synthase activity compared with MPH. Whether MSPH modifies the availability of the nitric oxide synthase cofactor tetrahydrobiopterin is unknown. We investigated whether MSPH-associated lower umbilical vein vascular reactivity results from reduced bioavailability of tetrahydrobiopterin. Total cholesterol <7.2mmol/L was considered as maternal physiological hypercholesterolemia (n=72 women) and ≥7.2mmol/L as MSPH (n=35 women). Umbilical veins rings were used for vascular reactivity assays (wire myography), and primary cultures of human umbilical vein endothelial cells (HUVECs) to measure nitric oxide synthase, GTP cyclohydrolase 1, and dihydrofolate reductase expression and activity, as well as tetrahydrobiopterin content. MSPH reduced the umbilical vein rings relaxation caused by calcitonine gene-related peptide, a phenomenon partially improved by incubation with sepiapterin. HUVECs from MSPH showed lower nitric oxide synthase activity (l-citrulline synthesis from l-arginine) without changes in its protein abundance, as well as reduced tetrahydrobiopterin level compared with MPH, a phenomenon reversed by incubation with sepiapterin. Expression and activity of GTP cyclohydrolase 1 was lower in MSPH, without changes in dihydrofolate reductase expression. MSPH is a pathophysiological condition reducing human umbilical vein reactivity due to lower bioavailability of tetrahydrobiopterin leading to lower NOS activity in the human umbilical vein endothelium. PMID:26826019

  7. Expression of TRPC homologs in endothelial cells and smooth muscle layers of human arteries.

    PubMed

    Yip, Ham; Chan, Wing-Yee; Leung, Pan-Cheung; Kwan, Hiu-Yee; Liu, Cuiling; Huang, Yu; Michel, Villaz; Yew, David Tai-Wai; Yao, Xiaoqiang

    2004-12-01

    TRPC channels are a group of Ca(2+)-permeable nonselective cation channels that mediate store-operated and/or agonist-stimulated Ca(2+) influx in a variety of cell types. In this study, we extensively examined the expression patterns of TRPC homologs in human vascular tissues. RT-PCR amplified cDNA fragments of TRPC1 (505 bp), TRPC3 (372 bp), TRPC4 (499 bp), TRPC5 (325 bp), TRPC6 (509 bp), and TRPC7 (187 bp) from RNA isolated from cultured human coronary artery endothelial cells. In situ hybridization yielded strong labeling of TRPC1,3-6 in the endothelial and smooth muscle cells of human coronary and cerebral arteries. TRPC7 labeling was exclusively found in endothelial cells but not in smooth muscle cells. Results from immunohistochemical staining were consistent with those from in situ hybridization. Similar expression patterns of TRPC homologs were also observed in arterioles and vaso vasora. In conclusion, our study indicates that TRPC homologs are widely expressed in human vessels of all calibers, including medium-sized coronary arteries and cerebral arteries, smaller-sized resistance arteries, and vaso vasora. These results suggest a ubiquitous role of TRPC homologs in regulating blood supply to different regions and in controlling arterial blood pressure. PMID:15538613

  8. Stimulation of angiogenesis and survival of endothelial cells by human monoclonal Tie2 receptor antibody.

    PubMed

    Hwang, Byungtae; Lee, Sang-Hyun; Kim, Jang-Seong; Moon, Ji Hyun; Jeung, In Cheul; Lee, Na Geum; Park, Jongjin; Hong, Hyo Jeong; Cho, Young-Lai; Jung, Haiyoung; Park, Young-Jun; Lee, Seon-Jin; Lee, Hee Gu; Kim, Won Kon; Han, Baek Soo; Bae, Kwang-Hee; Chung, Sang J; Kwon, Young-Guen; Lee, Sang Chul; Kim, Sang Jik; Min, Jeong-Ki

    2015-05-01

    Angiopoietin-1 (Ang1) and its endothelium-specific receptor, tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2), play critical roles in vascular development. Although the Ang1/Tie2 system has been considered a promising target for therapeutic neovascularization, several imitations of large-scale production have hampered the development of recombinant Ang1 for therapeutics. In this study, we produced a fully human agonistic antibody against Tie2, designated 1-4h, and tested the applicability of 1-4h as an alternative to native Ang1 in therapeutic angiogenesis. 1-4h significantly enhanced the phosphorylation of Tie2 in a dose- and time-dependent manner in human Tie2-expressing HEK293 cells and human umbilical vein endothelial cells. Moreover, 1-4h induced the activation of Tie2-mediated intracellular signaling such as AKT, eNOS, MAPK, and Focal Adhesion Kinase p125(FAK). In addition, 1-4h increased the chemotactic motility and capillary-like tube formation of endothelial cells in vitro and enhanced the survival of serum-deprived endothelial cells. Taken together, our data clearly suggest that a human Tie2 agonistic antibody is a potentially useful therapeutic approach for the treatment of several ischemic diseases including delayed-wound healing and ischemic heart and limb diseases. PMID:25771003

  9. Ginkgolide B protects human umbilical vein endothelial cells against xenobiotic injuries via PXR activation

    PubMed Central

    Zhou, Tao; You, Wen-ting; Ma, Zeng-chun; Liang, Qian-de; Tan, Hong-ling; Xiao, Cheng-rong; Tang, Xiang-lin; Zhang, Bo-li; Wang, Yu-guang; Gao, Yue

    2016-01-01

    Aim: Pregnane X receptor (PXR) is a nuclear receptor that regulates a number of genes encoding drug metabolism enzymes and transporters and plays a key role in xeno- and endobiotic detoxification. Ginkgolide B has shown to increase the activity of PXR. Here we examined whether ginkgolide B activated PXR and attenuated xenobiotic-induced injuries in endothelial cells. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with ginkgolide B. The expression of PXR, CYP3A4, MDR1, VCAM-1, E-selectin and caspase-3 were quantified with qRT-PCR and Western blot analysis. Cell apoptosis was analyzed with flow cytometry. Fluorescently labeled human acute monocytic leukemia cells (THP-1 cells) were used to examine cell adhesion. Results: Ginkgolide B (30–300 μmol/L) did not change the mRNA and protein levels of PXR in the cells, but dose-dependently increased nuclear translocation of PXR protein. Ginkgolide B increased the expression of CYP3A4 and MDR1 in the cells, which was partially reversed by pretreatment with the selective PXR signaling antagonist sulforaphane, or transfection with PXR siRNA. Functionally, ginkgolide B dose-dependently attenuated doxorubicin- or staurosporine-induced apoptosis, which was reversed by transfection with PXR siRNA. Moreover, ginkgolide B suppressed TNF-α-induced THP-1 cell adhesion and TNF-α-induced expression of vascular adhesion molecule 1 (VCAM-1) and E-selectin in the cells, which was also reversed by transfection with PXR siRNA. Conclusion: Ginkgolide B exerts anti-apoptotic and anti-inflammatory effects on endothelial cells via PXR activation, suggesting that a PXR-mediated endothelial detoxification program may be important for protecting endothelial cells from xeno- and endobiotic-induced injuries. PMID:26775663

  10. Biomaterials trigger endothelial cell activation when co-incubated with human whole blood.

    PubMed

    Herklotz, Manuela; Hanke, Jasmin; Hänsel, Stefanie; Drichel, Juliane; Marx, Monique; Maitz, Manfred F; Werner, Carsten

    2016-10-01

    Endothelial cell activation resulting from biomaterial contact or biomaterial-induced blood activation may in turn also affect hemostasis and inflammatory processes in the blood. Current in vitro hemocompatibility assays typically ignore these modulating effects of the endothelium. This study describes a co-incubation system of human whole blood, biomaterial and endothelial cells (ECs) that was developed to overcome this limitation. First, human endothelial cells were characterized in terms of their expression of coagulation- and inflammation-relevant markers in response to various activators. Subsequently, their capacity to regulate hemostasis as well as complement and granulocyte activation was monitored in a hemocompatibility assay. After blood contact, quiescent ECs exhibited anticoagulant and anti-inflammatory properties. When they were co-incubated with surfaces exhibiting pro-coagulant or pro-inflammatory characteristics, the ECs down-regulated coagulation but not complement or leukocyte activation. Analysis of intracellular levels of the endothelial activation markers E-selectin and tissue factor showed that co-incubation with model surfaces and blood significantly increased the activation state of ECs. Finally, the coagulation- and inflammation-modulating properties of the ECs were tested after blood/biomaterial exposure. Pre-activation of ECs by biomaterials in the blood induced a pro-coagulant and pro-inflammatory state of the ECs, wherein the pro-coagulant response was higher for biomaterial/blood pre-activated ECs than for TNF-α-pre-activated cells. This work provides evidence that biomaterials, even without directly contacting the endothelium, affect the endothelial activation state with and have consequences for plasmatic and cellular reactions in the blood. PMID:27472163

  11. Microrheology and ROCK Signaling of Human Endothelial Cells Embedded in a 3D Matrix

    PubMed Central

    Panorchan, Porntula; Lee, Jerry S. H.; Kole, Thomas P.; Tseng, Yiider; Wirtz, Denis

    2006-01-01

    Cell function is profoundly affected by the geometry of the extracellular environment confining the cell. Whether and how cells plated on a two-dimensional matrix or embedded in a three-dimensional (3D) matrix mechanically sense the dimensionality of their environment is mostly unknown, partly because individual cells in an extended matrix are inaccessible to conventional cell-mechanics probes. Here we develop a functional assay based on multiple particle tracking microrheology coupled with ballistic injection of nanoparticles to measure the local intracellular micromechanical properties of individual cells embedded inside a matrix. With our novel assay, we probe the mechanical properties of the cytoplasm of individual human umbilical vein endothelial cells (HUVECs) embedded in a 3D peptide hydrogel in the presence or absence of vascular endothelial growth factor (VEGF). We found that VEGF treatment, which enhances endothelial migration, increases the compliance and reduces the elasticity of the cytoplasm of HUVECs in a matrix. This VEGF-induced softening response of the cytoplasm is abrogated by specific Rho-kinase (ROCK) inhibition. These results establish combined particle-tracking microrheology and ballistic injection as the first method able to probe the micromechanical properties and mechanical response to agonists and/or drug treatments of individual cells inside a matrix. These results suggest that ROCK plays an essential role in the regulation of the intracellular mechanical response to VEGF of endothelial cells in a 3D matrix. PMID:16891369

  12. Human Umbilical Vein Endothelial Cell Interaction with Fluorine-Incorporated Amorphous Carbon Films

    NASA Astrophysics Data System (ADS)

    Yoshimoto, Yukihiro; Hasebe, Terumitsu; Nagashima, So; Kamijo, Aki; Nakatani, Tatsuyuki; Yamagami, Takuji; Kitamura, Noriko; Kitagawa, Tomoya; Hotta, Atsushi; Takahashi, Koki; Suzuki, Tetsuya

    2012-09-01

    A major clinical concern in coronary intervention for cardiovascular disease is late stent thrombosis after the implantation of drug eluting stents (DES). DES widely used in clinical settings currently utilize polymer coatings, which can induce persistent arterial wall inflammation and delayed vascular healing, resulting in impaired endothelialization. We examined the viability of human umbilical vein endothelial cells (HUVECs) for fluorine-incorporated amorphous carbon (a-C:H:F) coatings, which are known to be anti-thrombogenic. a-C:H:F and a-C:H were synthesized on the tissue culture dishes using radio frequency plasma enhanced chemical vapor deposition by varying the ratio of hexafluoroethane and acetylene. HUVECs were seeded on coated dishes for 6 days. The results indicate that the a-C:H:F surface does not disturb HUVEC proliferation in 6 days of culture and is promising for stent materials that allows the preservation of endothelialization, even if the fluorine concentration of a-C:H surface affects the early adhesion of endothelial cells.

  13. Human neural stem cell-induced endothelial morphogenesis requires autocrine/paracrine and juxtacrine signaling.

    PubMed

    Chou, Chung-Hsing; Modo, Michel

    2016-01-01

    Transplanted neural stem cells (NSC) interact with the host brain microenvironment. A neovascularization is commonly observed in the vicinity of the cell deposit, which is correlated with behavioral improvements. To elucidate the signaling mechanisms between human NSCs and endothelial cells (ECs), these were cocultured in an in vitro model in which NSC-induced endothelial morphogenesis produced a neurovascular environment. Soluble (autocrine/paracrine) and contact-mediated (juxtacrine) signaling molecules were evaluated for two conditionally immortalized fetal NSC lines derived from the cortical anlage (CTXOE03) and ganglionic eminence (STROC05), as well as an adult EC line (D3) derived from the cerebral microvasculature of a hippocampal biopsy. STROC05 were 4 times as efficient to induce endothelial morphogenesis compared to CTXOE03. The cascade of reciprocal interactions between NSCs and ECs in this process was determined by quantifying soluble factors, receptor mapping, and immunocytochemistry for extracellular matrix molecules. The mechanistic significance of these was further evaluated by pharmacological blockade. The sequential cell-specific regulation of autocrine/paracrine and juxtacrine signaling accounted for the differential efficiency of NSCs to induce endothelial morphogenesis. These in vitro studies shed new light on the reciprocal interactions between NSCs and ECs, which are pivotal for our mechanistic understanding of the efficacy of NSC transplantation. PMID:27374240

  14. p27 Nuclear localization and growth arrest caused by perlecan knockdown in human endothelial cells

    SciTech Connect

    Sakai, Katsuya; Oka, Kiyomasa; Matsumoto, Kunio; Nakamura, Toshikazu

    2010-02-12

    Perlecan, a secreted heparan sulfate proteoglycan, is a major component of the vascular basement membrane and participates in angiogenesis. Here, we used small interference RNA-mediated knockdown of perlecan expression to investigate the regulatory function of perlecan in the growth of human vascular endothelial cells. Basic fibroblast growth factor (bFGF)-induced ERK phosphorylation and cyclin D1 expression were unchanged by perlecan deficiency in endothelial cells; however, perlecan deficiency inhibited the Rb protein phosphorylation and DNA synthesis induced by bFGF. By contrast to cytoplasmic localization of the cyclin-dependent kinase inhibitor p27 in control endothelial cells, p27 was localized in the nucleus and its expression increased in perlecan-deficient cells, which suggests that p27 mediates inhibition of Rb phosphorylation. In addition to the well-characterized function of perlecan as a co-receptor for heparin-binding growth factors such as bFGF, our results suggest that perlecan plays an indispensible role in endothelial cell proliferation and acts through a mechanism that involves subcellular localization of p27.

  15. Irradiation induced expression of CD31, ICAM-1 and VCAM-1 in human microvascular endothelial cells.

    PubMed

    Quarmby, S; Hunter, R D; Kumar, S

    The adherence and migration of leukocytes through the endothelium of blood vessels is an important early event which occurs in normal tissues following ionizing irradiation but the underlying mechanisms are not fully understood. ICAM-1, VCAM-1 and CD31 are membrane proteins of endothelial cells, mediate this process when the vasculature is exposed to other inflammatory stimuli. In this study, expression of ICAM-1, VCAM-1 and CD31 on human dermal microvascular endothelial cells (HDMECs) at 72 hours post-irradiation using flow cytometry and northern analysis was determined. Dose-dependent increases in the surface expression and mRNA of ICAM-1 and CD31 were observed. In contrast VCAM-1 was practically undetectable on both control and irradiated HDMECs but was strongly expressed in TNF-alpha activated positive control HDMECs. The upregulation in ICAM-1 and CD31 was independent of radiation-induced changes in cell size, number and cell cycle stage. We suggest that ICAM-1 is active over a prolonged period whereas VCAM-1 acts only transiently in leukocyte-endothelial interactions in the irradiated microvasculature. The late upregulation of CD31 is a novel finding and may have a function in radiation-induced leukocyte extravasation, platelet adherence to the vascular wall and abnormal endothelial cell proliferation. Both ICAM-1 and CD31 seem to be therapeutic targets for the amelioration of radiation-induced normal tissue damage. PMID:11131637

  16. Human neural stem cell-induced endothelial morphogenesis requires autocrine/paracrine and juxtacrine signaling

    PubMed Central

    Chou, Chung-Hsing; Modo, Michel

    2016-01-01

    Transplanted neural stem cells (NSC) interact with the host brain microenvironment. A neovascularization is commonly observed in the vicinity of the cell deposit, which is correlated with behavioral improvements. To elucidate the signaling mechanisms between human NSCs and endothelial cells (ECs), these were cocultured in an in vitro model in which NSC-induced endothelial morphogenesis produced a neurovascular environment. Soluble (autocrine/paracrine) and contact–mediated (juxtacrine) signaling molecules were evaluated for two conditionally immortalized fetal NSC lines derived from the cortical anlage (CTXOE03) and ganglionic eminence (STROC05), as well as an adult EC line (D3) derived from the cerebral microvasculature of a hippocampal biopsy. STROC05 were 4 times as efficient to induce endothelial morphogenesis compared to CTXOE03. The cascade of reciprocal interactions between NSCs and ECs in this process was determined by quantifying soluble factors, receptor mapping, and immunocytochemistry for extracellular matrix molecules. The mechanistic significance of these was further evaluated by pharmacological blockade. The sequential cell-specific regulation of autocrine/paracrine and juxtacrine signaling accounted for the differential efficiency of NSCs to induce endothelial morphogenesis. These in vitro studies shed new light on the reciprocal interactions between NSCs and ECs, which are pivotal for our mechanistic understanding of the efficacy of NSC transplantation. PMID:27374240

  17. Shear stress reduces protease activated receptor-1 expression in human endothelial cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

  18. Advanced oxidation protein products induce endothelial-to-mesenchymal transition in human renal glomerular endothelial cells through induction of endoplasmic reticulum stress.

