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Sample records for affect endothelial cell

  1. Inhibition of cyclooxygenase (COX)-2 affects endothelial progenitor cell proliferation

    SciTech Connect

    Colleselli, Daniela; Bijuklic, Klaudija; Mosheimer, Birgit A.; Kaehler, Christian M. . E-mail: C.M.Kaehler@uibk.ac.at

    2006-09-10

    Growing evidence indicates that inducible cyclooxygenase-2 (COX-2) is involved in the pathogenesis of inflammatory disorders and various types of cancer. Endothelial progenitor cells recruited from the bone marrow have been shown to be involved in the formation of new vessels in malignancies and discussed for being a key point in tumour progression and metastasis. However, until now, nothing is known about an interaction between COX and endothelial progenitor cells (EPC). Expression of COX-1 and COX-2 was detected by semiquantitative RT-PCR and Western blot. Proliferation kinetics, cell cycle distribution and rate of apoptosis were analysed by MTT test and FACS analysis. Further analyses revealed an implication of Akt phosphorylation and caspase-3 activation. Both COX-1 and COX-2 expression can be found in bone-marrow-derived endothelial progenitor cells in vitro. COX-2 inhibition leads to a significant reduction in proliferation of endothelial progenitor cells by an increase in apoptosis and cell cycle arrest. COX-2 inhibition leads further to an increased cleavage of caspase-3 protein and inversely to inhibition of Akt activation. Highly proliferating endothelial progenitor cells can be targeted by selective COX-2 inhibition in vitro. These results indicate that upcoming therapy strategies in cancer patients targeting COX-2 may be effective in inhibiting tumour vasculogenesis as well as angiogenic processes.

  2. New thiazolidinediones affect endothelial cell activation and angiogenesis.

    PubMed

    Rudnicki, Martina; Tripodi, Gustavo L; Ferrer, Renila; Boscá, Lisardo; Pitta, Marina G R; Pitta, Ivan R; Abdalla, Dulcineia S P

    2016-07-01

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor-γ (PPARγ) agonists used in treating type 2 diabetes that may exhibit beneficial pleiotropic effects on endothelial cells. In this study, we characterized the effects of three new TZDs [GQ-32 (3-biphenyl-4-ylmethyl-5-(4-nitro-benzylidene)-thiazolidine-2,4-dione), GQ-169 (5-(4-chloro-benzylidene)-3-(2,6-dichloro-benzyl)-thiazolidine-2,4-dione), and LYSO-7 (5-(5-bromo-1H-indol-3-ylmethylene)-3-(4-chlorobenzyl)-thiazolidine-2,4-dione)] on endothelial cells. The effects of the new TZDs were evaluated on the production of nitric oxide (NO) and reactive oxygen species (ROS), cell migration, tube formation and the gene expression of adhesion molecules and angiogenic mediators in human umbilical vein endothelial cells (HUVECs). PPARγ activation by new TZDs was addressed with a reporter gene assay. The three new TZDs activated PPARγ and suppressed the tumor necrosis factor α-induced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. GQ-169 and LYSO-7 also inhibited the glucose-induced ROS production. Although NO production assessed with 4-amino-5-methylamino-2',7'-difluorofluorescein-FM probe indicated that all tested TZDs enhanced intracellular levels of NO, only LYSO-7 treatment significantly increased the release of NO from HUVEC measured by chemiluminescence analysis of culture media. Additionally, GQ-32 and GQ-169 induced endothelial cell migration and tube formation by the up-regulation of angiogenic molecules expression, such as vascular endothelial growth factor A and interleukin 8. GQ-169 also increased the mRNA levels of basic fibroblast growth factor, and GQ-32 enhanced transforming growth factor-β expression. Together, the results of this study reveal that these new TZDs act as partial agonists of PPARγ and modulate endothelial cell activation and endothelial dysfunction besides to stimulate migration and tube formation. PMID:27108791

  3. Relationship between Microtubule Network Structure and Intracellular Transport in Cultured Endothelial Cells Affected by Shear Stress

    NASA Astrophysics Data System (ADS)

    Kudo, Susumu; Ikezawa, Kenji; Ikeda, Mariko; Tanishita, Kazuo

    Endothelial cells (ECs) that line the inner surface of blood vessels are barriers to the transport of various substances into or from vessel walls, and are continuously exposed to shear stress induced by blood flow in vivo. Shear stress affects the cytoskeleton (e.g., microtubules, microfilaments, intermediate filaments), and affects the transport of macromolecules. Here, the relationship between the microtubule network structure and this transport process for albumin uptake within cultured aortic endothelial cells affected by shear stress was studied. Based on fluorescent images of albumin uptake obtained by using confocal laser scanning microscopy (CLSM), both the microtubule network and albumin uptake in ECs were disrupted by colchicine and were affected by shear stress loading.