    PubMed

    Liang, Xiujie; Duan, Na; Wang, Yue; Shu, Shuangshuang; Xiang, Xiaohong; Guo, Tingting; Yang, Lei; Zhang, Shaojie; Tang, Xun; Zhang, Jun

    2016-01-01

    Endothelial-to-mesenchymal transition (EndMT) in renal glomerular endothelial cells plays a critical role in the pathogenesis of diabetic nephropathy (DN). Furthermore, advanced oxidation protein products (AOPPs) have been shown to contribute to the progression of DN. However, whether AOPPs induce EndMT in renal glomerular endothelial cells remains unclear. Thus, we investigated the effect of AOPPs on human renal glomerular endothelial cells (HRGECs) and the mechanisms underlying the effects. Our results showed that AOPP treatment lowered the expression of vascular endothelial cadherin, CD31, and claudin 5 and induced the overexpression of α-smooth muscle actin, vimentin, and fibroblast-specific protein 1, which indicated that AOPPs induced EndMT in HRGECs. Furthermore, AOPP stimulation increased the expression of glucose-regulated protein 78 and CCAAT/enhancer-binding protein-homologous protein, which suggested that AOPPs triggered endoplasmic reticulum (ER) stress in HRGECs. Notably, the aforementioned AOPP effects were reversed following the treatment of cells with salubrinal, an inhibitor of ER stress, whereas the effects were reproduced after exposure to thapsigargin, an inducer of ER stress. Collectively, our results indicate that AOPPs trigger EndMT in HRGECs through the induction of ER stress. These findings suggest novel therapeutic strategies for inhibiting renal fibrosis by targeting ER stress. PMID:26861949

  19. Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿

    PubMed Central

    Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed

    2007-01-01

    Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment. PMID:17875991

  20. Effects of chronic noradrenaline on the nitric oxide pathway in human endothelial cells.

    PubMed

    Bachetti, T; Comini, L; Agnoletti, L; Pedersini, P; Gaia, G; Cargnoni, A; Bellet, M; Curello, S; Ferrari, R

    1998-08-01

    Altered endothelium-dependent vasodilation has been observed in congestive heart failure (CHF), a disease characterized by a sustained adrenergic activation. The purpose of our study was to test the hypothesis that chronically elevated catecholamines influence the nitric oxide (NO) pathway in the human endothelium. Human umbilical vein endothelial cells (HUVEC) were exposed for 7 days to a concentration of noradrenaline (NA, 1 ng/mL) similar to that found in the blood of patients with CHF. Kinetics of endothelial constitutive NO synthase (ecNOS) and inducible NO synthase (iNOS) activity, measured by [3H]L-arginine to [3H]L-citrulline conversion, and protein expression of ecNOS and iNOS, assessed by Western blot analysis, were unaffected by chronic NA treatment. Furthermore, no changes in subcellular fraction-associated ecNOS were found; this indirectly shows that chronic NA did not cause phosphorylation of the enzyme. Moreover, [3H]L-arginine transport through the plasma membrane was conserved in chronically NA-treated cells. The data demonstrate that prolonged in vitro exposure to pathologic CHF-like NA does not affect the L-arginine: NO pathway in human endothelial cells. PMID:9782366

  1. Vascular Endothelial Growth Factor-A (VEGF-A) Mediates Activin A-Induced Human Trophoblast Endothelial-Like Tube Formation.

    PubMed

    Li, Yan; Zhu, Hua; Klausen, Christian; Peng, Bo; Leung, Peter C K

    2015-11-01

    Remodeling of maternal spiral arteries during pregnancy requires a subpopulation of extravillous cytotrophoblasts (EVTs) to differentiate into endovascular EVTs. Activin A, which is abundantly expressed at the maternal-fetal interface, has been shown to promote trophoblast invasion, but its role in endovascular differentiation remains unknown. Vascular endothelial growth factor-A (VEGF-A) is well recognized as a key regulator in trophoblast endovascular differentiation. Whether and how activin A might regulate VEGF-A production in human trophoblasts and its relationship to endovascular differentiation have yet to be determined. In the present study, we found that activin A increased VEGF-A production in primary and immortalized (HTR8/SVneo) human EVT cells. In addition, activin A enhanced HTR8/SVneo endothelial-like tube formation, and these effects were attenuated by pretreatment with small interfering RNA targeting VEGF-A or the VEGF receptor 1/2 inhibitor SU4312. Pretreatment with the activin/TGF-β type 1 receptor (ALK4/5/7) inhibitor SB431542 abolished the stimulatory effects of activin A on phosphorylated mothers against decapentaplegic (SMAD)-2/3 phosphorylation, VEGF-A production, and endothelial-like tube formation. Moreover, small interfering RNA-mediated down-regulation of SMAD2, SMAD3, or common SMAD4 abolished the effects of activin A on VEGF-A production and endothelial-like tube formation. In conclusion, activin A may promote human trophoblast cell endothelial-like tube formation by up-regulating VEGF-A production in an SMAD2/3-SMAD4-dependent manner. These findings provide insight into the cellular and molecular events regulated by activin A during human implantation. PMID:26327470

  2. Effects of Recombinant Human Erythropoietin on Resistance Artery Endothelial Function in Stage 4 Chronic Kidney Disease

    PubMed Central

    Briet, Marie; Barhoumi, Tlili; Mian, Muhammad Oneeb Rehman; Sierra, Cristina; Boutouyrie, Pierre; Davidman, Michael; Bercovitch, David; Nessim, Sharon J.; Frisch, Gershon; Paradis, Pierre; Lipman, Mark L.; Schiffrin, Ernesto L.

    2013-01-01

    Background Recent studies have raised concern about the safety of erythropoiesis‐stimulating agents because of evidence of increased risk of hypertension and cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients. In the present study, we investigated the effects of recombinant human erythropoietin (EPO) on endothelial function of gluteal subcutaneous resistance arteries isolated from 17 stage 4 patients (estimated glomerular filtration rate 21.9±7.4 mL/min per 1.73 m2) aged 63±13 years. Methods and Results Arteries were mounted on a pressurized myograph. EPO impaired endothelium‐dependent relaxation in a concentration‐dependent manner. The maximal response to acetylcholine with EPO at 1, 10, and 20 IU/mL was reduced by 12%, 34%, and 43%, respectively, compared with the absence of EPO (P<0.001). EPO‐induced endothelial dysfunction was significantly associated with carotid stiffness and history of cardiovascular events. EPO had no effect on norepinephrine‐induced vasoconstriction or sodium nitroprusside–induced relaxation. ABT‐627, an endothelin type A receptor antagonist, and tempol, a superoxide dismutase mimetic, partially reversed the altered endothelial function in the presence of EPO (P<0.01). Increased expression of endothelin‐1 was found in the vessel wall after incubation with EPO. Conclusions EPO alters endothelial function of resistance arteries in CKD patients via a mechanism involving in part oxidative stress and signaling through an endothelin type A receptor. EPO‐induced endothelial dysfunction could contribute to deleterious effects of EPO described in large interventional trials. PMID:23584809

  3. The effect of hyperglycaemia on permeability and the expression of junctional complex molecules in human retinal and choroidal endothelial cells.

    PubMed

    Saker, S; Stewart, E A; Browning, A C; Allen, C L; Amoaku, W M

    2014-04-01

    Diabetic retinopathy is the leading cause of preventable blindness in the working population and its prevalence continues to increase as the worldwide prevalence of diabetes grows. Diabetic choroidopathy is less well studied and occurs in the late stages of diabetic eye disease. The main cause of visual loss in diabetic eye disease is diabetic macular oedema caused by an increase in microvascular endothelial permeability. Endothelial cell permeability is influenced by multiple factors which have not been fully elucidated, particularly in human models. In addition, the gene and protein expression between retinal and choroidal endothelial cells, even in humans, has been shown to be heterogeneous. The aim of this project was to determine, in vitro, the effect of high glucose (25 mM) on human paracellular permeability in retinal and choroidal endothelial cells. The expression of selected tight junction molecules (Occludin, Claudin-5, JAM-A and JAM-C) and adheren junction (VE-Cadherin) molecules was also compared between retinal and choroidal endothelial cells and with high glucose. High glucose conditions significantly increased the permeability in both retinal and choroidal endothelial cells monolayers although the increase was higher in retinal endothelial cells. Under normal glucose culture conditions microarray analysis determined that occludin and claudin-5 gene expression was higher in retinal endothelial cells than choroidal endothelial cells, and western blotting indicated that claudin-5 protein expression was also higher in retinal endothelial cells whilst JAM-A, and C and VE-Cadherin levels were similar. In retinal endothelial cells exposed to high glucose claudin-5, occludin and JAM-A was found to be reduced, whereas the expression of VE-Cadherin and JAM-C was unchanged when evaluated with western blotting, immunofluorescence and qPCR. None of the proteins were significantly decreased by high glucose in choroidal endothelial cells. The increase in retinal

  4. Stretch-induced human myometrial cytokines enhance immune cell recruitment via endothelial activation

    PubMed Central

    Lee, Yu-Hui; Shynlova, Oksana; Lye, Stephen J

    2015-01-01

    Spontaneous term labour is associated with amplified inflammatory events in the myometrium including cytokine production and leukocyte infiltration; however, potential mechanisms regulating such events are not fully understood. We hypothesized that mechanical stretch of the uterine wall by the growing fetus facilitates peripheral leukocyte extravasation into the term myometrium through the release of various cytokines by uterine myocytes. Human myometrial cells (hTERT-HM) were subjected to static mechanical stretch; stretch-conditioned media was collected and analysed using 48-plex Luminex assay and ELISA. Effect of stretch-conditioned media on cell adhesion molecule expression of human uterine microvascular endothelial cells (UtMVEC-Myo) was detected by quantitative polymerase chain reaction (qPCR) and flow cytometry; functional assays testing leukocyte–endothelial interactions: adhesion of leukocytes to endothelial cells and transendothelial migration of calcein-labelled primary human neutrophils as well as migration of THP-1 monocytic cells were assessed by fluorometry. The current in vitro study demonstrated that mechanical stretch (i) directly induces secretion of multiple cytokines and chemokines by hTERT-HM cells (IL-6, CXCL8, CXCL1, migration inhibitory factor (MIF), VEGF, G-CSF, IL-12p70, bFGF and platelet-derived growth factor subunit B (PDGF-bb), P<0.05); stretch-induced cytokines (ii) enhance leukocyte adhesion to the endothelium of the surrounding uterine microvasculature by (iii) inducing the expression of endothelial cell adhesion molecules and (iv) directing the transendothelial migration of peripheral leukocytes. (vi) Chemokine-neutralizing antibodies and broad-spectrum chemokine inhibitor block leukocyte migration. Our data provide a proof of mechanical regulation for leukocyte recruitment from the uterine blood vessels to the myometrium, suggesting a putative mechanism for the leukocyte infiltrate into the uterus during labour and postpartum

  5. An immortalized human blood-nerve barrier endothelial cell line for in vitro permeability studies

    PubMed Central

    Yosef, Nejla; Ubogu, Eroboghene E.

    2012-01-01

    Solute and macromolecular transport studies may elucidate nutritional requirements and drug effects in healthy and diseased peripheral nerves. Endoneurial endothelial cells are specialized microvascular cells that form the restrictive blood-nerve barrier (BNB). Primary human endoneurial endothelial cells (pHEndECs) are difficult to isolate, limiting their widespread availability for biomedical research. We developed a simian virus-40 large T-antigen (SV40-LTA) immortalized human BNB cell line via stable transfection of low passage pHEndECs and observed continuous growth in culture for >45 population doublings. As observed with pHEndECs, the immortalized BNB endothelial cells were Ulex Europaeus agglutinin-1 (UEA-1)-positive and endocytosed low density lipoprotein, but lost von Willebrand factor (vWF) expression. Glucose transporter-1 (GLUT-1), P-glycoprotein (P-gp), γ-glutamyl transpeptidase (γ-GT), large neutral amino acid transporter-1 (LAT-1), creatine transporter (CRT) and monocarboxylate transporter-1 (MCT-1) mRNA expression were retained at all passages with loss of alkaline phosphatase (AP) expression after passages 16-20. Compared with an SV40-LTA immortalized human blood-brain barrier (BBB) endothelial cell line, there was increased γ-GT protein expression, equivalent expression of organic anion transporting polypeptide-C (OATP-C), organic anion transporter 3 (OAT-3), MCT-1 and LAT-1, and reduced expression of AP, CRT and P-gp by the BNB cell line at passage 20. Further studies demonstrated lower transendothelial electrical resistance (TEER: ~181 Ω.cm2 vs. 191 Ω.cm2), equivalent permeability to fluoresceinated sodium (4.84% vs. 4.39%) and lower permeability to fluoresceinated high molecular weight (70 kDa) dextran (0.39% vs. 0.52%) by the BNB cell line. This cell line retained essential molecular and biophysical properties suitable for in vitro peripheral nerve permeability studies. PMID:23104242

  6. A novel adipocytokine, chemerin exerts anti-inflammatory roles in human vascular endothelial cells

    SciTech Connect

    Yamawaki, Hideyuki; Kameshima, Satoshi; Usui, Tatsuya; Okada, Muneyoshi; Hara, Yukio

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Chemerin is a novel adipocytokine with almost unknown function in vasculature. Black-Right-Pointing-Pointer Chemerin activates Akt/eNOS/NO pathways in endothelial cells. Black-Right-Pointing-Pointer Chemerin inhibits TNF-{alpha}-induced monocyte adhesion to endothelial cells. Black-Right-Pointing-Pointer Chemerin inhibits TNF-induced VCAM-1 via suppressing NF-{kappa}B and p38 signal. Black-Right-Pointing-Pointer Chemerin is anti-inflammatory through producing NO in vascular endothelium. -- Abstract: Chemerin is a recently identified adipocytokine which plays a role on inflammation and adipocytes metabolism. However, its function in vasculature is largely unknown. We examined the effects of chemerin on vascular endothelial inflammatory states. Treatment of human umbilical vein endothelial cells with chemerin (300 ng/ml, 20 min) induced phosphorylation of Akt (Ser473) and endothelial nitric oxide (NO) synthase (eNOS) (Ser1177). Consistently, chemerin increased intracellular cyclic GMP content. Pretreatment with chemerin (1-300 ng/ml, 24 h) significantly inhibited phosphorylation of nuclear factor (NF)-{kappa}B p65 (Ser536) and p38 as well as vascular cell adhesion molecule (VCAM)-1 expression induced by tumor necrosis factor (TNF)-{alpha} (5 ng/ml, 20 min-6 h). Inhibitor of NF-{kappa}B or p38 significantly inhibited the TNF-{alpha}-induced VCAM-1 expression. Chemerin also inhibited TNF-{alpha}-induced VCAM-1 expression in rat isolated aorta. Moreover, chemerin significantly inhibited monocytes adhesion to TNF-{alpha}-stimulated endothelial cells. The inhibitory effect of chemerin on TNF-{alpha}-induced VCAM-1 was reversed by a NOS inhibitor. Conversely, an NO donor, sodium nitroprusside significantly inhibited TNF-{alpha}-induced VCAM-1. The present results for the first time demonstrate that chemerin plays anti-inflammatory roles by preventing TNF-{alpha}-induced VCAM-1 expression and monocytes adhesion in vascular

  7. The human peripheral lymph node vascular addressin. An inducible endothelial antigen involved in lymphocyte homing.

    PubMed Central

    Michie, S. A.; Streeter, P. R.; Bolt, P. A.; Butcher, E. C.; Picker, L. J.

    1993-01-01

    The extravasation of blood-borne lymphocytes into organized lymphoid tissues and sites of chronic inflammation is directed in part by interactions of lymphocyte surface adhesion molecules, known as homing receptors, with tissue-selective endothelial ligands called vascular addressins. In mice and humans, lymphocyte L-selectin and the peripheral lymph node addressin (PNAd) form a homing receptor-endothelial ligand pair involved in lymphocyte traffic to peripheral lymph node (PLN). We have examined the tissue distribution and function of human PNAd, using monoclonal antibody MECA-79 and in vitro assays of L-selectin-dependent lymphocyte binding. We demonstrate that PNAd is expressed by human high endothelial venules (HEV) in lymphoid tissues which support lymphocyte adhesion via a PLN-associated recognition system. MECA-79 inhibits adhesion to these HEV of a cell line that binds predominantly via the PLN-homing receptor, L-selectin, but has no effect on adhesion by a mucosal HEV-binding cell line. Furthermore, MECA-79 blocks binding of human peripheral blood mononuclear cells to both PLN and tonsil HEV, but not significantly to HEV in the appendix. In addition, we demonstrate PNAd induction on venules at chronic inflammatory sites in humans, particularly sites with severe or long-standing chronic inflammatory involvement. These results confirm that PNAd functions as a PLN vascular addressin in humans, and that in addition to directing normal lymphocyte recirculation to lymph nodes and tonsils, this addressin likely participates in lymphocyte recruitment to sites of chronic inflammation. Images Figure 1 Figure 4 PMID:8256856

  8. Adhesion and migration of polymorphonuclear leukocytes across human brain microvessel endothelial cells are differentially regulated by endothelial cell adhesion molecules and modulate monolayer permeability.

    PubMed

    Wong, Donald; Prameya, Rukmini; Dorovini-Zis, Katerina

    2007-03-01

    The mechanisms by which polymorphonuclear leukocytes (PMN) cross the human blood-brain barrier have not been fully elucidated. Using a well characterized in vitro model of the human BBB, we examined the role of endothelial cell adhesion molecules on the adhesion and transendothelial migration of PMN across primary cultures of human brain microvessel endothelial cells (HBMEC). A small number of PMN (0.06%) adhered to unstimulated HBMEC, and the basal adhesion was not affected by anti-adhesion molecule antibodies. Treatment of HBMEC with tumor necrosis factor (TNF)-alpha resulted in increased PMN adhesion that was significantly inhibited by blocking antibodies to E-selectin and ICAM-1, but not VCAM-1 or PECAM-1. A very small number of adherent PMN migrated across unstimulated HBMEC monolayers. Migration increased 2 to 20 fold following stimulation of HBMEC with TNF-alpha. Monoclonal antibody blocking studies showed that PMN used ICAM-1, but not VCAM-1, E-selectin or PECAM-1 to move across activated monolayers. Anti-adhesion molecule antibodies did not diminish the basal PMN migration. Ultrastructurally, PMN often aggregated on top and between adjacent endothelial cells and adhered by first extending pseudopodia along the apical endothelial surface. They then flattened and inserted themselves between endothelial cells in order to migrate across the monolayers. At the end of the migration period, the cultures resumed their continuity with no evidence of disruption. Transendothelial migration of PMN decreased the transendothelial electrical resistance and increased the permeability to horseradish peroxidase, which penetrated alongside the migrating leukocytes. A blocking antibody to ICAM-1 that greatly decreased migration, had no effect on the permeability changes. These studies provide insights into the mechanisms that regulate the entry of PMN into the brain and the increased permeability of the BBB in CNS inflammation. PMID:17291598

  9. A role for GPR55 in human placental venous endothelial cells

    PubMed Central

    Kremshofer, Julia; Siwetz, Monika; Berghold, Veronika M.; Lang, Ingrid; Huppertz, Berthold; Gauster, Martin

    2015-01-01

    Endocannabinoids and their G protein-coupled receptors have been suggested to play a key role in human pregnancy, by regulating important aspects such as implantation, decidualization, placentation and labour. G protein-coupled receptor 55 (GPR55) was previously postulated to be another cannabinoid receptor, since specific cannabinoids were shown to act independently of the classical cannabinoid receptors CB1 or CB2. Current knowledge about GPR55 expression and function in human placenta is very limited and motivated us to evaluate human placental GPR55 expression in relation to other human peripheral tissues and to analyze spatiotemporal GPR55 expression in human placenta. Gene expression analysis revealed low GPR55 levels in human placenta, when compared to spleen and lung, the organs showing highest GPR55 expression. Moreover, expression analysis showed 5.8 fold increased placental GPR55 expression at term compared to first trimester. Immunohistochemistry located GPR55 solely at the fetal endothelium of first trimester and term placenta. qPCR and immunocytochemistry consistently confirmed GPR55 expression in isolated primary placental arterial and venous endothelial cells. Incubation with L-α-lysophosphatidylinositol (LPI), the specific and functional ligand for GPR55, at a concentration of 1 μM, significantly enhanced migration of venous, but not arterial endothelial cells. LPI enhanced migration was inhibited by the GPR55 antagonist O-1918, suggesting a role of the LPI-GPR55 axis in placental venous endothelium function. PMID:25869640

  10. Endothelin B receptors on human endothelial and smooth-muscle cells show equivalent binding pharmacology.

    PubMed

    Flynn, M A; Haleen, S J; Welch, K M; Cheng, X M; Reynolds, E E

    1998-07-01

    We have described the pharmacologic profiles of endothelin B receptors in human endothelial cells and vascular and nonvascular smooth-muscle cells. First, by amplifying endothelin B receptor numbers through the use of phosphoramidon and intact cell-binding techniques, we demonstrated the presence of these receptors in human umbilical vein endothelial cells (100% endothelin B receptors), human aortic smooth-muscle cells (22% endothelin B, 78% endothelin A receptors), and human bronchial smooth-muscle cells (55% endothelin B, 45% endothelin A receptors) by using [125I]-endothelin-1 radioligand binding. The typical binding profiles of the endothelin B receptors were established through competition binding curve analysis with endothelin-1, endothelin-3, sarafotoxin 6c, and the endothelin A receptor-selective antagonist BQ-123. In the presence of BQ-123, a diverse group of antagonists, including PD 142893, BQ-788, SB 209670, and Ro 47-0203, were used to probe for binding differences indicative of multiple endothelin B-receptor subtypes. The results indicate a rank order of potency for the antagonists of BQ-788 > SB 209670 > PD 142893 > Ro 47-0203 for each cell line, and that between any of these human cell lines, measurements of [125I]-endothelin-1-binding antagonism for each of the four test compounds differed by less than twofold. Although this study cannot discount the possibility of more than one endothelin B-receptor subtype in humans, it does indicate that these tissues express receptors that show equivalent binding pharmacology. PMID:9676729

  11. A role for GPR55 in human placental venous endothelial cells.

    PubMed

    Kremshofer, Julia; Siwetz, Monika; Berghold, Veronika M; Lang, Ingrid; Huppertz, Berthold; Gauster, Martin

    2015-07-01

    Endocannabinoids and their G protein-coupled receptors have been suggested to play a key role in human pregnancy, by regulating important aspects such as implantation, decidualization, placentation and labor. G protein-coupled receptor 55 (GPR55) was previously postulated to be another cannabinoid receptor, since specific cannabinoids were shown to act independently of the classical cannabinoid receptors CB1 or CB2. Current knowledge about GPR55 expression and function in human placenta is very limited and motivated us to evaluate human placental GPR55 expression in relation to other human peripheral tissues and to analyze spatiotemporal GPR55 expression in human placenta. Gene expression analysis revealed low GPR55 levels in human placenta, when compared to spleen and lung, the organs showing highest GPR55 expression. Moreover, expression analysis showed 5.8 fold increased placental GPR55 expression at term compared to first trimester. Immunohistochemistry located GPR55 solely at the fetal endothelium of first trimester and term placentas. qPCR and immunocytochemistry consistently confirmed GPR55 expression in isolated primary placental arterial and venous endothelial cells. Incubation with L-α-lysophosphatidylinositol (LPI), the specific and functional ligand for GPR55, at a concentration of 1 µM, significantly enhanced migration of venous, but not arterial endothelial cells. LPI-enhanced migration was inhibited by the GPR55 antagonist O-1918, suggesting a role of the LPI-GPR55 axis in placental venous endothelium function. PMID:25869640

  12. Characterization of the response of human bone marrow endothelial cells to in vitro irradiation.

    PubMed

    Gaugler, M H; Squiban, C; Claraz, M; Schweitzer, K; Weksler, B; Gourmelon, P; Van der Meeren, A

    1998-12-01

    Endothelial cell dysfunction is a classic consequence of radiation damage. Bone marrow endothelial cells (BMEC) are a critical component of the stroma in the regulation of haemopoiesis. In animal models, radiation-induced injury of BMEC has been described and a role for BMEC in haemopoietic regeneration after irradiation has been suggested. However, functions of BMEC involved in the haemopoietic regeneration have not been assessed. Therefore we studied the functional response of human BMEC to irradiation using the transformed human BMEC line (TrHBMEC) irradiated with 2. 5 or 10Gy. Our results showed a time- and a dose-dependent increase in damage to irradiated TrHBMEC measured by a decreased number of adherent cells which correlated with increased apoptosis and augmented release of soluble ICAM-1 and von Willebrand factor. 2 Gy irradiated TrHBMEC expressed more ICAM-1 on their surface than non-irradiated cells, whereas no change in VCAM-1, E-selectin and PECAM-1 expression was observed. An increased production of G-CSF, GM-CSF, IL-8, IL-6, IL-1alpha, IL-11, MIP-1alpha and SCF and no production of LIF, TNF-alpha, TPO and IL-3 by 2 Gy irradiated TrHBMEC was observed. The haemopoietic supportive function of TrHBMEC was not altered after a 2 Gy exposure. These results suggest that although radiation induces endothelial cell damage, irradiated cells still support the proliferation and the differentiation of CD34+ haemopoietic cells. PMID:9886309

  13. Anti-angiogenic action of plasma hyaluronan binding protein in human umbilical vein endothelial cells.

    PubMed

    Jeon, Ji Won; Song, Hyun Seok; Moon, Eun-Joung; Park, Shi-Young; Son, Myung Jin; Jung, Seung Youn; Kim, Ji Tae; Nam, Do-Hyun; Choi-Miura, Nam-Ho; Kim, Kyu-Won; Kim, Yung-Jin

    2006-07-01

    The kringle domain is a triple loop structure present in angiostatin and endostatin. The disulfide bond-linked kringle architectures have been known to be essential for anti-angiogenic activity. Plasma hyaluronan binding protein (PHBP) is a novel serine protease which consists of three epidermal growth factor (EGF) domains, a kringle domain, and a serine protease domain. PHBP can be cleaved autocatalytically to generate activity and is highly expressed in the human blood and liver. To determine the anti-angiogenic activities of PHBP, we purified recombinant mouse PHBP from stable cell line overexpressing PHBP and used protein in vivo and in vitro angiogenesis assays. We found that recombinant PHBP inhibits not only angiogenesis in vivo in chorioallantoic membrane (CAM) assay but also the basic fibroblast growth factor (bFGF)-induced proliferation, invasion and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependant manner. Moreover, we found that the kringle domain of PHBP was essential for the anti-angiogenic action of PHBP by the deletion mutants. These findings unravel a new function of PHBP as an inhibitor of the proangiogenic phenotype of vascular endothelial cells and demonstrate that the kringle domain of PHBP might be a potent novel inhibitor of activated endothelial cells in vitro and in vivo. PMID:16773202

  14. Micropatterned 3-Dimensional Hydrogel System to Study Human Endothelial-Mesenchymal Stem Cell Interactions

    PubMed Central

    Trkov, Sasa; Eng, George; di Liddo, Rosa; Parnigotto, Pier Paolo; Vunjak-Novakovic, Gordana

    2009-01-01

    The creation of vascularized engineered tissues of clinically relevant size is a major challenge of tissue engineering. While it is known that endothelial and mural vascular cells are integral to the formation of stable blood vessels, the specific cell type and optimal conditions for engineered vascular networks are poorly understood. To this end, we investigated the vasculogenic potential of human mesenchymal stem cell (MSC) populations derived from three different sources: (i) bone marrow aspirates, (ii) perivascular cells from umbilical cord vein, and (iii) perivascular cells from umbilical cord artery. Cell populations were isolated and identified as MSCs according to their phenotypes and differentiation potential. Human umbilical vein endothelial cells (HUVEC) were used as a standard for endothelial cells. A novel co-culture system was developed to study cell-cell interactions in a spatially controlled three-dimensional (3D) fibrin hydrogel model. Using microfluidic patterning, it was possible to localize hydrogel-encapsulated HUVECs and MSCs within separate channels spaced at 500, 1000 or 2000 μm. All three MSC populations had similar expression profiles of mesenchymal cell markers, and similar capacity for osteogenic and adipogenic differentiation. However, bone marrow-derived MSCs (but not umbilical vein or artery derived MSCs) showed strong distance-dependent migration toward HUVECs and supported the formation of stable vascular networks resembling capillary-like vasculature. The presented approach provides a simple and robust model to study cell-cell communication of relevance to engineering vascularized tissues. PMID:19998330

  15. Therapeutic transdifferentiation of human fibroblasts into endothelial cells using forced expression of lineage-specific transcription factors

    PubMed Central

    Wong, Wing Tak; Cooke, John P

    2016-01-01

    Transdifferentiation is the direct conversion from one somatic cell type into another desired somatic cell type. This reprogramming method offers an attractive approach for regenerative medicine. Here, we demonstrate that neonatal fibroblasts can be transdifferentiated into endothelial cells using only four endothelial transcription factors, namely, ETV2, FLI1, GATA2, and KLF4. We observed a significant up-regulation of endothelial genes including KDR, CD31, CD144, and vWF in human neonatal foreskin (BJ) fibroblasts infected with the lentiviral construct encoding the open reading frame of the four transcription factors. We observed morphological changes in BJ fibroblasts from the fibroblastic spindle shape into a more endothelial-like cobblestone structures. Fluorescence-activated cell sorting analysis revealed that ~16% of the infected cells with the lentiviral constructs encoding 4F expressed CD31. The sorted cells were allowed to expand for 2 weeks and these cells were immunostained and found to express endothelial markers CD31. The induced endothelial cells also incorporated fluorescence-labeled acetylated low-density lipoprotein and efficiently formed capillary-like networks when seeded on Matrigel. These results suggested that the induced endothelial cells were functional in vitro. Taken together, we successfully demonstrated the direct conversion of human neonatal fibroblasts into endothelial cells by transduction of lentiviral constructs encoding endothelial lineage-specific transcription factors ETV2, FLI1, GATA2, and KLF4. The directed differentiation of fibroblasts into endothelial cells may have significant utility in diseases characterized by fibrosis and loss of microvasculature. PMID:27081470

  16. Interferon-γ inhibits group B Streptococcus survival within human endothelial cells

    PubMed Central

    Lione, Viviane de Oliveira Freitas; dos Santos, Michelle Hanthequeste Bittencourt; de Oliveira, Jessica Silva Santos; Mattos-Guaraldi, Ana Luiza; Nagao, Prescilla Emy

    2014-01-01

    Endothelial dysfunction is a major component of the pathophysiology of septicaemic group B Streptococcus (GBS) infections. Although cytokines have been shown to activate human umbilical vein endothelial cells (HUVECs), the capacity of interferon (IFN)-γ to enhance the microbicidal activity of HUVECs against GBS has not been studied. We report that the viability of intracellular bacteria was reduced in HUVECs activated by IFN-γ. Enhanced fusion of lysosomes with bacteria-containing vacuoles was observed by acid phosphatase and the colocalisation of Rab-5, Rab-7 and lysosomal-associated membrane protein-1 with GBS in IFN-γ-activated HUVECs. IFN-γ resulted in an enhancement of the phagosome maturation process in HUVECs, improving the capacity to control the intracellular survival of GBS. PMID:25410999

  17. Magnetic particle spectroscopy allows precise quantification of nanoparticles after passage through human brain microvascular endothelial cells

    NASA Astrophysics Data System (ADS)

    Gräfe, C.; Slabu, I.; Wiekhorst, F.; Bergemann, C.; von Eggeling, F.; Hochhaus, A.; Trahms, L.; Clement, J. H.

    2016-06-01

    Crossing the blood–brain barrier is an urgent requirement for the treatment of brain disorders. Superparamagnetic iron oxide nanoparticles (SPIONs) are a promising tool as carriers for therapeutics because of their physical properties, biocompatibility, and their biodegradability. In order to investigate the interaction of nanoparticles with endothelial cell layers in detail, in vitro systems are of great importance. Human brain microvascular endothelial cells are a well-suited blood–brain barrier model. Apart from generating optimal conditions for the barrier-forming cell units, the accurate detection and quantification of SPIONs is a major challenge. For that purpose we use magnetic particle spectroscopy to sensitively and directly quantify the SPION-specific iron content. We could show that SPION concentration depends on incubation time, nanoparticle concentration and location. This model system allows for further investigations on particle uptake and transport at cellular barriers with regard to parameters including particles’ shape, material, size, and coating.

  18. Magnetic particle spectroscopy allows precise quantification of nanoparticles after passage through human brain microvascular endothelial cells.

    PubMed

    Gräfe, C; Slabu, I; Wiekhorst, F; Bergemann, C; von Eggeling, F; Hochhaus, A; Trahms, L; Clement, J H

    2016-06-01

    Crossing the blood-brain barrier is an urgent requirement for the treatment of brain disorders. Superparamagnetic iron oxide nanoparticles (SPIONs) are a promising tool as carriers for therapeutics because of their physical properties, biocompatibility, and their biodegradability. In order to investigate the interaction of nanoparticles with endothelial cell layers in detail, in vitro systems are of great importance. Human brain microvascular endothelial cells are a well-suited blood-brain barrier model. Apart from generating optimal conditions for the barrier-forming cell units, the accurate detection and quantification of SPIONs is a major challenge. For that purpose we use magnetic particle spectroscopy to sensitively and directly quantify the SPION-specific iron content. We could show that SPION concentration depends on incubation time, nanoparticle concentration and location. This model system allows for further investigations on particle uptake and transport at cellular barriers with regard to parameters including particles' shape, material, size, and coating. PMID:27163489

  19. Molecular Characterization of NF-HEV, a Nuclear Factor Preferentially Expressed in Human High Endothelial Venules

    PubMed Central

    Baekkevold, Espen S.; Roussigné, Myriam; Yamanaka, Takeshi; Johansen, Finn-Eirik; Jahnsen, Frode L.; Amalric, François; Brandtzaeg, Per; Erard, Monique; Haraldsen, Guttorm; Girard, Jean-Philippe

    2003-01-01

    Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). In this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyers’s patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs. Finally, threading and molecular modeling studies suggested that the amino-terminal part of NF-HEV (aa 1–60) corresponds to a novel homeodomain-like Helix-Turn-Helix (HTH) DNA-binding domain. Similarly to the atypical homeodomain transcription factor Prox-1, which plays a critical role in the induction of the lymphatic endothelium phenotype, NF-HEV may be one of the key nuclear factors that controls the specialized HEV phenotype. PMID:12819012

  20. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    NASA Astrophysics Data System (ADS)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.

    2011-11-01

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  1. Klotho modulates FGF23-mediated NO synthesis and oxidative stress in human coronary artery endothelial cells.

    PubMed

    Richter, Beatrice; Haller, Jacqueline; Haffner, Dieter; Leifheit-Nestler, Maren

    2016-09-01

    Chronic kidney disease (CKD) is a state of Klotho deficiency and excess of the phosphaturic hormone fibroblast growth factor 23 (FGF23). Both dysregulations were shown to be associated with endothelial dysfunction in humans, but direct vascular effects of FGF23 remain largely elusive. In vitro experiments were performed to assess the effects of FGF23 (10 ng/mL) in relation to its co-receptor Klotho on nitric oxide (NO) synthesis and reactive oxygen species (ROS) formation and detoxification in human coronary artery endothelial cells (HCAEC). Membrane-bound Klotho is expressed in HCAEC, and FGF23 increases the expression of the Klotho shedding protease ADAM17, and consequently the secretion of soluble Klotho. FGF23 activates FGF receptor 1 and stimulates NO release via Akt-dependent activation of endothelial NO synthase (eNOS). Both FGF receptor (FGFR)-dependent ROS formation via activation of NADPH oxidase 2 (Nox2) as well as ROS degradation via superoxide dismutase 2 (SOD2) and catalase (CAT) is stimulated by FGF23. Pre-incubation with a Klotho inhibitor blunts the FGF23-stimulated Akt-eNOS activation and NO synthesis, and decreases ROS degradation by blocking SOD2 and CAT enzymes, whereas FGF23-stimulated ROS synthesis via Nox2 is unaffected, resulting in low NO bioavailability and increased oxidative stress. Our data indicate that in the presence of Klotho, FGF23 induces NO release in HCAEC and its stimulating effects on ROS production are counterbalanced by increased ROS degradation. In states of Klotho deficiency, e.g., CKD, FGF23-mediated NO synthesis is blunted and ROS formation overrules ROS degradation. Thus, FGF23 excess may primarily promote oxidative stress and thus endothelial dysfunction. PMID:27448998

  2. Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

    PubMed

    Quan, S; Yang, L; Abraham, N G; Kappas, A

    2001-10-01

    Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin. PMID:11593038

  3. Cross-reactivity of cell-selective CRRETAWAC peptide with human and porcine endothelial cells.

    PubMed

    Dudash, Lynn A; Kligman, Faina L; Bastijanic, Jennifer M; Kottke-Marchant, Kandice; Marchant, Roger E

    2014-08-01

    We report on the cross-reactivity of the cell adhesive peptide CRRETAWAC between human and porcine endothelial cells (ECs). CRRETAWAC is a phage display derived peptide which has been shown to bind the α5 β1 receptor on human ECs, but does not bind platelets and thus could be incorporated into a coating for cardiovascular biomaterials that resists platelet adhesion and thrombosis, while allowing for endothelialization. To determine the cross-reactivity of the peptide, attachment and growth of human and porcine ECs on CRRETAWAC fluorosurfactant polymer (FSP) coated surfaces was explored. CRRETAWAC FSP was synthesized and characterized by mass spectrometry, NMR, and IR spectroscopy. pEC attachment and growth on CRRETAWAC FSP was similar to the positive controls, human fibronectin and RGD FSP, achieving confluence in 72 h. Initial adhesion on CRRETAWAC FSP was also similar for porcine and human ECs. Blocking with soluble CRRETAWAC peptide reduced adhesion to CRRETAWAC coated surfaces by over 50%, indicating that the pECs specifically bind CRRETAWAC peptide. With this study, we have demonstrated that CRRETAWAC peptide coated surfaces are capable of binding porcine ECs in a specific manner and supporting a confluent layer of pECs. In vitro validation of the porcine model was critical for ensuring the best chance of success for the in vivo testing of CRRETAWAC coated ePTFE vascular grafts. PMID:24123752

  4. Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

    PubMed Central

    Reed, Daniel M.; Foldes, Gabor; Gatheral, Timothy; Paschalaki, Koralia E.; Lendvai, Zsuzsanna; Bagyura, Zsolt; Nemeth, Tamas; Skopal, Judit; Merkely, Bela; Telcian, Aurica G.; Gogsadze, Leila; Edwards, Michael R.; Gough, Peter J.; Bertin, John; Johnston, Sebastian L.; Harding, Sian E.; Mitchell, Jane A.