  4. Hyaluronic acid hydrogel stiffness and oxygen tension affect cancer cell fate and endothelial sprouting

    PubMed Central

    Shen, Yu-I; Abaci, Hasan E.; Krupsi, Yoni; Weng, Lien-Chun; Burdick, Jason A.; Gerecht, Sharon

    2014-01-01

    Three-dimensional (3D) tissue culture models may recapitulate aspects of the tumorigenic microenvironment in vivo, enabling the study of cancer progression in vitro. Both hypoxia and matrix stiffness are known to regulate tumor growth. Using a modular culture system employing an acrylated hyaluronic acid (AHA) hydrogel, three hydrogel matrices with distinctive degrees of viscoelasticity — soft (78±16 Pa), medium (309± 57 Pa), and stiff (596± 73 Pa) — were generated using the same concentration of adhesion ligands. Oxygen levels within the hydrogel in atmospheric (21 %), hypoxic (5 %), and severely hypoxic (1 %) conditions were assessed with a mathematical model. HT1080 fibrosarcoma cells, encapsulated within the AHA hydrogels in high densities, generated nonuniform oxygen distributions, while lower cell densities resulted in more uniform oxygen distributions in the atmospheric and hypoxic environments. When we examined how varying viscoelasticity in atmospheric and hypoxic environments affects cell cycles and the expression of BNIP3 and BNIP3L (autophagy and apoptosis genes), and GLUT-1 (a glucose transport gene), we observed that HT1080 cells in 3D hydrogel adapted better to hypoxic conditions than those in a Petri dish, with no obvious correlation to matrix viscoelasticity, by recovering rapidly from possible autophagy/apoptotic events and alternating metabolism mechanisms. Further, we examined how HT1080 cells cultured in varying viscoelasticity and oxygen tension conditions affected endothelial sprouting and invasion. We observed that increased matrix stiffness reduced endothelial sprouting and invasion in atmospheric conditions; however, we observed increased endothelial sprouting and invasion under hypoxia at all levels of matrix stiffness with the upregulation of vascular endothelial growth factor (VEGF) and angiopoeitin-1 (ANG-1). Overall, HT1080 cells encapsulated in the AHA hydrogels under hypoxic stress recovered better from apoptosis and

  5. Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) affects RhoA activation in endothelial cells.

    PubMed

    Suzuki, Takahiro; Abe, Mayumi; Miyashita, Hiroki; Kobayashi, Toshimitsu; Sato, Yasufumi

    2007-06-01

    Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) expressed in endothelial cells (ECs) plays an important role in angiogenesis due to its involvement in migration, proliferation and network formation. Here we examined the biological function of PILSAP with respect to EC morphogenesis and the related intracellular signaling for this process. When mouse endothelial MSS31 cells were cultured, a dominant negative PILSAP mutant converted cell shape to disk-like morphology, blocked stress fiber formation, and augmented membrane ruffling in random directions. These phenotypic changes led us to test whether PILSAP affected activities of Rho family small G-proteins. Abrogation of PILSAP enzymatic activity or its expression attenuated RhoA but not Rac1 activation during cell adhesion. This attenuation of RhoA activation was also evident when G-protein coupled receptors such as proteinase-activated receptor or lysophosphatidic acid receptor were activated in ECs. These results indicate that PILSAP affects RhoA activation and that influences the proper function of ECs.

  6. Cyclic Stretch Affects Pulmonary Endothelial Cell Control of Pulmonary Smooth Muscle Cell Growth

    PubMed Central

    Ochoa, Cristhiaan D.; Baker, Haven; Hasak, Stephen; Matyal, Robina; Salam, Aleya; Hales, Charles A.; Hancock, William; Quinn, Deborah A.