    2014-01-01

    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. PMID:24690886

  5. Markers of early endothelial dysfunction in intrauterine growth restriction-derived human umbilical vein endothelial cells revealed by 2D-DIGE and mass spectrometry analyses.

    PubMed

    Caniuguir, Andres; Krause, Bernardo J; Hernandez, Cherie; Uauy, Ricardo; Casanello, Paola

    2016-05-01

    Intrauterine growth restriction (IUGR) associates with fetal and placental vascular dysfunction, and increased cardiovascular risk later on life. We hypothesize that endothelial cells derived from IUGR umbilical veins present significant changes in the proteome which could be involved in the endothelial dysfunction associated to this conditions. To address this the proteome profile of human umbilical endothelial cells (HUVEC) isolated from control and IUGR pregnancies was compared by 2D-Differential In Gel Electrophoresis (DIGE) and further protein identification by MALDI-TOF MS. Using 2D-DIGE 124 spots were identified as differentially expressed between control and IUGR HUVEC, considering a cut-off of 2 fold change, which represented ∼10% of the total spots detected. Further identification by MALDI-TOF MS and in silico clustering of the proteins showed that those differentially expressed proteins between control and IUGR HUVEC were mainly related with cytoskeleton organization, proteasome degradation, oxidative stress response, mRNA processing, chaperones and vascular function. Finally Principal Component analysis of the identified proteins showed that differentially expressed proteins allow distinguishing between control and IUGR HUVEC based on their proteomic profile. This study demonstrates for the first time that IUGR-derived HUVEC maintained in primary culture conditions present an altered proteome profile, which could reflect an abnormal programming of endothelial function in this fetal condition. PMID:27208404

  6. Molecular mimicry of human endothelial cell antigen by autoantibodies to nonstructural protein 1 of dengue virus.

    PubMed

    Liu, I-Ju; Chiu, Chien-Yu; Chen, Yun-Ching; Wu, Han-Chung

    2011-03-18

    The pathogenesis of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), both serious complications of dengue virus (DV) infection, remains unclear. In this study, we found that anti-DV NS1 (nonstructural protein 1) polyclonal antibodies cross-reacted with human umbilical vein endothelial cells (HUVECs). We further identified a complex-specific mAb, DB16-1, which could recognize DV NS1 and cross-react with HUVECs and human blood vessels. The target protein of DB16-1 was further purified by immunoaffinity chromatography. LC-MS/MS analysis and co-immunoprecipitation revealed that the target protein of DB16-1 was human LYRIC (lysine-rich CEACAM1 co-isolated). Our newly generated anti-LYRIC mAbs bound to HUVECs in a pattern similar to that of DB16-1. The B-cell epitope of DB16-1 displayed a consensus motif, Lys-X-Trp-Gly (KXWG), which corresponded to amino acid residues 116-119 of DV NS1 and mimicked amino acid residues 334-337 in LYRIC. Moreover, the binding activity of DB16-1 in NS1 of DV-2 and in LYRIC disappeared after the KXWG epitope was deleted in each. In conclusion, DB16-1 targeted the same epitope in DV NS1 and LYRIC protein on human endothelial cells, suggesting that it might play a role in the pathogenesis of DHF/DSS. Future studies on the role of the anti-NS1 antibody in causing vascular permeability will undoubtedly be performed on sera collected from individuals before, during, and after the endothelial cell malfunction phase of a dengue illness. PMID:21233208

  7. Effects of interferon-gamma on primary cultures of human brain microvessel endothelial cells.

    PubMed Central

    Huynh, H. K.; Dorovini-Zis, K.

    1993-01-01

    Primary cultures of human brain microvessel endothelial cells were used to study the effects of human recombinant interferon-gamma (IFN-gamma) on cerebral endothelium in vitro. Incubation of monolayers with various concentrations of IFN-gamma (10 to 200 U/ml) for 12 to 96 hours induced surface expression of class II major histocompatibility complex (Ia) antigen in a time- and concentration-dependent manner. In immunogold-stained cultures, labeling was observed as early as 12 hours, was maximal after 48 hours, and persisted at plateau levels in the continuous presence of the cytokine. Expression was blocked by coincubation with anti-IFN-gamma antibody and was reversed 4 days following removal of IFN-gamma from the culture media. Endothelial cells treated with IFN-gamma for 3 to 4 days became spindle-shaped, extensively overlapped, and frequently formed cellular whorls. These changes did not occur in the presence of anti-IFN-gamma antibody and reversed upon removal of IFN-gamma from the media. The morphological alterations were associated with increased permeability of confluent monolayers to macromolecules as compared with untreated cultures. The results of these studies indicate that human brain microvessel endothelial cells respond to in vitro cytokine stimulation by undergoing profound morphological, functional, and permeability changes. We conclude that cerebral endothelium may play an important role in the initiation and regulation of lymphocyte traffic across the blood-brain barrier in inflammatory disorders of the human central nervous system. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 8 PMID:8475997

  8. Human vascular endothelial cells transport foreign exosomes from cow's milk by endocytosis.

    PubMed

    Kusuma, Rio Jati; Manca, Sonia; Friemel, Taylor; Sukreet, Sonal; Nguyen, Christopher; Zempleni, Janos

    2016-05-15

    Encapsulation of microRNAs in exosomes confers protection against degradation and a vehicle for shuttling of microRNAs between cells and tissues, and cellular uptake by endocytosis. Exosomes can be found in foods including milk. Humans absorb cow's milk exosomes and deliver the microRNA cargo to peripheral tissues, consistent with gene regulation by dietary nucleic acids across species boundaries. Here, we tested the hypothesis that human vascular endothelial cells transport milk exosomes by endocytosis, constituting a step crucial for the delivery of dietary exosomes and their cargo to peripheral tissues. We tested this hypothesis by using human umbilical vein endothelial cells and fluorophore-labeled exosomes isolated from cow's milk. Exosome uptake followed Michaelis-Menten kinetics (Vmax = 0.057 ± 0.004 ng exosome protein × 40,000 cells/h; Km = 17.97 ± 3.84 μg exosomal protein/200 μl media) and decreased by 80% when the incubation temperature was lowered from 37°C to 4°C. When exosome surface proteins were removed by treatment with proteinase K, or transport was measured in the presence of the carbohydrate competitor d-galactose or measured in the presence of excess unlabeled exosomes, transport rates decreased by 45% to 80% compared with controls. Treatment with an inhibitor of endocytosis, cytochalasin D, caused a 50% decrease in transport. When fluorophore-labeled exosomes were administered retro-orbitally, exosomes accumulated in liver, spleen, and lungs in mice. We conclude that human vascular endothelial cells transport bovine exosomes by endocytosis and propose that this is an important step in the delivery of dietary exosomes and their cargo to peripheral tissues. PMID:26984735

  9. Characterization of calcium signals provoked by lysophosphatidylinositol in human microvascular endothelial cells.

    PubMed

    Al Suleimani, Y M; Hiley, C R

    2016-01-01

    The lipid molecule, lysophosphatidylinositol (LPI), is hypothesised to form part of a novel lipid signalling system that involves the G protein-coupled receptor GPR55 and distinct intracellular signalling cascades in endothelial cells. This work aimed to study the possible mechanisms involved in LPI-evoked cytosolic Ca(2+) mobilization in human brain microvascular endothelial cells. Changes in intracellular Ca(2+) concentrations were measured using cell population Ca(2+) assay. LPI evoked biphasic elevation of intracellular calcium concentration, a rapid phase and a sustained phase. The rapid phase was attenuated by the inhibitor of PLC (U 73122), inhibitor of IP(3) receptors, 2-APB and the depletor of endoplasmic reticulum Ca(2+) store, thapsigargin. The sustained phase, on the other hand, was enhanced by U 73122 and abolished by the RhoA kinase inhibitor, Y-27632. In conclusion, the Ca(2+) signal evoked by LPI is characterised by a rapid phase of Ca(2+) release from the endoplasmic reticulum, and requires activation of the PLC-IP(3) signalling pathway. The sustained phase mainly depends on RhoA kinase activation. LPI acts as novel lipid signalling molecule in endothelial cells, and elevation of cytosolic Ca(2+) triggered by it may present an important intracellular message required in gene expression and controlling of vascular tone. PMID:26596318

  10. Sphingosine kinase-1 is a hypoxia-regulated gene that stimulates migration of human endothelial cells

    SciTech Connect

    Schwalm, Stephanie; Doell, Frauke; Roemer, Isolde; Bubnova, Svetlana

    2008-04-18

    Sphingosine kinases (SK) catalyze the production of sphingosine-1-phosphate which in turn regulates cell responses such as proliferation and migration. Here, we show that exposure of the human endothelial cell line EA.hy 926 to hypoxia stimulates a increased SK-1, but not SK-2, mRNA, protein expression, and activity. This effect was due to stimulated SK-1 promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation was abrogated. Furthermore, hypoxia upregulated the expression of HIF-1{alpha} and HIF-2{alpha}, and both contributed to SK-1 gene transcription as shown by selective depletion of HIF-1{alpha} or HIF-2{alpha} by siRNA. The hypoxia-stimulated SK-1 upregulation was functionally coupled to increased migration since the selective depletion of SK-1, but not of SK-2, by siRNAs abolished the migratory response. In summary, these data show that hypoxia upregulates SK-1 activity and results in an accelerated migratory capacity of endothelial cells. SK-1 may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression.

  11. Potential in vitro effects of carbon nanotubes on human aortic endothelial cells

    SciTech Connect

    Walker, Valerie G.; Li Zheng; Hulderman, Tracy; Schwegler-Berry, Diane; Kashon, Michael L.; Simeonova, Petia P.

    2009-05-01

    Respiratory exposure of mice to carbon nanotubes induces pulmonary toxicity and adverse cardiovascular effects associated with atherosclerosis. We hypothesize that the direct contact of carbon nanotubes with endothelial cells will result in dose-dependent effects related to altered cell function and cytotoxicity which may play a role in potential adverse pulmonary and cardiovascular outcomes. To test this hypothesis, we examined the effects of purified single- and multi-walled carbon nanotubes (SWCNT and MWCNT) on human aortic endothelial cells by evaluating actin filament integrity and VE-cadherin distribution by fluorescence microscopy, membrane permeability by measuring the lactate dehydrogenase (LDH) release, proliferation/viability by WST-1 assay, and overall functionality by tubule formation assay. Marked actin filament and VE-cadherin disruption, cytotoxicity, and reduced tubule formation occurred consistently at 24 h post-exposure to the highest concentrations [50-150 {mu}g/10{sup 6} cells (1.5-4.5 {mu}g/ml)] for both SWCNT and MWCNT tested in our studies. These effects were not observed with carbon black exposure and carbon nanotube exposure in lower concentrations [1-10 {mu}g/10{sup 6} cells (0.04-0.4 {mu}g/ml)] or in any tested concentrations at 3 h post-exposure. Overall, the results indicate that SWCNT and MWCNT exposure induce direct effects on endothelial cells in a dose-dependent manner.

  12. Human pulmonary artery endothelial cells in the model of mucopolysaccharidosis VI present a prohypertensive phenotype

    PubMed Central

    Golda, Adam; Jurecka, Agnieszka; Gajda, Karolina; Tylki-Szymańska, Anna; Lalik, Anna

    2015-01-01

    Background Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a deficient activity of N-acetylgalactosamine-4-sulfatase (ARSB). Pulmonary hypertension (PH) occurs in MPS VI patients and is a marker of bad prognosis. Malfunction of endothelium, which regulates vascular tonus and stimulates angiogenesis, can contribute to the occurrence of PH in MPS VI. Aim The aim of the study was to establish a human MPS VI cellular model of pulmonary artery endothelial cells (HPAECs) and evaluate how it affects factors that may trigger PH such as proliferation, apoptosis, expression of endothelial nitric oxide synthase (eNOS), natriuretic peptide type C (NPPC), and vascular endothelial growth factor A (VEGFA). Results Increasing concentrations of dermatan sulfate (DS) reduce the viability of the cells in both ARSB deficiency and controls, but hardly influence apoptosis. The expression of eNOS in HPAECs is reduced up to two thirds in the presence of DS. NPPC shows a biphasic expression reaction with an increase at 50 μg/mL DS and reduction at 0 and 100 μg/mL DS. The expression of VEGFA decreases with increasing DS concentrations and absence of elastin, and increases with increasing DS in the presence of elastin. Conclusion Our data suggest that MPS VI endothelium presents a prohypertensive phenotype due to the reduction of endothelium's proliferation ability and expression of vasorelaxing factors. PMID:26937388

  13. Effects of statins on nitric oxide/cGMP signaling in human umbilical vein endothelial cells.

    PubMed

    Meda, Claudia; Plank, Christian; Mykhaylyk, Olga; Schmidt, Kurt; Mayer, Bernd

    2010-01-01

    Human umbilical vein endothelial cells (HUVECs) were established as in vitro models for the modulation of endothelial function and cell viability by statins. Emphasis was placed on the biphasic effects of the drugs on nitric oxide (NO) bioavailability and cytotoxicity, as well as drug interference with the interaction of endothelial NO synthase (eNOS) with caveolin-1 (Cav-1). Incubation of HUVECs with fluvastatin, lovastatin or cerivastatin for 24 h caused an approximately 3-fold upregulation of eNOS expression that was associated with increased eNOS activity and accumulation of cGMP. Cerivastatin exhibited the highest potency with an EC50 of 13.8 +/- 2 nM after 24 h, while having no effect after only 30 min. The effects of statins on eNOS expression were similar in control and Cav-1 knockdown cells, but the increase in eNOS activity was less pronounced in Cav-1-deficient cells. Statin-triggered cytotoxicity occurred at approximately 10-fold higher drug concentrations (maximal toxicity at 1-10 microM), was sensitive to mevalonate, and was significantly enhanced in the presence of NG-nitro-L-arginine. The overexpression of eNOS induced by clinically relevant concentrations of statins may contribute to the beneficial vascular effects of the drugs in patients. Stimulation of NO synthesis and cytotoxicity appear to share a common initial mechanism but involve distinct downstream signaling cascades that exhibit differential sensitivity to HMG-CoA reductase inhibition. PMID:20360620

  14. Differentiation of immature DCs into endothelial-like cells in human esophageal carcinoma tissue homogenates.

    PubMed

    Lu, Jing; Bai, Ruihua; Qin, Zhenzhu; Zhang, Yanyan; Zhang, Xiaoyan; Jiang, Yanan; Yang, Hongyan; Huang, Youtian; Li, Gang; Zhao, Mingyao; Dong, Ziming

    2013-08-01

    We previously reported endothelial-like differentiation (ELD) of immature dendritic cells (iDCs) in the microenvironment derived from EC9706 human esophageal squamous cell carcinoma conditioned medium (CM). However, the CM is far different from the esophageal carcinoma tissue of patients. In addition, the potential role of peri-esophageal carcinoma in the ELD of iDCs is also unknown. In the present study, we showed that the tumor microenvironment derived from esophageal carcinoma homogenate promoted iDCs to differentiate from the DC pathway toward endothelial cells, while the peri-esophageal carcinoma homogenate did not have this function. During the course of ELD, ERK signaling pathway and CREB were activated. Blocking MEK, both the phosphorylation of ERK and CREB, and the ELD of iDCs were inhibited. These data suggest that esophageal carcinoma tissue, not peri-esophageal carcinoma tissue, can drive iDCs to differentiate into endothelial-like cells, instead of differentiation into mature DCs, thereby losing the ability of antigen presentation. PMID:23708958

  15. Effects of macro- versus nanoporous silicon substrates on human aortic endothelial cell behavior

    PubMed Central

    2014-01-01

    Human aortic endothelial cells play a key role in the pathogenesis of atherosclerosis, which is a common, progressive, and multifactorial disease that is the clinical endpoint of an inflammatory process and endothelial dysfunction. Study and development of new therapies against cardiovascular disease must be tested in vitro cell models, prior to be evaluated in vivo. To this aim, new cell culture platforms are developed that allow cells to grow and respond to their environment in a realistic manner. In this work, the cell adhesion and morphology of endothelial cells are investigated on functionalized porous silicon substrates with two different pore size configurations: macroporous and nanoporous silicon. Herein, we modified the surfaces of porous silicon substrates by aminopropyl triethoxysilane, and we studied how different pore geometries induced different cellular response in the cell morphology and adhesion. The cell growth over the surface of porous silicon becomes an attractive field, especially for medical applications. Surface properties of the biomaterial are associated with cell adhesion and as well as, with proliferation, migration and differentiation. PMID:25246859

  16. Differing transcriptional responses to pulsed or continuous estradiol exposure in human umbilical vein endothelial cells.

    PubMed

    Li, Jin; Wang, Hongliang; Johnson, Suzanne M; Horner-Glister, Emma; Thompson, John; White, Ian N H; Al-Azzawi, Farook

    2008-07-01

    This study used human umbilical vein endothelial cells (HUVECs) that were treated with 17beta-estradiol for 5 days as 1h pulse or 24h continuous treatment at concentrations such that the 24h exposure (concentration x time) was identical in both conditions. Cell proliferation was studied and gene expression profiling was carried out using the Affymetrix GeneChip microarray analysis. Changes in morphology and apoptosis in HUVECs were examined with electron microscopy. Time-course studies of expression of genes vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were performed by quantitative PCR. We observed that cell proliferation was significantly decreased over days 3-5 with pulsed estradiol treatment relative to constant exposure. Microarray results showed that after 5 days, 801 genes differed (P<0.05) between continuous versus pulsed estradiol treatment. Functional analysis showed a significant number of genes to be associated with apoptosis and cell cycle pathways. We did not find any evidence of apoptosis from flow cytometry or electron microscopy examination. Our study highlights a large number of significantly different molecular responses to estradiol depending upon the mode of administration of estradiol. Significant changes were observed in genes involved in apoptosis and proliferation including VEGF, IGF receptors, and tumor protein p53. PMID:18562193

  17. Mechanism of the toxicity induced by natural humic acid on human vascular endothelial cells.

    PubMed

    Kihara, Yusuke; Yustiawati; Tanaka, Masato; Gumiri, Sulmin; Ardianor; Hosokawa, Toshiyuki; Tanaka, Shunitz; Saito, Takeshi; Kurasaki, Masaaki

    2014-08-01

    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 cells, as well as the mechanisms behind these effects. It was found that 50 mg/L HA showed cytotoxicity, which we considered to be mediated by apoptosis through the mitochondrial pathway because of an increase in the expression of caspases 6 and 9 in response to HA administration. In addition, this cytotoxicity was enhanced when cells in this experimental system were exposed to oxidative stress, while it was decreased by the addition of vitamin C. Thus, we conclude that the apoptosis induced by HA depends upon oxidative stress. Furthermore, an iron chelator, DFO, showed a tendency to decrease HA-induced cytotoxicity, suggesting that iron may potentially mediate HA-induced oxidative stress. In conclusion, long-term consumption of HA-rich water obtained from our study area may cause damage to endothelial cells and subsequent chronic health problems. PMID:23042718

  18. Endothelial to Mesenchymal Transition (EndoMT) in the Pathogenesis of Human Fibrotic Diseases

    PubMed Central

    Piera-Velazquez, Sonsoles; Mendoza, Fabian A.; Jimenez, Sergio A.

    2016-01-01

    Fibrotic diseases encompass a wide spectrum of clinical entities including systemic fibrotic diseases such as systemic sclerosis, sclerodermatous graft versus host disease, nephrogenic systemic fibrosis, and IgG4-associated sclerosing disease, as well as numerous organ-specific disorders including radiation-induced fibrosis, and cardiac, pulmonary, liver, and kidney fibrosis. Although their causative mechanisms are quite diverse, these diseases share the common feature of an uncontrolled and progressive accumulation of fibrous tissue macromolecules in affected organs leading to their dysfunction and ultimate failure. The pathogenesis of fibrotic diseases is complex and despite extensive investigation has remained elusive. Numerous studies have identified myofibroblasts as the cells responsible for the establishment and progression of the fibrotic process. Tissue myofibroblasts in fibrotic diseases originate from several sources including quiescent tissue fibroblasts, circulating CD34+ fibrocytes, and the phenotypic conversion of various cell types including epithelial and endothelial cells into activated myofibroblasts. However, the role of the phenotypic transition of endothelial cells into mesenchymal cells (Endothelial to Mesenchymal Transition or EndoMT) in the pathogenesis of fibrotic disorders has not been fully elucidated. Here, we review the evidence supporting EndoMT’s contribution to human fibrotic disease pathogenesis. PMID:27077889

  19. Fever temperature enhances mechanisms of survival of Streptococcus agalactiae within human endothelial cells.

    PubMed

    Freitas Lione, Viviane Oliveira; Bittencourt Dos Santos, Michelle Hanthequeste; Ulisses Carvalho, Técia Maria; Hirata, Raphael; Mattos-Guaraldi, Ana Luiza; Arruda Mortara, Renato; Nagao, Prescilla Emy

    2010-10-01

    Group B streptococci (GBS) are the most common cause of pneumonia and sepsis during the neonatal period. However, the pathogenesis of invasive infection is poorly understood. We investigated the ability of GBS grown at 37 degrees C and 40 degrees C to adhere and invade human umbilical vein endothelial cells (HUVECs) at different periods of incubation (0, 0.5, 1, 2, 18 and 24 h). All strains tested, except strain 88641-vagina survived for 24 h in the intracellular environment at 40 degrees C. For serotype III grown at 40 degrees C, both strains (80340-vagina and 90356-liquor) showed increased adherence and intracellular survival when compared to bacteria grown at 37 degrees C (P<0.01). GBS serotype V strains (88641-vagina and 90186-blood) showed ability to survive inside HUVECs until 2 and 24 h post-infection at 40 degrees C and 37 degrees C, respectively (P<0.01). Influence of growth temperature in bacterial interaction with endothelial cells was partially dependent of serotypes and the clinical origin of strains. Serotypes III and V strains grown at both temperatures remained viable within acidic endothelial vacuoles which acquired Rab7 and LAMP-1 endosomal markers. The data emphasize the influence of temperature on cellular events of phagocytosis and pathogenesis of GBS diseases. PMID:20818490

  20. Uptake and Transport of Superparamagnetic Iron Oxide Nanoparticles through Human Brain Capillary Endothelial Cells

    PubMed Central

    2013-01-01

    The blood–brain barrier (BBB) formed by brain capillary endothelial cells (BCECs) constitutes a firm physical, chemical, and immunological barrier, making the brain accessible to only a few percent of potential drugs intended for treatment inside the central nervous system. With the purpose of overcoming the restraints of the BBB by allowing the transport of drugs, siRNA, or DNA into the brain, a novel approach is to use superparamagnetic iron oxide nanoparticles (SPIONs) as drug carriers. The aim of this study was to investigate the ability of fluorescent SPIONs to pass through human brain microvascular endothelial cells facilitated by an external magnet. The ability of SPIONs to penetrate the barrier was shown to be significantly stronger in the presence of an external magnetic force in an in vitro BBB model. Hence, particles added to the luminal side of the in vitro BBB model were found in astrocytes cocultured at a remote distance on the abluminal side, indicating that particles were transported through the barrier and taken up by astrocytes. Addition of the SPIONs to the culture medium did not negatively affect the viability of the endothelial cells. The magnetic force-mediated dragging of SPIONs through BCECs may denote a novel mechanism for the delivery of drugs to the brain. PMID:23919894

  1. Effects of macro- versus nanoporous silicon substrates on human aortic endothelial cell behavior

    NASA Astrophysics Data System (ADS)

    Formentín, Pilar; Alba, María; Catalán, Úrsula; Fernández-Castillejo, Sara; Pallarès, Josep; Solà, Rosà; Marsal, Lluís F.

    2014-08-01

    Human aortic endothelial cells play a key role in the pathogenesis of atherosclerosis, which is a common, progressive, and multifactorial disease that is the clinical endpoint of an inflammatory process and endothelial dysfunction. Study and development of new therapies against cardiovascular disease must be tested in vitro cell models, prior to be evaluated in vivo . To this aim, new cell culture platforms are developed that allow cells to grow and respond to their environment in a realistic manner. In this work, the cell adhesion and morphology of endothelial cells are investigated on functionalized porous silicon substrates with two different pore size configurations: macroporous and nanoporous silicon. Herein, we modified the surfaces of porous silicon substrates by aminopropyl triethoxysilane, and we studied how different pore geometries induced different cellular response in the cell morphology and adhesion. The cell growth over the surface of porous silicon becomes an attractive field, especially for medical applications. Surface properties of the biomaterial are associated with cell adhesion and as well as, with proliferation, migration and differentiation.

  2. Identification of DNA-binding proteins on human umbilical vein endothelial cell plasma membrane.

    PubMed Central

    Chan, T M; Frampton, G; Cameron, J S

    1993-01-01

    The binding of anti-DNA antibodies to the endothelial cell is mediated through DNA, which forms a bridge between the immunoglobulin and the plasma membrane. We have shown that 32P-labelled DNA bound to the plasma membrane of human umbilical vein endothelial cells (HUVEC) by a saturable process, which could be competitively inhibited by non-radiolabelled DNA. In addition, DNA-binding was enhanced in HUVEC that had been treated with IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha). DNA-binding proteins of mol. wt 46,000, 92,000, and 84,000 were identified by the binding of 32P-labelled DNA to plasma membrane proteins separated on SDS-PAGE. DNA-binding proteins of mol. wt 46,000 and 84,000 were also present in the cytosol and nucleus. Murine anti-DNA MoAb410 bound to a single band, at mol. wt 46,000, of plasma membrane protein, in the presence of DNA. Our results showed that DNA-binding proteins are present in different cellular fractions of endothelial cells. DNA-binding proteins on the cell membrane could participate in the in situ formation of immune deposits; and their presence in the cell nucleus suggests a potential role in the modulation of cell function. Images Fig. 3 Fig. 4 PMID:8419070

  3. Human Neutrophil Peptides Mediate Endothelial-Monocyte Interaction, Foam Cell Formation, and Platelet Activation

    PubMed Central

    Quinn, Kieran L.; Henriques, Melanie; Tabuchi, Arata; Han, Bing; Yang, Hong; Cheng, Wei-Erh; Tole, Soumitra; Yu, Hanpo; Luo, Alice; Charbonney, Emmanuel; Tullis, Elizabeth; Lazarus, Alan; Robinson, Lisa A.; Ni, Heyu; Peterson, Blake R.; Kuebler, Wolfgang M.; Slutsky, Arthur S.; Zhang, Haibo

    2016-01-01

    Objective Neutrophils are involved in the inflammatory responses during atherosclerosis. Human neutrophil peptides (HNPs) released from activated neutrophils exert immune modulating properties. We hypothesized that HNPs play an important role in neutrophil-mediated inflammatory cardiovascular responses in atherosclerosis. Methods and Results We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor–related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation. Conclusion HNPs exert proatherosclerotic properties that appear to be mediated through LRP8 signaling pathways, suggesting an important role for HNPs in the development of inflammatory cardiovascular diseases. PMID:21817096

  4. Human papillomavirus causes an angiogenic switch in keratinocytes which is sufficient to alter endothelial cell behavior

    SciTech Connect

    Chen, W.; Li, F.; Mead, L.; White, H.; Walker, J.; Ingram, D.A.; Roman, A.

    2007-10-10

    One of the requirements for tumor growth is the ability to recruit a blood supply, a process known as angiogenesis. Angiogenesis begins early in the progression of cervical disease from mild to severe dysplasia and on to invasive cancer. We have previously reported that expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7) proteins in primary foreskin keratinocytes (HFKs) decreases expression of two inhibitors and increases expression of two angiogenic inducers [Toussaint-Smith, E., Donner, D.B., Roman, A., 2004. Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors. Oncogene 23, 2988-2995]. Here we report that HPV-induced early changes in the keratinocyte phenotype are sufficient to alter endothelial cell behavior both in vitro and in vivo. Conditioned media from HPV16 E6E7 expressing HFKs as well as from human cervical keratinocytes containing the intact HPV16 were able to stimulate proliferation and migration of human microvascular endothelial cells. In addition, introduction of the conditioned media into immunocompetent mice using a Matrigel plug model resulted in a clear angiogenic response. These novel data support the hypothesis that HPV proteins contribute not only to the uncontrolled keratinocyte growth seen following HPV infection but also to the angiogenic response needed for tumor formation.

  5. Sulfation of chlorotyrosine and nitrotyrosine by human lung endothelial and epithelial cells: Role of the human SULT1A3

    SciTech Connect

    Yasuda, Shin; Yasuda, Tomoko; Liu, Ming-Yih; Shetty, Sreerama; Idell, Steven; Boggaram, Vijayakumar; Suiko, Masahito; Sakakibara, Yoichi; Fu Jian; Liu, Ming-Cheh

    2011-03-01

    During inflammation, potent reactive oxidants formed may cause chlorination and nitration of both free and protein-bound tyrosine. In addition to serving as biomarkers of inflammation-mediated oxidative stress, elevated levels of chlorotyrosine and nitrotyrosine have been linked to the pathogenesis of lung and vascular disorders. The current study was designed to investigate whether the lung cells are equipped with mechanisms for counteracting these tyrosine derivatives. By metabolic labeling, chlorotyrosine O-[{sup 35}S]sulfate and nitrotyrosine O-[{sup 35}S]sulfate were found to be generated and released into the labeling media of human lung endothelial and epithelial cells labeled with [{sup 35}S]sulfate in the presence of added chlorotyrosine and nitrotyrosine. Enzymatic assays using the eleven known human cytosolic sulfotransferases (SULTs) revealed SULT1A3 as the enzyme responsible for catalyzing the sulfation of chlorotyrosine and nitrotyrosine. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated the expression of SULT1A3 in the lung endothelial and epithelial cells used in this study. Kinetic constants of the sulfation of chlorotyrosine and nitrotyrosine by SULT1A3 were determined. Collectively, these results suggest that sulfation by SULT1A3 in lung endothelial and epithelial cells may play a role in the inactivation and/or disposal of excess chlorotyrosine and nitrotyrosine generated during inflammation.

  6. Biophysical Properties of Scaffolds Modulate Human Blood Vessel Formation from Circulating Endothelial Colony-Forming Cells

    NASA Astrophysics Data System (ADS)

    Critser, Paul J.; Yoder, Mervin C.

    A functional vascular system forms early in development and is continually remodeled throughout the life of the organism. Impairment to the regeneration or repair of this system leads to tissue ischemia, dysfunction, and disease. The process of vascular formation and remodeling is complex, relying on local microenvironmental cues, cytokine signaling, and multiple cell types to function properly. Tissue engineering strategies have attempted to exploit these mechanisms to develop functional vascular networks for the generation of artificial tissues and therapeutic strategies to restore tissue homeostasis. The success of these strategies requires the isolation of appropriate progenitor cell sources which are straightforward to obtain, display high proliferative potential, and demonstrate an ability to form functional vessels. Several populations are of interest including endothelial colony-forming cells, a subpopulation of endothelial progenitor cells. Additionally, the development of scaffolds to deliver and support progenitor cell survival and function is crucial for the formation of functional vascular networks. The composition and biophysical properties of these scaffolds have been shown to modulate endothelial cell behavior and vessel formation. However, further investigation is needed to better understand how these mechanical properties and biophysical properties impact vessel formation. Additionally, several other cell populations are involved in neoangiogenesis and formation of tissue parenchyma and an understanding of the potential impact of these cell populations on the biophysical properties of scaffolds will also be needed to advance these strategies. This chapter examines how the biophysical properties of matrix scaffolds can influence vessel formation and remodeling and, in particular, the impact on in vivo human endothelial progenitor cell vessel formation.

  7. Spatial segregation of transport and signalling functions between human endothelial caveolae and lipid raft proteomes

    PubMed Central

    Sprenger, Richard R.; Fontijn, Ruud D.; van Marle, Jan; Pannekoek, Hans; Horrevoets, Anton J. G.

    2006-01-01

    Lipid rafts and caveolae are biochemically similar, specialized domains of the PM (plasma membrane) that cluster specific proteins. However, they are morphologically distinct, implying different, possibly complementary functions. Two-dimensional gel electrophoresis preceding identification of proteins by MS was used to compare the relative abundance of proteins in DRMs (detergent-resistant membranes) isolated from HUVEC (human umbilical-vein endothelial cells), and caveolae immunopurified from DRM fractions. Various signalling and transport proteins were identified and additional cell-surface biotinylation revealed the majority to be exposed, demonstrating their presence at the PM. In resting endothelial cells, the scaffold of immunoisolated caveolae consists of only few resident proteins, related to structure [CAV1 (caveolin-1), vimentin] and transport (V-ATPase), as well as the GPI (glycosylphosphatidylinositol)-linked, surface-exposed protein CD59. Further quantitative characterization by immunoblotting and confocal microscopy of well-known [eNOS (endothelial nitric oxide synthase) and CAV1], less known [SNAP-23 (23 kDa synaptosome-associated protein) and BASP1 (brain acid soluble protein 1)] and novel [C8ORF2 (chromosome 8 open reading frame 2)] proteins showed different subcellular distributions with none of these proteins being exclusive to either caveolae or DRM. However, the DRM-associated fraction of the novel protein C8ORF2 (∼5% of total protein) associated with immunoseparated caveolae, in contrast with the raft protein SNAP-23. The segregation of caveolae from lipid rafts was visually confirmed in proliferating cells, where CAV1 was spatially separated from eNOS, SNAP-23 and BASP1. These results provide direct evidence for the previously suggested segregation of transport and signalling functions between specialized domains of the endothelial plasma membrane. PMID:16886909

  8. An Innovative Ultrasound Technique for Evaluation of Tumor Vascularity in Breast Cancers: Superb Micro-Vascular Imaging

    PubMed Central

    Park, Ah Young; Cha, Sang Hoon; Yeom, Suk Keu; Lee, Seung Wha; Chung, Hwan Hoon

    2016-01-01

    Tumor vascularity is an important indicator for differential diagnosis, tumor growth, and prognosis. Superb micro-vascular imaging (SMI) is an innovative ultrasound technique for vascular examination that uses a multidimensional filter to eliminate clutter and preserve extremely low-velocity flows. Theoretically, SMI could depict more vessels and more detailed vascular morphology, due to the increased sensitivity of slow blood flow. Here, we report the early experience of using SMI in 21 breast cancer patients. We evaluated tumor vascular features in breast cancer and compared SMI and conventional color or power Doppler imaging. SMI was superior to color or power Doppler imaging in detecting tumor vessels, the details of vessel morphology, and both peripheral and central vascular distribution. In conclusion, SMI is a promising ultrasound technique for evaluating microvascular information of breast cancers.

  9. Resident Endothelial Progenitor Cells From Human Placenta Have Greater Vasculogenic Potential Than Circulating Endothelial Progenitor Cells From Umbilical Cord Blood

    PubMed Central

    Rapp, Brian M.; Saadatzedeh, M. Reza; Ofstein, Richard H.; Bhavsar, Janak R.; Tempel, Zachary S.; Moreno, Oscar; Morone, Peter; Booth, Dana A.; Traktuev, Dmitry O.; Dalsing, Michael C.; Ingram, David A.; Yoder, Mervin C.; March, Keith L.; Murphy, Michael P.