    2008-01-01

    Endothelial cells are subjected to mechanical forces in the form of cyclic stretch resulting from blood pulsatility. Pulmonary artery endothelial cells (PAECs) produce factors that stimulate and inhibit pulmonary artery smooth muscle cell (PASMC) growth. We hypothesized that PAECs exposed to cyclic stretch secrete proteins that inhibit PASMC growth. Media from PAECs exposed to cyclic stretch significantly inhibited PASMC growth in a time-dependent manner. Lyophilized material isolated from stretched PAEC-conditioned media significantly inhibited PASMC growth in a dose-dependent manner. This inhibition was reversed by trypsin inactivation, which is consistent with the relevant factor being a protein(s). To identify proteins that inhibited cell growth in conditioned media from stretched PAECs, we used proteomic techniques and found that thrombospondin (TSP)-1, a natural antiangiogenic factor, was up-regulated by stretch. In vitro, exogenous TSP-1 inhibited PASMC growth. TSP-1–blocking antibodies reversed conditioned media–induced inhibition of PASMC growth. Cyclic stretched PAECs secrete protein(s) that inhibit PASMC proliferation. TSP-1 may be, at least in part, responsible for this inhibition. The complete identification and understanding of the secreted proteome of stretched PAECs may lead to new insights into the pathophysiology of pulmonary vascular remodeling. PMID:18314539

  7. Polyphenolic composition of grape stem extracts affects antioxidant activity in endothelial and muscle cells

    PubMed Central

    GOUTZOURELAS, NIKOLAOS; STAGOS, DIMITRIOS; SPANIDIS, YPATIOS; LIOSI, MARIA; APOSTOLOU, ANNA; PRIFTIS, ALEXANDROS; HAROUTOUNIAN, SERKO; SPANDIDOS, DEMETRIOS A.; TSATSAKIS, ARISTIDIS M.; KOURETAS, DEMETRIOS

    2015-01-01

    The aim of the present study was the assessment of the antioxidant effects of polyphenolic extracts derived from the stems of three Greek grape varieties (Moshomayro, Mavrotragano and Mandilaria) in endothelial (EA.hy926) and muscle (C2C12) cells. We also investigated the effects of the polyphenolic composition on the antioxidant effects of the grape stem extracts. For this purpose, the endothelial and muscle cells were treated with low non-cytotoxic concentrations of the extracts for 24 h in order to assess the effects of the extracts on cellular redox status using oxidative stress biomarkers. The oxidative stress markers were thiobarbituric acid reactive substances (TBARS), protein carbonyl (CARB) levels, reactive oxygen species (ROS) levels and glutathione (GSH) levels. The results revealed that treatment of the EA.hy926 cells with Mandilaria extract significantly decreased the TBARS levels by 14.8% and the CARB levels by 25.9 %, while it increased the GSH levels by 15.8% compared to the controls. Moreover, treatment of the EA.hy926 cells with Mavrotragano extract significantly increased the GSH levels by 20.2%, while it significantly decreased the TBARS and CARB levels by 12.5% and 16.6%, respectively. Treatment of the C2C12 cells with Mandilaria extract significantly decreased the TBARS levels by 47.3 %, the CARB levels by 39.0 % and the ROS levels by 21.8%, while it increased the GSH levels by 22.6% compared to the controls. Moreover, treatment of the C2C12 cells with Mavrotragano significantly decreased the TBARS, CARB and ROS levels by 36.2%, 35.9% and 16.5%, respectively. In conclusion, to the best of our knowledgel, our results demonstrate for the first time that treatment with grape stem extracts at low concentrations improves the redox status of endothelial and muscle cells. Thus, grape stem extracts may be used for developing antioxidant food supplements or biofunctional foods. However, it was also found that the polyphenolic composition of grape stem

  8. Polyphenolic composition of grape stem extracts affects antioxidant activity in endothelial and muscle cells.

    PubMed

    Goutzourelas, Nikolaos; Stagos, Dimitrios; Spanidis, Ypatios; Liosi, Maria; Apostolou, Anna; Priftis, Alexandros; Haroutounian, Serko; Spandidos, Demetrios A; Tsatsakis, Aristidis M; Kouretas, Demetrios

    2015-10-01

    The aim of the present study was the assessment of the antioxidant effects of polyphenolic extracts derived from the stems of three Greek grape varieties (Moshomayro, Mavrotragano and Mandilaria) in endothelial (EA.hy926) and muscle (C2C12) cells. We also investigated the effects of the polyphenolic composition on the antioxidant effects of the grape stem extracts. For this purpose, the endothelial and muscle cells were treated with low non-cytotoxic concentrations of the extracts for 24 h in order to assess the effects of the extracts on cellular redox status using oxidative stress biomarkers. The oxidative stress markers were thiobarbituric acid reactive substances (TBARS), protein carbonyl (CARB) levels, reactive oxygen species (ROS) levels and glutathione (GSH) levels. The results revealed that treatment of the EA.hy926 cells with Mandilaria extract significantly decreased the TBARS levels by 14.8% and the CARB levels by 25.9 %, while it increased the GSH levels by 15.8% compared to the controls. Moreover, treatment of the EA.hy926 cells with Mavrotragano extract significantly increased the GSH levels by 20.2%, while it significantly decreased the TBARS and CARB levels by 12.5% and 16.6%, respectively. Treatment of the C2C12 cells with Mandilaria extract significantly decreased the TBARS levels by 47.3 %, the CARB levels by 39.0 % and the ROS levels by 21.8%, while it increased the GSH levels by 22.6% compared to the controls. Moreover, treatment of the C2C12 cells with Mavrotragano significantly decreased the TBARS, CARB and ROS levels by 36.2%, 35.9% and 16.5%, respectively. In conclusion, to the best of our knowledgel, our results demonstrate for the first time that treatment with grape stem extracts at low concentrations improves the redox status of endothelial and muscle cells. Thus, grape stem extracts may be used for developing antioxidant food supplements or biofunctional foods. However, it was also found that the polyphenolic composition of grape stem

  9. Polyphenolic composition of grape stem extracts affects antioxidant activity in endothelial and muscle cells.

    PubMed

    Goutzourelas, Nikolaos; Stagos, Dimitrios; Spanidis, Ypatios; Liosi, Maria; Apostolou, Anna; Priftis, Alexandros; Haroutounian, Serko; Spandidos, Demetrios A; Tsatsakis, Aristidis M; Kouretas, Demetrios

    2015-10-01

    The aim of the present study was the assessment of the antioxidant effects of polyphenolic extracts derived from the stems of three Greek grape varieties (Moshomayro, Mavrotragano and Mandilaria) in endothelial (EA.hy926) and muscle (C2C12) cells. We also investigated the effects of the polyphenolic composition on the antioxidant effects of the grape stem extracts. For this purpose, the endothelial and muscle cells were treated with low non-cytotoxic concentrations of the extracts for 24 h in order to assess the effects of the extracts on cellular redox status using oxidative stress biomarkers. The oxidative stress markers were thiobarbituric acid reactive substances (TBARS), protein carbonyl (CARB) levels, reactive oxygen species (ROS) levels and glutathione (GSH) levels. The results revealed that treatment of the EA.hy926 cells with Mandilaria extract significantly decreased the TBARS levels by 14.8% and the CARB levels by 25.9 %, while it increased the GSH levels by 15.8% compared to the controls. Moreover, treatment of the EA.hy926 cells with Mavrotragano extract significantly increased the GSH levels by 20.2%, while it significantly decreased the TBARS and CARB levels by 12.5% and 16.6%, respectively. Treatment of the C2C12 cells with Mandilaria extract significantly decreased the TBARS levels by 47.3 %, the CARB levels by 39.0 % and the ROS levels by 21.8%, while it increased the GSH levels by 22.6% compared to the controls. Moreover, treatment of the C2C12 cells with Mavrotragano significantly decreased the TBARS, CARB and ROS levels by 36.2%, 35.9% and 16.5%, respectively. In conclusion, to the best of our knowledgel, our results demonstrate for the first time that treatment with grape stem extracts at low concentrations improves the redox status of endothelial and muscle cells. Thus, grape stem extracts may be used for developing antioxidant food supplements or biofunctional foods. However, it was also found that the polyphenolic composition of grape stem