    2012-01-01

    Endothelial colony-forming cells (ECFCs) isolated from umbilical cord blood (CBECFCs) are highly proliferative and form blood vessels in vivo. The purpose of this investigation was to isolate and characterize a population of resident ECFCs from the chorionic villi of term human placenta and provide a comparative analysis of their proliferative and vasculogenic potential with CBECFCs. ECFCs were isolated from umbilical cord blood and chorionic villi from placentas obtained by caesarean deliveries. Placental ECFCs (PECFCs) expressed CD144, CD31, CD105, and KDR and were negative for CD45 and CD34, consistent with other ECFC phenotypes. PECFCs were capable of 28.6 ± 6.0 population doublings before reaching senescence (vs. 47.4 ± 3.2 for CBECFCs, p < 0.05, n = 4). In single cell assays, 46.5 ± 1.2% underwent at least one division (vs. 51.0 ± 1.8% of CBECFCs, p = 0.07, n = 6), and of those dividing PECFCs, 71.8 ± 0.9% gave rise to colonies of >500 cells (highly proliferative potential clones) over 14 days (vs. 69.4 ± 0.7% of CBECFCs, p = 0.07, n = 9). PECFCs formed 5.2 ± 0.8 vessels/mm2 in collagen/fibronectin plugs implanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, whereas CBECFCs formed only 1.7 ± 1.0 vessels/mm2 (p < 0.05, n = 4). This study demonstrates that circulating CBECFCs and resident PECFCs are identical phenotypically and contain equivalent quantities of high proliferative potential clones. However, PECFCs formed significantly more blood vessels in vivo than CBECFCs, indicating that differences in vasculogenic potential between circulating and resident ECFCs exist. PMID:27004134

  10. Effect of Thrombus Composition and Viscosity on Sonoreperfusion Efficacy in a Model of Micro-Vascular Obstruction.

    PubMed

    Black, John J; Yu, Francois T H; Schnatz, Rick G; Chen, Xucai; Villanueva, Flordeliza S; Pacella, John J

    2016-09-01

    Distal embolization of micro-thrombi during stenting for myocardial infarction causes micro-vascular obstruction (MVO). We have previously shown that sonoreperfusion (SRP), a microbubble (MB)-mediated ultrasound (US) therapy, resolves MVO from venous micro-thrombi in vitro in saline. However, blood is more viscous than saline, and arterial thrombi that embolize during stenting are mechanically distinct from venous clot. Therefore, we tested the hypothesis that MVO created with arterial micro-thrombi are more resistant to SRP therapy compared with venous micro-thrombi, and higher viscosity further increases the US requirement for effective SRP in an in vitro model of MVO. Lipid MBs suspended in plasma with adjusted viscosity (1.1 cP or 4.0 cP) were passed through tubing bearing a mesh with 40-μm pores to simulate a micro-vascular cross-section; upstream pressure reflected thrombus burden. To simulate MVO, the mesh was occluded with either arterial or venous micro-thrombi to increase upstream pressure to 40 mmHg ± 5 mmHg. Therapeutic long-tone-burst US was delivered to the occluded area for 20 min. MB activity was recorded with a passive cavitation detector. MVO caused by arterial micro-thrombi at either blood or plasma viscosity resulted in less effective SRP therapy compared to venous thrombi. Higher viscosity further reduced the effectiveness of SRP therapy. The passive cavitation detector showed a decrease in inertial cavitation when viscosity was increased, while stable cavitation was affected in a more complex manner. Overall, these data suggest that arterial thrombi may require higher acoustic pressure US than venous thrombi to achieve similar SRP efficacy; increased viscosity decreases SRP efficacy; and both inertial and stable cavitation are implicated in observed SRP efficacy. PMID:27207018

  11. The isolation and culture of endothelial colony-forming cells from human and rat lungs.

    PubMed

    Alphonse, Rajesh S; Vadivel, Arul; Zhong, Shumei; Zong, Shumei; McConaghy, Suzanne; Ohls, Robin; Yoder, Mervin C; Thébaud, Bernard

    2015-11-01

    Blood vessels are crucial for the normal development, lifelong repair and homeostasis of tissues. Recently, vascular progenitor cell-driven 'postnatal vasculogenesis' has been suggested as an important mechanism that contributes to new blood vessel formation and organ repair. Among several described progenitor cell types that contribute to blood vessel formation, endothelial colony-forming cells (ECFCs) have received widespread attention as lineage-specific 'true' vascular progenitors. Here we describe a protocol for the isolation of pulmonary microvascular ECFCs from human and rat lung tissue. Our technique takes advantage of an earlier protocol for the isolation of circulating ECFCs from the mononuclear cellular fraction of peripheral blood. We adapted the earlier protocol to isolate resident ECFCs from the distal lung tissue. After enzymatic dispersion of rat or human lung samples into a cellular suspension, CD31-expressing cells are positively selected using magnetic-activated cell sorting and plated in endothelial-specific growth conditions. The colonies arising after 1-2 weeks in culture are carefully separated and expanded to yield pure ECFC cultures after a further 2-3 weeks. The resulting cells demonstrate the defining characteristics of ECFCs such as (i) 'cobblestone' morphology of cultured cell monolayers; (ii) acetylated low-density lipoprotein uptake and Ulex europaeus lectin binding; (iii) tube-like network formation in Matrigel; (iv) expression of endothelial cell-specific surface markers and the absence of hematopoietic or myeloid surface antigens; (v) self-renewal potential displayed by the most proliferative cells; and (vi) contribution to de novo vessel formation in an in vivo mouse implant model. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 4 weeks. Isolation and culture of lung vascular ECFCs will allow assessment of the functional state of these cells in experimental and human

  12. Calcitonin Gene-related Peptide Inhibits Chemokine Production by Human Dermal Microvascular Endothelial Cells

    PubMed Central

    Huang, Jing; Stohl, Lori L.; Zhou, Xi; Ding, Wanhong; Granstein, Richard D.

    2011-01-01

    This study examined whether the sensory neuropeptide calcitonin gene-related peptide (CGRP) inhibits release of chemokines by dermal microvascular endothelial cells. Dermal blood vessels are associated with nerves containing CGRP, suggesting that CGRP-containing nerves may regulate cutaneous inflammation through effects on vessels. We examined CGRP effects on stimulated chemokine production by a human dermal microvascular endothelial cell line (HMEC-1) and primary human dermal microvascular endothelial cells (pHDMECs). HMEC-1 cells and pHDMECs expressed mRNA for components of the CGRP and adrenomedullin receptors and CGRP inhibited LPS-induced production of the chemokines CXCL8, CCL2, and CXCL1 by both HMEC-1 cells and pHDMECs. The receptor activity-modifying protein (RAMP)1/calcitonin receptor-like receptor (CL)-specific antagonists CGRP8-37 and BIBN4096BS, blocked this effect of CGRP in a dose-dependent manner. CGRP prevented LPS-induced IκBα degradation and NF-κB binding to the promoters of CXCL1, CXCL8 and CCL2 in HMEC-1 cells and Bay 11-7085, an inhibitor of NF-κB activation, suppressed LPS-induced production of CXCL1, CXCL8 and CCL2. Thus, the NF-κB pathway appears to be involved in CGRP-mediated suppression of chemokine production. Accordingly, CGRP treatment of LPS-stimulated HMEC-1 cells inhibited their ability to chemoattract human neutrophils and mononuclear cells. Elucidation of this pathway may suggest new avenues for therapeutic manipulation of cutaneous inflammation. PMID:21334428

  13. Human β-NGF gene transferred to cat corneal endothelial cells

    PubMed Central

    Luo, Wen-Juan; Liu, Min; Zhao, Gui-Qiu; Wang, Chuan-Fu; Hu, Li-Ting; Liu, Xiang-Ping

    2016-01-01

    AIM To transfect the cat corneal endothelial cells (CECs) with recombinant human β-nerve growth factor gene adeno-associated virus (AAV-β-NGF) and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs. METHODS The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco's modified Eagle's medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope. RESULTS The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result

  14. Human endothelial cell-based assay for endotoxin as sensitive as the conventional Limulus Amebocyte Lysate assay.

    PubMed

    Unger, Ronald E; Peters, Kirsten; Sartoris, Anne; Freese, Christian; Kirkpatrick, C James

    2014-03-01

    Endotoxin, also known as lipopolysaccharide (LPS) produced by bacteria can be present in any liquid or on any biomaterial even if the material is sterile. Endotoxin in mammals can cause fever, inflammation, cell and tissue damage and irreversible septic shock and death. In the body, endothelial cells making up the blood vasculature and endothelial cells in vitro rapidly react to minute amounts of endotoxin resulting in a rapid induction of the cell adhesion molecule E-selectin. In this study we have used immunofluorescent staining to evaluate the expression of E-selectin on human microvascular endothelial cells from the skin (HDMEC) and human umbilical vein endothelial cells (HUVEC) exposed to various concentrations of LPS. In addition, the sensitivity of detection was compared with the most widely used assay for the presence of endotoxin, the Limulus Amebocyte Lysate assay (LAL). The detection of E-selectin on endothelial cells in the presence of LPS for 4 h was found to be at least as sensitive in detecting the same concentration using the LAL assay. A cell adhesion molecule-enzyme immunosorbent assay was also developed and used to quantify LPS using the endothelial cell model. A comparison of LAL and the immunofluorescent staining method was carried out with solutions, nanoparticles, biomaterial extracts and endothelial cells grown directly on biomaterials. Under all conditions, the endothelial/E-selectin model system was positive for the test samples that were positive by LAL. Thus, we propose the use of this highly sensitive, rapid, reproducible assay for the routine testing of endotoxin in all steps in the manufacturing process of materials destined for use in humans. This can give a rapid feedback and localization of bacterial contamination sources with the LAL being reserved for the testing of the final product. PMID:24456607

  15. Short and prolonged exposure to hyperglycaemia in human fibroblasts and endothelial cells: metabolic and osmotic effects.

    PubMed

    Moruzzi, Noah; Del Sole, Marianna; Fato, Romana; Gerdes, Jantje M; Berggren, Per-Olof; Bergamini, Christian; Brismar, Kerstin

    2014-08-01

    High blood glucose levels are the main feature of diabetes. However, the underlying mechanism linking high glucose concentration to diabetic complications is still not fully elucidated, particularly with regard to human physiology. Excess of glucose is likely to trigger a metabolic response depending on the cell features, activating deleterious pathways involved in the complications of diabetes. In this study, we aim to elucidate how acute and prolonged hyperglycaemia alters the biology and metabolism in human fibroblasts and endothelial cells. We found that hyperglycaemia triggers a metabolic switch from oxidative phosphorylation to glycolysis that is maintained over prolonged time. Moreover, osmotic pressure is a major factor in the early metabolic response, decreasing both mitochondrial transmembrane potential and cellular proliferation. After prolonged exposure to hyperglycaemia we observed decreased mitochondrial steady-state and uncoupled respiration, together with a reduced ATP/ADP ratio. At the same time, we could not detect major changes in mitochondrial transmembrane potential and reactive oxygen species. We suggest that the physiological and metabolic alterations observed in healthy human primary fibroblasts and endothelial cells are an adaptive response to hyperglycaemia. The severity of metabolic and bioenergetics impairment associated with diabetic complications may occur after longer glucose exposure or due to interactions with cell types more sensitive to hyperglycaemia. PMID:24814290

  16. Development of Blood and Lymphatic Endothelial Cells in Embryonic and Fetal Human Skin.

    PubMed

    Schuster, Christopher; Mildner, Michael; Botta, Albert; Nemec, Lucas; Rogojanu, Radu; Beer, Lucian; Fiala, Christian; Eppel, Wolfgang; Bauer, Wolfgang; Petzelbauer, Peter; Elbe-Bürger, Adelheid

    2015-09-01

    Blood and lymphatic vessels provide nutrients for the skin and fulfill important homeostatic functions, such as the regulation of immunologic processes. In this study, we investigated the development of blood and lymphatic endothelial cells in prenatal human skin in situ using multicolor immunofluorescence and analyzed angiogenic molecules by protein arrays of lysates and cell culture supernatants. We found that at 8 to 10 weeks of estimated gestational age, CD144(+) vessels predominantly express the venous endothelial cell marker PAL-E, whereas CD144(+)PAL-E(-) vessels compatible with arteries only appear at the end of the first trimester. Lymphatic progenitor cells at 8 weeks of estimated gestational age express CD31, CD144, Prox1, and temporary PAL-E. At that developmental stage not all lymphatic progenitor cells express podoplanin or Lyve-1, which are acquired with advancing gestational age in a stepwise fashion. Already in second-trimester human skin, the phenotype of blood and lymphatic vessels roughly resembles the one in adult skin. The expression pattern of angiogenic molecules in lysates and cell culture supernatants of prenatal skin did not reveal the expected bent to proangiogenic molecules, indicating a complex regulation of angiogenesis during ontogeny. In summary, this study provides enticing new insights into the development and phenotypic characteristics of the vascular system in human prenatal skin. PMID:26188132

  17. Patterning human stem cells and endothelial cells with laser printing for cardiac regeneration.

    PubMed

    Gaebel, Ralf; Ma, Nan; Liu, Jun; Guan, Jianjun; Koch, Lothar; Klopsch, Christian; Gruene, Martin; Toelk, Anita; Wang, Weiwei; Mark, Peter; Wang, Feng; Chichkov, Boris; Li, Wenzhong; Steinhoff, Gustav

    2011-12-01

    Recent study showed that mesenchymal stem cells (MSC) could inhibit apoptosis of endothelial cells in hypoxic condition, increase their survival, and stimulate the angiogenesis process. In this project we applied Laser-Induced-Forward-Transfer (LIFT) cell printing technique and prepared a cardiac patch seeded with human umbilical vein endothelial cells (HUVEC) and human MSC (hMSC) in a defined pattern for cardiac regeneration. We seeded HUVEC and hMSC in a defined pattern on a Polyester urethane urea (PEUU) cardiac patch. On control patches an equal amount of cells was randomly seeded without LIFT. Patches were cultivated in vitro or transplanted in vivo to the infarcted zone of rat hearts after LAD-ligation. Cardiac performance was measured by left ventricular catheterization 8 weeks post infarction. Thereafter hearts were perfused with fluorescein tomato lectin for the assessment of functional blood vessels and stored for histology analyses. We demonstrated that LIFT-derived cell seeding pattern definitely modified growth characteristics of co-cultured HUVEC and hMSC leading to increased vessel formation and found significant functional improvement of infarcted hearts following transplantation of a LIFT-tissue engineered cardiac patch. Further, we could show enhanced capillary density and integration of human cells into the functionally connected vessels of murine vascular system. LIFT-based Tissue Engineering of cardiac patches for the treatment of myocardial infarction might improve wound healing and functional preservation. PMID:21911255

  18. Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells.

    PubMed

    Stebbins, Matthew J; Wilson, Hannah K; Canfield, Scott G; Qian, Tongcheng; Palecek, Sean P; Shusta, Eric V

    2016-05-15

    The blood-brain barrier (BBB) is a critical component of the central nervous system (CNS) that regulates the flux of material between the blood and the brain. Because of its barrier properties, the BBB creates a bottleneck to CNS drug delivery. Human in vitro BBB models offer a potential tool to screen pharmaceutical libraries for CNS penetration as well as for BBB modulators in development and disease, yet primary and immortalized models respectively lack scalability and robust phenotypes. Recently, in vitro BBB models derived from human pluripotent stem cells (hPSCs) have helped overcome these challenges by providing a scalable and renewable source of human brain microvascular endothelial cells (BMECs). We have demonstrated that hPSC-derived BMECs exhibit robust structural and functional characteristics reminiscent of the in vivo BBB. Here, we provide a detailed description of the methods required to differentiate and functionally characterize hPSC-derived BMECs to facilitate their widespread use in downstream applications. PMID:26518252

  19. A boost of BMP4 accelerates the commitment of human embryonic stem cells to the endothelial lineage.

    PubMed

    Goldman, Orit; Feraud, Olivier; Boyer-Di Ponio, Julie; Driancourt, Catherine; Clay, Denis; Le Bousse-Kerdiles, Marie-Caroline; Bennaceur-Griscelli, Annelise; Uzan, Georges

    2009-08-01

    Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133(+)KDR(+) population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144(+)KDR(+) population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144(+)KDR(+) peak was shifted to day 7. Most of these cells, however, were CD31(-), becoming CD31(+) in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144(+)KDR(+)CD31(-) population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12. PMID:19544443

  20. Human immunodeficiency virus type 1 transport across the in vitro mouse brain endothelial cell monolayer.

    PubMed

    Nakaoke, Ryota; Ryerse, Jan S; Niwa, Masami; Banks, William A

    2005-05-01

    Human immunodeficiency virus type 1 (HIV-1) is associated with a neuroinflammatory dementia. Cognitive impairment remains a common complication of late-stage HIV-1 infection. Previous studies have shown that entry of HIV-1 into the central nervous system (CNS) occurs soon after infection. For these reasons, it is important to understand how HIV-1 crosses the BBB. We used primary mouse brain microvessel endothelial cell (MBEC) monolayer models to study interactions between brain endothelial cells and radioactively labeled HIV-1 CL4 (131I-HIV-1), which had been rendered noninfectious with aldithiol, and compared to radioactively labeled bovine serum albumin (131I-BSA or 125I-BSA) and detected HIV-1 on MBEC monolayer with electron microscopic analysis. The permeability of the monolayers to HIV-1 was measured by determining the percent material transported (PMT). Luminal to abluminal PMT of 131I-HIV-1 was 4.65 times greater than that of the much smaller 131I-BSA, showing that the MBEC monolayer is more permeable to HIV-1 than to BSA. Electron microscopy showed that HIV-1 was transported through a trans-cellular pathway from luminal side to basolateral space with some virus associated with the nucleus. Unlabeled HIV-1 did not affect the transport of 131I-HIV-1 or break down the MBEC monolayer. Wheatgerm agglutinin (WGA) increased 131I-HIV-1 penetration across the MBEC monolayer, consistent with absorptive endocytosis as the mechanism for HIV-1 penetration. The enhanced transport of HIV-1 was unidirectional, as the abluminal to luminal PMT of 131I-HIV-1 was not different from that of BSA nor enhanced by WGA. Characterization of the radioactivity transported from the luminal to abluminal chamber on Sepharose 4B-200 columns showed the transported radioactivity represented intact virus. MBEC monolayers preloaded from the luminal surface with 131I-HIV-1 showed most of the virus was retained by the endothelial cells, while the remainder was effluxed mainly to the luminal

  1. Sphingosine 1-phosphate induces platelet/endothelial cell adhesion molecule-1 phosphorylation in human endothelial cells through cSrc and Fyn.

    PubMed

    Huang, Yu-Ting; Chen, Shee-Uan; Chou, Chia-Hong; Lee, Hsinyu

    2008-08-01

    Sphingosine 1-phosphate (S1P) is a multifunctional phospholipid which acts through a specific family of G protein-coupled receptors. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) form trans-homophilic binding at lateral cell border. Upon stimulation, its cytoplasmic tyrosine residues could be phosphorylated and interact with various downstream signaling molecules. In this study, we demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in human umbilical cord vein cells (HUVECs). By pharmacological inhibitors, it was suggested that G(i) and Src family kinases were involved in PECAM-1 phosphorylation. Moreover, cSrc and Fyn siRNA significantly suppressed S1P-induced PECAM-1 phosphorylation. These results suggested that S1P-induced PECAM-1 phosphorylation through G(i) and subsequent cSrc and Fyn. Our findings provide further understanding of S1P and PECAM-1 signaling as well as their functions in endothelial cells. PMID:18502612

  2. Human corneal endothelial cell sheets for transplantation: thermo-responsive cell culture carriers to meet cell-specific requirements.

    PubMed

    Teichmann, J; Valtink, M; Gramm, S; Nitschke, M; Werner, C; Funk, R H W; Engelmann, K

    2013-02-01

    Corneal endothelial diseases lead to severe vision impairment, motivating the transplantation of donor corneae or corneal endothelial lamellae, which is, however, impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. The current study presents a novel thermo-responsive carrier based on simultaneous electron beam immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces, which allows one to adjust layer thickness, stiffness, switching amplitude and functionalization with bioactive molecules to meet cell type specific requirements. The efficacy of this approach for HCEC, which require elaborate cell culture conditions and are strongly adherent to the substratum, is demonstrated. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options. PMID:23099299

  3. The coffee diterpene kahweol inhibits tumor necrosis factor-{alpha}-induced expression of cell adhesion molecules in human endothelial cells

    SciTech Connect

    Kim, Hyung Gyun; Kim, Ji Young; Hwang, Yong Pil; Lee, Kyung Jin; Lee, Kwang Youl; Kim, Dong Hee; Kim, Dong Hyun; Jeong, Hye Gwang . E-mail: hgjeong@chosun.ac.kr

    2006-12-15

    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 TNF{alpha}-induced monocytes to endothelial cells and suppressed the TNF{alpha}-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNF{alpha}-induced JAK2-PI3K/Akt-NF-{kappa}B 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.