  10. Extracellular S100A4 affects endothelial cell integrity and stimulates transmigration of A375 melanoma cells.

    PubMed

    Herwig, Nadine; Belter, Birgit; Pietzsch, Jens

    2016-09-01

    High extracellular S100A4 level proves a specific characteristic of some cancer cases, including malignant melanoma. Concerning the latter, extracellular S100A4 in an autocrine manner was shown to promote prometastatic activation of A375 cells by interaction with the receptor for advanced glycation endproducts (RAGE). We hypothesized that interaction of extracellular S100A4 with RAGE in a paracrine manner will affect endothelial cell (EC) integrity thus further promoting melanoma metastasis. We investigated the influence of recombinant and cell (A375)-derived S100A4 on junction protein expression and EC (hCMEC/D3) integrity by measuring transendothelial electrical resistance (TEER). Decrease of TEER and diminished expression of both occludin and VE-cadherin revealed the loss of EC integrity. Transmigration of transgenic A375 cells (A375-hS100A4/A375-hRAGE) through the EC monolayer was significantly higher compared to wild-type A375 cells, and was substantially decreased by sRAGE. A pilot study in mice, intracardially injected with A375-hS100A4 or A375-hRAGE cells, showed lower survival rates and a higher incidence of metastases compared to wild-type A375 cells. Tumor development was mostly located in the brain, bones, and ovaries. These findings provide further evidence on extracellular S100A4 as paracrine mediator of prometastatic endothelial dysfunction involving its interaction with RAGE.

  11. Pericyte abundance affects sucrose permeability in cultures of rat brain microvascular endothelial cells.

    PubMed

    Parkinson, Fiona E; Hacking, Cindy

    2005-07-01

    The blood-brain barrier is a physical and metabolic barrier that restricts diffusion of blood-borne substances into brain. In vitro models of the blood-brain barrier are used to characterize this structure, examine mechanisms of damage and repair and measure permeability of test substances. The core component of in vitro models of the blood-brain barrier is brain microvascular endothelial cells. We cultured rat brain microvascular endothelial cells (RBMEC) from isolated rat cortex microvessels. After 2-14 days in vitro (DIV), immunohistochemistry of these cells showed strong labeling for zona occludens 1 (ZO-1), a tight junction protein expressed in endothelial cells. Pericytes were also present in these cultures, as determined by expression of alpha-actin. The present study was performed to test different cell isolation methods and to compare the resulting cell cultures for abundance of pericytes and for blood-brain barrier function, as assessed by 14C-sucrose flux. Two purification strategies were used. First, microvessels were preabsorbed onto uncoated plastic for 4 h, then unattached microvessels were transferred to coated culture ware. Second, microvessels were incubated with an antibody to platelet-endothelial cell adhesion molecule 1 (PECAM-1; CD31) precoupled to magnetic beads, and a magnetic separation procedure was performed. Our results indicate that immunopurification, but not preadsorption, was an effective method to purify microvessels and reduce pericyte abundance in the resulting cultures. This purification significantly reduced 14C-sucrose fluxes across cell monolayers. These data indicate that pericytes can interfere with the development of blood-brain barrier properties in in vitro models that utilize primary cultures of RBMECs.

  12. Duration of microbead seeding on endothelial cells significantly affects their response to magnetic excitation

    NASA Astrophysics Data System (ADS)

    Reichenberg, Yaniv; Lanir, Yoram

    2012-04-01

    Our investigation of endothelial cell rheology using optical magnetic twisting cytometry revealed that with time following incubation of ferromagnetic beads on the cells, beads were sinking into the cells and an increasing number of beads demonstrated apparent absurd negative rheological properties. In parallel, the beads’ average rheological response changed considerably over time, both in magnitude and in distribution. It was hypothesized that the apparent negative rheological response was related to the above sinking process of seeded beads into the cells, resulting in an elevation of the beads’ rotation axis, thus causing a reversal of the beads’ lateral movement direction in response to twisting external magnetic excitation. The results suggest that microbead-based rheological characterization of cells should be interpreted with caution, while considering the time of data acquisition.

  13. Hydrostatic pressure and shear stress affect endothelin-1 and nitric oxide release by endothelial cells in bioreactors.

    PubMed

    Vozzi, Federico; Bianchi, Francesca; Ahluwalia, Arti; Domenici, Claudio

    2014-01-01

    Abundant experimental evidence demonstrates that endothelial cells are sensitive to flow; however, the effect of fluid pressure or pressure gradients that are used to drive viscous flow is not well understood. There are two principal physical forces exerted on the blood vessel wall by the passage of intra-luminal blood: pressure and shear. To analyze the effects of pressure and shear independently, these two stresses were applied to cultured cells in two different types of bioreactors: a pressure-controlled bioreactor and a laminar flow bioreactor, in which controlled levels of pressure or shear stress, respectively, can be generated. Using these bioreactor systems, endothelin-1 (ET-1) and nitric oxide (NO) release from human umbilical vein endothelial cells were measured under various shear stress and pressure conditions. Compared to the controls, a decrease of ET-1 production by the cells cultured in both bioreactors was observed, whereas NO synthesis was up-regulated in cells under shear stress, but was not modulated by hydrostatic pressure. These results show that the two hemodynamic forces acting on blood vessels affect endothelial cell function in different ways, and that both should be considered when planning in vitro experiments in the presence of flow. Understanding the individual and synergic effects of the two forces could provide important insights into physiological and pathological processes involved in vascular remodeling and adaptation.

  14. [Endothelial cell adhesion molecules].

    PubMed

    Ivanov, A N; Norkin, I A; Puchin'ian, D M; Shirokov, V Iu; Zhdanova, O Iu

    2014-01-01

    The review presents current data concerning the functional role of endothelial cell adhesion molecules belonging to different structural families: integrins, selectins, cadherins, and the immunoglobulin super-family. In this manuscript the regulatory mechanisms and factors of adhesion molecules expression and distribution on the surface of endothelial cells are discussed. The data presented reveal the importance of adhesion molecules in the regulation of structural and functional state of endothelial cells in normal conditions and in pathology. Particular attention is paid to the importance of these molecules in the processes of physiological and pathological angiogenesis, regulation of permeability of the endothelial barrier and cell transmigration.

  15. Pulmonary arterial endothelial cells affect the redox status of coenzyme Q0.

    PubMed

    Audi, Said H; Zhao, Hongtao; Bongard, Robert D; Hogg, Neil; Kettenhofen, Nicholas J; Kalyanaraman, Balaraman; Dawson, Christopher A; Merker, Marilyn P

    2003-04-01

    The pulmonary endothelium is capable of reducing certain redox-active compounds as they pass from the systemic venous to the arterial circulation. This may have important consequences with regard to the pulmonary and systemic disposition and biochemistry of these compounds. Because quinones comprise an important class of redox-active compounds with a range of physiological, toxicological, and pharmacological activities, the objective of the present study was to determine the fate of a model quinone, coenzyme Q0 (Q), added to the extracellular medium surrounding pulmonary arterial endothelial cells in culture, with particular attention to the effect of the cells on the redox status of Q in the medium. Spectrophotometry, electron paramagnetic resonance (EPR), and high-performance liquid chromatography (HPLC) demonstrated that, when the oxidized form Q is added to the medium surrounding the cells, it is rapidly converted to its quinol form (QH2) with a small concentration of semiquinone (Q*-) also detectable. The isolation of cell plasma membrane proteins revealed an NADH-Q oxidoreductase located on the outer plasma membrane surface, which apparently participates in the reduction process. In addition, once formed the QH2 undergoes a cyanide-sensitive oxidation by the cells. Thus, the actual rate of Q reduction by the cells is greater than the net QH2 output from the cells.

  16. Circulating endothelial progenitor cells are not affected by acute systemic inflammation

    PubMed Central

    Tura, Olga; Haeck, Marlieke L. A.; Short, Abigail; Freyer, Elizabeth; Barclay, G. Robin; Newby, David E.; Mills, Nicholas L.