  4. Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease.

    PubMed Central

    Burns, E H; Marciel, A M; Musser, J M

    1996-01-01

    Human umbilical vein endothelial cells (HUVECs) were used to gain insight into the molecular mechanism whereby the major extracellular protease from group A streptococci damages host tissue. HUVECs exposed to streptococcal cysteine protease (SCP) for various times exhibited cytopathic effect and cell detachment from the culture vessel. Gelatin substrate zymography showed that a time- and concentration-dependent increase in the level of activity of an approximately 66-kDa gelatinase occurred in culture medium taken from cells exposed to enzymatically active SCP. This gelatinase comigrated in gelatin zymograms with the activated form of purified recombinant matrix metalloprotease 2 (MMP-2) and had type IV collagenase activity. In contrast, medium taken from cells exposed to inactivated (boiled) SCP and cells exposed to SCP inhibited by treatment with N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone lacked the 66-kDa gelatinase. Appearance of the 66-kDa gelatinase activity was also prevented by 1,10-phenanthroline, a zinc chelator and MMP inhibitor. Inasmuch as proteolytically active SCP is required for the emergence of this gelatinase and MMP activation occurs by proteolytic processing, the 66-kDa gelatinase may be a proteolytic cleavage product of a latent MMP expressed extracellularly by HUVECs. Direct SCP treatment of culture supernatant taken from HUVECs not exposed to SCP also produced the 66-kDa gelatinase. The data show that SCP activates an MMP produced by human endothelial cells, a process that may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive group A streptococcal infection. PMID:8890235

  5. Isolation of Functional Human Endothelial Cells from Small Volumes of Umbilical Cord Blood

    PubMed Central

    Do Kang, Sa; Carlon, Tim A.; Jantzen, Alexandra E.; Lin, Fu-Hsiung; Ley, Melissa M.; Allen, Jason D.; Stabler, Thomas V.; Haley, N. Rebecca; Truskey, George A.; Achneck, Hardean E.

    2013-01-01

    Endothelial cells (ECs) isolated from endothelial progenitor cells in blood have great potential as a therapeutic tool to promote vasculogenesis and angiogenesis and treat cardiovascular diseases. However, current methods to isolate ECs are limited by a low yield with few colonies appearing during isolation. In order to utilize blood-derived ECs for therapeutic applications, a simple method is needed that can produce a high yield of ECs from small volumes of blood without the addition of animal-derived products. For the first time, we show that human endothelial cells can be isolated without the prior separation of blood components through the technique of diluted whole blood incubation (DWBI) utilizing commercially available human serum. We isolated ECs from small volumes of blood (~ 10 ml) via DWBI and characterized them with flow cytometry, immunohistochemistry, and uptake of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL). These ECs are functional as demonstrated by their ability to form tubular networks in Matrigel, adhere and align with flow under physiological fluid shear stress, and produce increased nitric oxide under fluid flow. An average of 7.0 ± 2.5 EC colonies that passed all functional tests described above were obtained per 10 ml of blood as compared to only 0.3 ± 0.1 colonies with the traditional method based on density centrifugation. The time until first colony appearance was 8.3 ± 1.2 days for ECs isolated with the DWBI method and 12 ± 1.4 days for ECs isolated with the traditional isolation method. A simplified method, such as DWBI, in combination with advances in isolation yield could enable the use of blood-derived ECs in clinical practice. PMID:23604849

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

    SciTech Connect

    Gao, Zhong-Xiu-Zi; Huang, Da-Yong; Li, Hai-Xia; Zhang, Li-Na; Lv, Yan-Hong; Cui, Hai-Dong; Zheng, Jin-Hua

    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 induced MMP-2

  7. Dengue Virus Induces Novel Changes in Gene Expression of Human Umbilical Vein Endothelial Cells

    PubMed Central

    Warke, Rajas V.; Xhaja, Kris; Martin, Katherine J.; Fournier, Marcia F.; Shaw, Sunil K.; Brizuela, Nathaly; de Bosch, Norma; Lapointe, David; Ennis, Francis A.; Rothman, Alan L.; Bosch, Irene

    2003-01-01

    Endothelial cells are permissive to dengue virus (DV) infection in vitro, although their importance as targets of DV infection in vivo remains a subject of debate. To analyze the virus-host interaction, we studied the effect of DV infection on gene expression in human umbilical vein endothelial cells (HUVECs) by using differential display reverse transcription-PCR (DD-RTPCR), quantitative RT-PCR, and Affymetrix oligonucleotide microarrays. DD identified eight differentially expressed cDNAs, including inhibitor of apoptosis-1, 2′-5′ oligoadenylate synthetase (OAS), a 2′-5′ OAS-like (OASL) gene, galectin-9, myxovirus protein A (MxA), regulator of G-protein signaling, endothelial and smooth muscle cell-derived neuropilin-like protein, and phospholipid scramblase 1. Microarray analysis of 22,000 human genes confirmed these findings and identified an additional 269 genes that were induced and 126 that were repressed more than fourfold after DV infection. Broad functional responses that were activated included the stress, defense, immune, cell adhesion, wounding, inflammatory, and antiviral pathways. These changes in gene expression were seen after infection of HUVECs with either laboratory-adapted virus or with virus isolated directly from plasma of DV-infected patients. Tumor necrosis factor alpha, OASL, and MxA and h-IAP1 genes were induced within the first 8 to 12 h after infection, suggesting a direct effect of DV infection. These global analyses of DV effects on cellular gene expression identify potentially novel mechanisms involved in dengue disease manifestations such as hemostatic disturbance. PMID:14557666

  8. Perturbation of Human Coronary Artery Endothelial Cell Redox State and NADPH Generation by Methylglyoxal

    PubMed Central

    Davies, Michael J.

    2014-01-01

    Diabetes is associated with elevated plasma glucose, increased reactive aldehyde formation, oxidative damage, and glycation/glycoxidation of biomolecules. Cellular detoxification of, or protection against, such modifications commonly requires NADPH-dependent reducing equivalents (e.g. GSH). We hypothesised that reactive aldehydes may modulate cellular redox status via the inhibition of NADPH-generating enzymes, resulting in decreased thiol and NADPH levels. Primary human coronary artery endothelial cells (HCAEC) were incubated with high glucose (25 mM, 24 h, 37°C), or methylglyoxal (MGO), glyoxal, or glycolaldehyde (100–500 µM, 1 h, 37°C), before quantification of intracellular thiols and NADPH-generating enzyme activities. Exposure to MGO, but not the other species examined, significantly (P<0.05) decreased total thiols (∼35%), further experiments with MGO showed significant losses of GSH (∼40%) and NADPH (∼10%); these changes did not result in an immediate loss of cell viability. Significantly decreased (∼10%) NADPH-producing enzyme activity was observed for HCAEC when glucose-6-phosphate or 2-deoxyglucose-6-phosphate were used as substrates. Cell lysate experiments showed significant MGO-dose dependent inhibition of glucose-6-phosphate-dependent enzymes and isocitrate dehydrogenase, but not malic enzyme. Analysis of intact cell or lysate proteins showed that arginine-derived hydroimidazolones were the predominant advanced glycation end-product (AGE) formed; lower levels of Nε-(carboxyethyl)lysine (CEL) and Nε-(carboxymethyl)lysine (CML) were also detected. These data support a novel mechanism by which MGO exposure results in changes in redox status in human coronary artery endothelial cells, via inhibition of NADPH-generating enzymes, with resultant changes in reduced protein thiol and GSH levels. These changes may contribute to the endothelial cell dysfunction observed in diabetes-associated atherosclerosis. PMID:24466151

  9. The cytotoxicity evaluation of magnetic iron oxide nanoparticles on human aortic endothelial cells

    NASA Astrophysics Data System (ADS)

    Ge, Gaoyuan; Wu, Hengfang; Xiong, Fei; Zhang, Yu; Guo, Zhirui; Bian, Zhiping; Xu, Jindan; Gu, Chunrong; Gu, Ning; Chen, Xiangjian; Yang, Di

    2013-05-01

    One major obstacle for successful application of nanoparticles in medicine is its potential nanotoxicity on the environment and human health. In this study, we evaluated the cytotoxicity effect of dimercaptosuccinic acid-coated iron oxide (DMSA-Fe2O3) using cultured human aortic endothelial cells (HAECs). Our results showed that DMSA-Fe2O3 in the culture medium could be absorbed into HAECs, and dispersed in the cytoplasm. The cytotoxicity effect of DMSA-Fe2O3 on HAECs was dose-dependent, and the concentrations no more than 0.02 mg/ml had little toxic effect which were revealed by tetrazolium dye assay. Meanwhile, the cell injury biomarker, lactate dehydrogenase, was not significantly higher than that from control cells (without DMSA-Fe2O3). However, the endocrine function for endothelin-1 and prostacyclin I-2, as well as the urea transporter function, was altered even without obvious evidence of cell injury in this context. We also showed by real-time PCR analysis that DMSA-Fe2O3 exposure resulted in differential effects on the expressions of pro- and anti-apoptosis genes of HAECs. Meanwhile, it was noted that DMSA-Fe2O3 exposure could activate the expression of genes related to oxidative stress and adhesion molecules, which suggested that inflammatory response might be evoked. Moreover, we demonstrated by in vitro endothelial tube formation that even a small amount of DMSA-Fe2O3 (0.01 and 0.02 mg/ml) could inhibit angiogenesis by the HAECs. Altogether, these results indicate that DMSA-Fe2O3 have some cytotoxicity that may cause side effects on normal endothelial cells.

  10. Low Dose Iron Treatments Induce a DNA Damage Response in Human Endothelial Cells within Minutes

    PubMed Central

    Mollet, Inês G.; Giess, Adam; Paschalaki, Koralia; Periyasamy, Manikandan; Lidington, Elaine C.; Mason, Justin C.; Jones, Michael D.; Game, Laurence; Ali, Simak; Shovlin, Claire L.

    2016-01-01

    Background Spontaneous reports from patients able to report vascular sequelae in real time, and recognition that serum non transferrin bound iron may reach or exceed 10μmol/L in the blood stream after iron tablets or infusions, led us to hypothesize that conventional iron treatments may provoke acute vascular injury. This prompted us to examine whether a phenotype could be observed in normal human endothelial cells treated with low dose iron. Methodology Confluent primary human endothelial cells (EC) were treated with filter-sterilized iron (II) citrate or fresh media for RNA sequencing and validation studies. RNA transcript profiles were evaluated using directional RNA sequencing with no pre-specification of target sequences. Alignments were counted for exons and junctions of the gene strand only, blinded to treatment types. Principal Findings Rapid changes in RNA transcript profiles were observed in endothelial cells treated with 10μmol/L iron (II) citrate, compared to media-treated cells. Clustering for Gene Ontology (GO) performed on all differentially expressed genes revealed significant differences in biological process terms between iron and media-treated EC, whereas 10 sets of an equivalent number of randomly selected genes from the respective EC gene datasets showed no significant differences in any GO terms. After 1 hour, differentially expressed genes clustered to vesicle mediated transport, protein catabolism, and cell cycle (Benjamini p = 0.0016, 0.0024 and 0.0032 respectively), and by 6 hours, to cellular response to DNA damage stimulus most significantly through DNA repair genes FANCG, BLM, and H2AFX. Comet assays demonstrated that 10μM iron treatment elicited DNA damage within 1 hour. This was accompanied by a brisk DNA damage response pulse, as ascertained by the development of DNA damage response (DDR) foci, and p53 stabilization. Significance These data suggest that low dose iron treatments are sufficient to modify the vascular endothelium

  11. Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease.

    PubMed

    Burns, E H; Marciel, A M; Musser, J M

    1996-11-01

    Human umbilical vein endothelial cells (HUVECs) were used to gain insight into the molecular mechanism whereby the major extracellular protease from group A streptococci damages host tissue. HUVECs exposed to streptococcal cysteine protease (SCP) for various times exhibited cytopathic effect and cell detachment from the culture vessel. Gelatin substrate zymography showed that a time- and concentration-dependent increase in the level of activity of an approximately 66-kDa gelatinase occurred in culture medium taken from cells exposed to enzymatically active SCP. This gelatinase comigrated in gelatin zymograms with the activated form of purified recombinant matrix metalloprotease 2 (MMP-2) and had type IV collagenase activity. In contrast, medium taken from cells exposed to inactivated (boiled) SCP and cells exposed to SCP inhibited by treatment with N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone lacked the 66-kDa gelatinase. Appearance of the 66-kDa gelatinase activity was also prevented by 1,10-phenanthroline, a zinc chelator and MMP inhibitor. Inasmuch as proteolytically active SCP is required for the emergence of this gelatinase and MMP activation occurs by proteolytic processing, the 66-kDa gelatinase may be a proteolytic cleavage product of a latent MMP expressed extracellularly by HUVECs. Direct SCP treatment of culture supernatant taken from HUVECs not exposed to SCP also produced the 66-kDa gelatinase. The data show that SCP activates an MMP produced by human endothelial cells, a process that may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive group A streptococcal infection. PMID:8890235

  12. Human endothelial and platelet septin SEPT11: cloning of novel variants and characterisation of interaction partners.

    PubMed

    Bartsch, Ingrid; Bläser, Susanne; Röseler, Sabrina; Sandrock, Kirstin; Busse, Anja; Huber, Michael; Rempp, Hansjörg; Lieber, Mareike; Horn, Julia; Brendle, Cornelia; Zieger, Barbara

    2010-12-01

    Septins are cytoskeletal GTPases forming heteropolymeric complexes involved in processes characterised by active membrane movement such as cytokinesis, vesicle trafficking, and exocytosis. Septins are expressed in non-mitotic cells such as neurons and platelets. SEPT11 belongs to the SEPT6 group and was identified as interaction partner of SEPT5. We cloned and characterised novel SEPT11 variants and investigated interaction partners of SEPT11 in platelets and human umbilical vein endothelial cells. An endothelial cell library was used for cloning novel SEPT11 variants. Using Northern analysis the different SEPT11 transcripts were illustrated. Interaction studies were performed using yeast two-hybrid system, precipitation, FRET, and immunofluorescence microscopy. We demonstrate that SEPT11 partners with SEPT2, SEPT4 and SEPT7 using yeast two-hybrid system and precipitation. The interaction of SEPT11 with SEPT7 is also demonstrated by FRET. In addition to the known SEPT11 transcript (SEPT11_v1) we identified a novel SEPT11 variant (SEPT11_v2) as interaction partner of SEPT4 and SEPT7. Library screening of an endothelial cell library also revealed the presence of this novel SEPT11_v2 transcript. In addition, a third SEPT11 variant (SEPT11_v3) was identified. Expression of SEPT11_v1 and of SEPT11_v2 and SEPT11_v3 in human brain regions was investigated by Northern analysis. Further interaction partners of SEPT11 are characterised using immunofluorescence. Co-localisation of SEPT2, SEPT4, SEPT7 and SEPT11 with tubulin and transferrin receptor (endocytotic marker) is demonstrated. In addition, co-localisation of SEPT4 and SEPT11 with the vesicle-associated protein synaptobrevin 1 (VAMP1), but not clearly with actin, was shown. Only SEPT2 and SEPT7 definitely co-localised with actin, but not clearly with VAMP1. PMID:20978712

  13. PAR-1 activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand

    PubMed Central

    Bae, Jong-Sup; Rezaie, Alireza R.

    2008-01-01

    Summary We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by its natural ligand activated protein C (APC)/protein C switches the protease activated receptor 1 (PAR-1)-dependent signaling specificity of thrombin from a disruptive to a protective effect in cultured human umbilical vein endothelial cells. Given the phenotypic differences between endothelial cells in venular and arterial beds, in this study we evaluated the signaling function of thrombin in human pulmonary artery endothelial cells (HPAECs) before and after treating them with PC-S195A which lacks catalytic activity but exhibits a normal affinity for EPCR. As expected, both thrombin and thrombin receptor agonist peptide (TRAP) enhanced the permeability barrier of HPAECs, however, both PAR-1 agonists exhibited a potent barrier protective effect when the cells were treated with PC-S195A prior to stimulation by the agonists. Interestingly, similar to APC, thrombin exhibited a potent cytoprotective activity in the LPS-induced permeability and TNF-α-induced apoptosis and adhesion assays in the PC-S195A treated HPAECs. Treatment of HPAECs with the cholesterol depleting molecule methyl-β-cyclodextrin eliminated the protective effect of both APC and thrombin. These results suggest that the occupancy of EPCR by its natural ligand recruits PAR-1 to a protective signaling pathway within lipid rafts of HPAECs. Based on these results we conclude that the activation of PAR-1 by thrombin would initiate a protective response in intact arterial vascular cells expressing EPCR. These findings may have important ramifications for understanding the mechanism of the participation of the vascular PAR-1 in pathophysiology of the inflammatory disorders. PMID:18612544

  14. Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage

    PubMed Central

    HORNER, A.; BISHOP, N. J.; BORD, S.; BEETON, C.; KELSALL, A. W.; COLEMAN, N.; COMPSTON, J. E.