    2010-01-01

    Vascular injury causes acute systemic inflammation and mobilizes endothelial progenitor cells (EPCs) and endothelial cell (EC) colony-forming units (EC-CFUs). Whether such mobilization occurs as part of a nonspecific acute phase response or is a phenomenon specific to vascular injury remains unclear. We aimed to determine the effect of acute systemic inflammation on EPCs and EC-CFU mobilization in the absence of vascular injury. Salmonella typhus vaccination was used as a model of acute systemic inflammation. In a double-blind randomized crossover study, 12 healthy volunteers received S. typhus vaccination or placebo. Phenotypic EPC populations enumerated by flow cytometry [CD34+VEGF receptor (VEGF)R-2+CD133+, CD14+VEGFR-2+Tie2+, CD45−CD34+, as a surrogate for late outgrowth EPCs, and CD34+CXCR-4+], EC-CFUs, and serum cytokine concentrations (high sensitivity C-reactive protein, IL-6, and stromal-derived factor-1) were quantified during the first 7 days. Vaccination increased circulating leukocyte (9.8 ± 0.6 vs. 5.1 ± 0.2 × 109 cells/l, P < 0.0001), serum IL-6 [0.95 (0–1.7) vs. 0 (0–0) ng/l, P = 0.016], and VEGF-A [60 (45–94) vs. 43 (21–64) pg/l, P = 0.006] concentrations at 6 h and serum high sensitivity C-reactive protein at 24 h [2.7 (1.4–3.6) vs. 0.4 (0.2–0.8) mg/l, P = 0.037]. Vaccination caused a 56.7 ± 7.6% increase in CD14+ cells at 6 h (P < 0.001) and a 22.4 ± 6.9% increase in CD34+ cells at 7 days (P = 0.04). EC-CFUs, putative vascular progenitors, and the serum stromal-derived factor-1 concentration were unaffected throughout the study period (P > 0.05 for all). In conclusion, acute systemic inflammation causes nonspecific mobilization of hematopoietic progenitor cells, although it does not selectively mobilize putative vascular progenitors. We suggest that systemic inflammation is not the primary stimulus for EPC mobilization after acute vascular injury. PMID:20382859

  17. Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite.

    PubMed

    Jones, Charles I; Han, Zhaosheng; Presley, Tennille; Varadharaj, Saradhadevi; Zweier, Jay L; Ilangovan, Govindasamy; Alevriadou, B Rita

    2008-07-01

    Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO(-)) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the "hyperoxic state" of 21% O(2), compared with more physiological O(2) tensions (Po(2)), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O(2)(*-)) generation leading to ONOO(-) formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O(2) consumption rates of bovine aortic ECs sheared (10 dyn/cm(2), 30 min) at 5%, 10%, or 21% O(2) or left static at 5% or 21% O(2). Respiration was inhibited to a greater extent when ECs were sheared at 21% O(2) than at lower Po(2) or left static at different Po(2). Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO(-) scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O(2)(*-) production was higher in ECs sheared at 21% than at 5% O(2), as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O(2) was modestly increased compared with ECs sheared at lower Po(2), suggesting that eNOS activity may be higher at 21% O(2). Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O(2)(*-) production, leads to enhanced ONOO(-) formation intramitochondrially and suppression of respiration.

  18. Endothelial cell micropatterning: Methods, effects, and applications

    PubMed Central

    Anderson, Deirdre E.J.; Hinds, Monica T.

    2012-01-01

    The effects of flow on endothelial cells have been widely examined for the ability of fluid shear stress to alter cell morphology and function; however, the effects of endothelial cell morphology without flow have only recently been observed. An increase in lithographic techniques in cell culture spurred a corresponding increase in research aiming to confine cell morphology. These studies lead to a better understanding of how morphology and cytoskeletal configuration affect the structure and function of the cells. This review examines endothelial cell micropatterning research by exploring both the many alternative methods used to alter endothelial cell morphology and the resulting changes in cellular shape and phenotype. Micropatterning induced changes in endothelial cell proliferation, apoptosis, cytoskeletal organization, mechanical properties, and cell functionality. Finally, the ways these cellular manipulation techniques have been applied to biomedical engineering research, including angiogenesis, cell migration, and tissue engineering, is discussed. PMID:21761242

  19. Hyperglycemia Does Not Affect Iron Mediated Toxicity of Cultured Endothelial and Renal Tubular Epithelial Cells: Influence of L-Carnosine.