    1999-01-01

    Angiogenesis is essential for the replacement of cartilage by bone during growth and repair. In order to obtain a better understanding of the mechanisms regulating vascular invasion at sites of endochondral ossification we have investigated the expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), by chondrocytes in human neonatal growth plates. VEGF was absent from chondrocytes in the resting zone and only weakly expressed by occasional chondrocytes in the proliferating region. In the hypertrophic zone the number of chondrocytes stained and the intensity of staining for VEGF increased with chondrocyte hypertrophy, maximum expression of VEGF being observed in chondrocytes in the lower hypertrophic and mineralised regions of the cartilage. These observations provide the first demonstration of the presence of VEGF in situ in developing human bone and are consistent with in vitro observations demonstrating the upregulation of proangiogenic growth factor production with increasing chondrocyte hypertrophy. The presence of numerous small blood vessels and vascular structures in the subchondral region where VEGF expression was maximal indicates that VEGF produced by hypertrophic chondrocytes may play a key role in the regulation of vascular invasion of the growth plate. PMID:10445820

  15. Human aortic endothelial cell morphology influenced by topography of porous silicon substrates.

    PubMed

    Formentín, Pilar; Catalán, Úrsula; Fernández-Castillejo, Sara; Alba, Maria; Baranowska, Malgorzata; Solà, Rosa; Pallarès, Josep; Marsal, Lluís F

    2015-10-01

    Porous silicon has received much attention because of its optical properties and for its usefulness in cell-based biosensing, drug delivery, and tissue engineering applications. Surface properties of the biomaterial are associated with cell adhesion and with proliferation, migration, and differentiation. The present article analyzes the behavior of human aortic endothelial cells in macro- and nanoporous collagen-modified porous silicon samples. On both substrates, cells are well adhered and numerous. Confocal microscopy and scanning electron microscopy were employed to study the effects of porosity on the morphology of the cells. On macroporous silicon, filopodia is not observed but the cell spreads on the surface, increasing the lamellipodia surface which penetrates the macropore. On nanoporous silicon, multiple filopodia were found to branch out from the cell body. These results demonstrate that the pore size plays a key role in controlling the morphology and growth rate of human aortic endothelial cells, and that these forms of silicon can be used to control cell development in tissue engineering as well as in basic cell biology research. PMID:26017716

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

    PubMed Central

    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 cell line from human aortic endothelial cells (HAECs). Fluorescence microscopy and flow cytometry were also applied to further explore DEP-induced cytotoxicity in HAECs. Results revealed that DEPs could negatively impair cell viability and alter membrane nanostructures and cytoskeleton components in a dosage- and a time-dependent manner; and analyses suggested that DEPs-induced hyperpolarization in HAECs appeared in a time-dependent manner, implying DEP treatment would lead to vasodilation, which could be supported by down-regulation of cell biophysical properties (e.g., cell elasticity). These findings are consistent with the conclusion that DEP exposure triggers important biochemical and biophysical changes that would negatively impact the pathological development of cardiovascular diseases. For example, DEP intervention would be one cause of vasodilation, which will expand understanding of biophysical aspects associated with DEP cytotoxicity in HAECs. PMID:22662129

  17. Recombinant human vascular endothelial growth factor receptor 1 effectively inhibits angiogenesis in vivo.

    PubMed

    Wang, Jinliang; Shi, Minglei; Xi, Yongyi; Gao, Lihua; Zhang, Guanyi; Shao, Yong; Chen, Huipeng; Hu, Xianwen

    2015-05-01

    Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. VEGF receptor‑1 (VEGFR‑1) acts as a decoy VEGF receptor that enables the regulation of VEGF on the vascular endothelium. In the present study, the recombinant human VEGFR1D1‑3/Fc (rhVEGFR‑1), which contains key domains for VEGF binding, was cloned and expressed in Chinese hamster ovary (CHO) cells. The rhVEGFR‑1 protein was purified using protein‑A affinity chromatography. The molecular weight of rhVEGFR‑1 was found to be ~162 and 81 kD in non‑reducing and reducing SDS‑PAGE, respectively. The majority of the final protein products were in the dimeric conformation. Western blot analysis revealed that rhVEGFR‑1 was only capable of binding to the full glycan form of rhVEGF‑165 and rhVEGF‑121. The dissociation constant for the binding of rhVEGFR‑1 to VEGF‑165, detected using Biacore, was 285 pM. In addition, rhVEGFR‑1 inhibited the proliferation and migration of human microvascular endothelial cells. In vivo experiments also demonstrated that rhVEGFR‑1 inhibited chicken chorioallantoic membrane neovascularization and angiogenesis in nude mice. In conclusion, an anti‑angiogenic recombinant soluble VEGFR was expressed (up to 5 mg/l) in CHO cells and was shown to be capable of inhibiting neovascularization in vivo and in vitro. PMID:25607471

  18. Effects of phthalates on the human corneal endothelial cell line B4G12.

    PubMed

    Krüger, Tanja; Cao, Yi; Kjærgaard, Søren K; Knudsen, Lisbeth E; Bonefeld-Jørgensen, Eva C

    2012-01-01

    Phthalates are industrial chemicals used in many cosmetics. We evaluated an in vitro model for eye irritancy testing using the human corneal endothelial cell line B4G12. Cell proliferation and toxicity were assessed after exposing to di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), di-2-ethylhexyl phthalate (DEHP), diisodecyl phthalate (DIDP), di-n-octyl phthalate (DnOP), and di-isononyl phthalate (DINP). Gene expression and secretion of inflammatory cytokines were evaluated after exposure to DBP. Decreased cell proliferation was observed for the phthalates DBP, BBP, and DEHP, and cell toxicity was observed for DBP and BBP. Upon DBP exposure at nontoxic concentrations, a significant increased gene expression and cytokine cell secretion were observed for interleukin-1β (IL-1β) and IL-8, and also an increased IL-6 secretion was observed. In conclusion, the human corneal endothelial cell line B4G12 may be a potential model for inflammatory eye irritancy testing of phthalates. PMID:22723514

  19. Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules

    PubMed Central

    Girard, Jean-Philippe; Baekkevold, Espen S.; Feliu, Jacques; Brandtzaeg, Per; Amalric, François

    1999-01-01

    High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na+-coupled anion transporters and exhibits 40–50% amino acid identity with the rat renal Na+/sulfate cotransporter, NaSi-1, as well as with the human and rat Na+/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na+-dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription–PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na+-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV. PMID:10535998

  20. Expression of amino acid transport systems in cultured human umbilical vein endothelial cells.

    PubMed

    Mann, G E; Pearson, J D; Sheriff, C J; Toothill, V J

    1989-03-01

    1. Nutrient transport in cultured human umbilical vein endothelial cells was characterized using a rapid dual-isotope dilution technique. Microcarrier beads with confluent endothelial cells were perfused in small columns, and uptake and efflux were assessed relative to D-mannitol (extracellular tracer) during a single transit through the column. 2. At tracer concentrations significant unidirectional uptakes were measured for L-leucine (53 +/- 2%), L-phenylalanine (73 +/- 2%), L-serine (40 +/- 4%), L-arginine (42 +/- 3%) and L-ornithine (26 +/- 3%). Uptake for L-proline, D-glucose, dopamine and serotonin was lower (6-10%), whereas uptake for the system A analogue 2-methylaminoisobutyric acid (2-MeAIB) was negligible. Uptakes rapidly decreased with time due to tracer efflux. 3. Endothelial cell transport of L-leucine was markedly inhibited during perfusion with 1 mM-BCH (beta-2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, system L analogue), L-leucine, D-leucine, L-phenylalanine, L-methionine and L-DOPA. 2-MeAIB, L-cysteine, glycine, L-proline, hydroxy-L-proline, L-aspartate and beta-alanine were poor inhibitors, while L-serine and the cationic substrates L-lysine and L-arginine inhibited uptake by 10-35%. 4. When the kinetics of L-leucine transport were examined over a wide range of substrate concentrations (0.025-1 mM) transport was saturable. A single entry site analysis gave a half-maximal saturation constant Kt = 0.24 +/- 0.08 mM (mean +/- S.E.M., n = 5) and a Vmax = 27.8 +/- 4.6 nmol/min per column (approximately 3 x 10(6) cells). 5. Removal of sodium from the perfusate inhibited tracer uptake of L-leucine, L-serine and L-arginine by respectively 20 +/- 5% (n = 3), 77 +/- 5% (n = 3) and 35 +/- 4% (n = 3). 6. Our results provide the first evidence that cultured human endothelial cells of venous origin express a saturable transport system for large neutral amino acids resembling system L described in brain microvascular endothelium. Detection of Na

  1. KLF4 Promotes Angiogenesis by Activating VEGF Signaling in Human Retinal Microvascular Endothelial Cells

    PubMed Central

    Wang, Yinan; Yang, Chuanhe; Gu, Qingqing; Sims, Michelle; Gu, Weiwang; Pfeffer, Lawrence M.; Yue, Junming

    2015-01-01

    The transcription factor Krüppel-like factor 4 (KLF4) has been implicated in regulating cell proliferation, migration and differentiation in a variety of human cells and is one of four factors required for the induction of pluripotent stem cell reprogramming. However, its role has not been addressed in ocular neovascular diseases. This study investigated the role of KLF4 in angiogenesis and underlying molecular mechanisms in human retinal microvascular endothelial cells (HRMECs). The functional role of KLF4 in HRMECs was determined following lentiviral vector mediated inducible expression and shRNA knockdown of KLF4. Inducible expression of KLF4 promotes cell proliferation, migration and tube formation. In contrast, silencing KLF4 inhibits cell proliferation, migration, tube formation and induces apoptosis in HRMECs. KLF4 promotes angiogenesis by transcriptionally activating VEGF expression, thus activating the VEGF signaling pathway in HRMECs. PMID:26075898

  2. The Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor Cediranib (Recentin; AZD2171) Inhibits Endothelial Cell Function and Growth of Human Renal Tumor Xenografts

    SciTech Connect

    Siemann, Dietmar W. Brazelle, W.D.; Juergensmeier, Juliane M.

    2009-03-01

    Purpose: The goal of this study was to examine the therapeutic potential of the vascular endothelial growth factor (VEGF) signaling inhibitor cediranib in a human model of renal cell carcinoma (Caki-1). Methods and Materials: The effects of cediranib treatment on in vitro endothelial cell function (proliferation, migration, and tube formation), as well as in vivo angiogenesis and tumor growth, were determined. Results: In vitro, cediranib significantly impaired the proliferation and migration of endothelial cells and their ability to form tubes, but had no effect on the proliferation of Caki-1 tumor cells. In vivo, cediranib significantly reduced Caki-1 tumor cell-induced angiogenesis, reduced tumor perfusion, and inhibited the growth of Caki-1 tumor xenografts. Conclusions: The present results are consistent with the notion that inhibition of VEGF signaling leads to an indirect (i.e., antiangiogenic) antitumor effect, rather than a direct effect on tumor cells. These results further suggest that inhibition of VEGF signaling with cediranib may impair the growth of renal cell carcinoma.

  3. Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells

    PubMed Central

    Ming, Xiu-Fen; Viswambharan, Hema; Barandier, Christine; Ruffieux, Jean; Kaibuchi, Kozo; Rusconi, Sandro; Yang, Zhihong

    2002-01-01

    Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB. PMID:12446767

  4. Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

    NASA Astrophysics Data System (ADS)

    Chiu, Brian; Z-M Wan, Jim; Abley, Doris; Akabutu, John

    2005-05-01

    Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity ( μg) may modulate the proliferation and differentiation. We investigated the application of μg to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated μg for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow-cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated μg may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.

  5. Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels

    PubMed Central

    Vieira, Monica L.; Alvarez-Flores, Miryam P.; Kirchgatter, Karin; Romero, Eliete C.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Chudzinski-Tavassi, Ana M.

    2013-01-01

    We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date. PMID:23478319

  6. Pro-inflammatory cytokines downregulate Hsp27 and cause apoptosis of human retinal capillary endothelial cells

    PubMed Central

    Nahomi, Rooban B.; Palmer, Allison; Roth, Katelyn E.; Fort, Patrice E.; Nagaraj, Ram H.

    2013-01-01

    The formation of acellular capillaries in the retina, a hallmark feature of diabetic retinopathy, is caused by apoptosis of endothelial cells and pericytes. The biochemical mechanism of such apoptosis remains unclear. Small heat shock proteins play an important role in the regulation of apoptosis. In the diabetic retina, pro-inflammatory cytokines are upregulated. In this study, we investigated the effects of pro-inflammatory cytokines on small heat shock protein 27 (Hsp27) in human retinal endothelial cells (HREC). In HREC cultured in the presence of cytokine mixtures (CM), a significant downregulation of Hsp27 at the protein and mRNA level occurred, with no effect on HSF-1, the transcription factor for Hsp27. The presence of high glucose (25 mM) amplified the effects of cytokines on Hsp27. CM activated indoleamine 2,3-dioxygenase (IDO) and enhanced the production of kynurenine and ROS. An inhibitor of IDO, 1-methyl tryptophan (MT), inhibited the effects of CM on Hsp27. CM also upregulated NOS2 and, consequently, nitric oxide (NO). A NOS inhibitor, L-NAME, and a ROS scavenger blocked the CM-mediated Hsp27 downregulation. While a NO donor in the culture medium did not decrease the Hsp27 content, a peroxynitrite donor and exogenous peroxynitrite did. The cytokines and high glucose-induced apoptosis of HREC were inhibited by MT and L-NAME. Downregulation of Hsp27 by a siRNA treatment promoted apoptosis in HREC. Together, these data suggest that pro-inflammatory cytokines induce the formation of ROS and NO, which, through the formation of peroxynitrite, reduce the Hsp27 content and bring about apoptosis of retinal capillary endothelial cells. PMID:24252613

  7. Effects of simulated microgravity on cell cycle in human endothelial cells

    NASA Astrophysics Data System (ADS)

    Sokolovskaya, Alisa A.; Ignashkova, Tatiana I.; Bochenkova, Anna V.; Moskovtsev, Aleksey A.; Baranov, Victor M.; Kubatiev, Aslan A.

    2014-06-01

    The aim of the current study is to investigate effects of simulated microgravity on the cell cycle of endothelial cells. We analyze changes in the cell cycle after exposure of endothelial-like EA.hy 926 cells to simulated microgravity using a Desktop random positioning machine (RPM). Cell cycle profiles determined by flow cytometry show, that the percentage of the cells in the G0/G1 phase after 24 and 96 h of RPM-simulated microgravity is significantly increased as compared to the control group. However, no significant difference is observed after 120 h of RPM-simulated microgravity. In regard to S phase, the percentage of cells is significantly decreased after 24 and 96 h of RPM, respectively; whereas 120 h later, the number of S-phase cells is comparable to the control group. Thus, we show that simulated microgravity inhibits cell cycle progression of human EA.hy 926 cells from the G0/G1 phase to the S phase. We observe an effect of a hibernation-like state, when the growth of the cells in the RPM group slows down, but does not stop. Our results further show that simulated microgravity can affect adhesion of endothelial cells, and alpha-tubulin expression, as most cells begin to detach from the surface of OptiCell unit after 24 h, form aggregates after 48 h, and exhibit accumulation of alpha-tubulin around the nucleus after 48 h of exposure to simulated microgravity conditions. Our results demonstrate a chance in the cell cycle in a low gravitational field.

  8. Adherence to human lung microvascular endothelial cells (HMVEC-L) of Plasmodium vivax isolates from Colombia

    PubMed Central

    2013-01-01

    Background For years Plasmodium vivax has been considered the cause of benign malaria. Nevertheless, it has been observed that this parasite can produce a severe disease comparable to Plasmodium falciparum. It has been suggested that some physiopathogenic processes might be shared by these two species, such as cytoadherence. Recently, it has been demonstrated that P. vivax-infected erythrocytes (Pv-iEs) have the capacity to adhere to endothelial cells, in which intercellular adhesion molecule-1 (ICAM-1) seems to be involved in this process. Methods Adherence capacity of 21 Colombian isolates, from patients with P. vivax mono-infection to a microvascular line of human lung endothelium (HMVEC-L) was assessed in static conditions and binding was evaluated at basal levels or in tumor necrosis factor (TNF) stimulated cells. The adherence specificity for the ICAM-1 receptor was determined through inhibition with an anti-CD54 monoclonal antibody. Results The majority of P. vivax isolates, 13 out of 21 (61.9%), adhered to the HMVEC-L cells, but P. vivax adherence was at least seven times lower when compared to the four P. falciparum isolates. Moreover, HMVEC-L stimulation with TNF led to an increase of 1.6-fold in P. vivax cytoadhesion, similar to P. falciparum isolates (1.8-fold) at comparable conditions. Also, blockage of ICAM-1 receptor with specific antibodies showed a significant 50% adherence reduction. Conclusions Plasmodium vivax isolates found in Colombia are also capable of adhering specifically in vitro to lung endothelial cells, via ICAM-1 cell receptor, both at basal state and after cell stimulation with TNF. Collectively, these findings reinforce the concept of cytoadherence for P. vivax, but here, to a different endothelial cell line and using geographical distinct isolates, thus contributing to understanding P. vivax biology. PMID:24080027

  9. Anti-atherosclerotic activity of platycodin D derived from roots of Platycodon grandiflorum in human endothelial cells.

    PubMed

    Wu, Jingtao; Yang, Guiwen; Zhu, Wenxing; Wen, Wujun; Zhang, Fumiao; Yuan, Jinduo; An, Liguo

    2012-01-01

    This study examined the effects of platycodin D (PD), a triterpene saponin from the the root of Platycodon grandiflorum A.DC on human umbilical vein endothelial cells (HUVECs) in vitro, which were pre-treated with PD (0.01, 0.15, 0.25 mg/mL), respectively, and treated with 50 mg/L oxidized low-density lipoprotein (oxLDL). The levels of nitric oxide (NO) and malonaldehyde (MAD) in the culture medium, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) mRNA expression in endothelium cells and the adhesion of monocytes to endothelial cells were measured. The results showed that PD increased NO concentration and decreased MDA level induced by oxLDL in the medium of endothelial cells. Moreover, PD significantly inhibited the oxLDL-induced increase in monocyte adhesion to endothelial cells as well as decreasing mRNA expression levels of VCAM-1 and ICAM-1 on these cells. Based on these results, it is suggested that PD is a promising anti-atherosclerotic activity, which is at least in part the result of its increasing NO concentration, reducing the oxLDL-induced cell adhesion molecule expression in endothelial cells and the endothelial adhesion to monocytes. PMID:22863916

  10. Overexpression of uncoupling protein 2 inhibits the high glucose-induced apoptosis of human umbilical vein endothelial cells

    PubMed Central

    HE, YING; LUAN, ZHOU; FU, XUNAN; XU, XUN

    2016-01-01

    Ectopic apoptosis of vascular cells plays a critical role in the early stage development of diabetic retinopathy (DR). Uncoupling protein 2 (UCP2) is a mitochondrial modulator which protects against endothelial dysfunction. However, the role which UCP2 plays in endothelial apoptosis and its association with DR was unclear. In the present study, we investigated whether UCP2 functioned as an inhibitor of DR in endothelial cells. Firstly, we noted that in UCP2-knockout mice retinal cell death and damage in vivo was similar to that of db/db diabetic mice. Additionally, UCP2 knockdown induced caspase-3 activation and exaggerated high glucose (HG)-induced apoptosis of human umbilical vein endothelial cells (HUVECs). Conversely, adenovirus-mediated UCP2 overexpression inhibited the apoptosis of HUVECs and HG-induced caspase-3 activation. Furthermore, HG treatment resulted in the opening of the permeability transition pore (PTP) and liberation of cytochrome c from mitochondria to the cytosol in HUVECs. Notably, UCP2 overexpression inhibited these processes. Furthermore, adenovirus-mediated UCP2 overexpression led to a significant increase in intracellular nitric oxide (NO) levels and a decrease in reactive oxygen species (ROS) generation in HUVECs. Collectively, these data suggest that UCP2 plays an anti-apoptotic role in endothelial cells. Thus, we suggest that approaches which augment UCP2 expression in vascular endothelial cells aid in preventing the early stage development and progression of DR. PMID:26846204