    PubMed

    Zhang, Shiqi; Ntasis, Emmanouil; Kabtni, Sarah; van den Born, Jaap; Navis, Gerjan; Bakker, Stephan J L; Krämer, Bernhard K; Yard, Benito A; Hauske, Sibylle J

    2016-01-01

    Iron has been suggested to affect the clinical course of type 2 diabetes (T2DM) as accompanying increased intracellular iron accumulation may provide an alternative source for reactive oxygen species (ROS). Although carnosine has proven its therapeutic efficacy in rodent models of T2DM, little is known about its efficacy to protect cells from iron toxicity. We sought to assess if high glucose (HG) exposure makes cultured human umbilical vein endothelial cells (HUVECs) and renal proximal tubular epithelial cells (PTECs) more susceptible to metal induced toxicity and if this is ameliorated by L-carnosine. HUVECs and PTECs, cultured under normal glucose (5 mM, NG) or HG (30 mM), were challenged for 24 h with FeCl3. Cell viability was not impaired under HG conditions nor did HG increase susceptibility to FeCl3. HG did not change the expression of divalent metal transporter 1 (DMT1), ferroportin (IREG), and transferrin receptor protein 1 (TFRC). Irrespective of glucose concentrations L-carnosine prevented toxicity in a dose-dependent manner, only if it was present during the FeCl3 challenge. Hence our study indicates that iron induced cytotoxicity is not enhanced under HG conditions. L-Carnosine displayed a strong protective effect, most likely by chelation of iron mediated toxicity.

  20. Liver Sinusoidal Endothelial Cells.

    PubMed

    Sørensen, Karen Kristine; Simon-Santamaria, Jaione; McCuskey, Robert S; Smedsrød, Bård

    2015-10-01

    The liver sinusoidal endothelial cell (LSEC) forms the fenestrated wall of the hepatic sinusoid and functions as a control post regulating and surveying the trafficking of molecules and cells between the liver parenchyma and the blood. The cell acts as a scavenger cell responsible for removal of potential dangerous macromolecules from blood, and is increasingly acknowledged as an important player in liver immunity. This review provides an update of the major functions of the LSEC, including its role in plasma ultrafiltration and regulation of the hepatic microcirculation, scavenger functions, immune functions, and role in liver aging, as well as issues that are either undercommunicated or confusingly dealt with in the literature. These include metabolic functions, including energy metabolic interplay between the LSEC and the hepatocyte, and adequate ways of identifying and distinguishing the cells.

  1. The challenging environment on board the International Space Station affects endothelial cell function by triggering oxidative stress through thioredoxin interacting protein overexpression: the ESA-SPHINX experiment.

    PubMed

    Versari, Silvia; Longinotti, Giulia; Barenghi, Livia; Maier, Jeanette Anne Marie; Bradamante, Silvia

    2013-11-01

    Exposure to microgravity generates alterations that are similar to those involved in age-related diseases, such as cardiovascular deconditioning, bone loss, muscle atrophy, and immune response impairment. Endothelial dysfunction is the common denominator. To shed light on the underlying mechanism, we participated in the Progress 40P mission with Spaceflight of Human Umbilical Vein Endothelial Cells (HUVECs): an Integrated Experiment (SPHINX), which consisted of 12 in-flight and 12 ground-based control modules and lasted 10 d. Postflight microarray analysis revealed 1023 significantly modulated genes, the majority of which are involved in cell adhesion, oxidative phosphorylation, stress responses, cell cycle, and apoptosis. Thioredoxin-interacting protein was the most up-regulated (33-fold), heat-shock proteins 70 and 90 the most down-regulated (5.6-fold). Ion channels (TPCN1, KCNG2, KCNJ14, KCNG1, KCNT1, TRPM1, CLCN4, CLCA2), mitochondrial oxidative phosphorylation, and focal adhesion were widely affected. Cytokine detection in the culture media indicated significant increased secretion of interleukin-1α and interleukin-1β. Nitric oxide was found not modulated. Our data suggest that in cultured HUVECs, microgravity affects the same molecular machinery responsible for sensing alterations of flow and generates a prooxidative environment that activates inflammatory responses, alters endothelial behavior, and promotes senescence.

  2. The challenging environment on board the International Space Station affects endothelial cell function by triggering oxidative stress through thioredoxin interacting protein overexpression: the ESA-SPHINX experiment.

    PubMed

    Versari, Silvia; Longinotti, Giulia; Barenghi, Livia; Maier, Jeanette Anne Marie; Bradamante, Silvia

    2013-11-01

    Exposure to microgravity generates alterations that are similar to those involved in age-related diseases, such as cardiovascular deconditioning, bone loss, muscle atrophy, and immune response impairment. Endothelial dysfunction is the common denominator. To shed light on the underlying mechanism, we participated in the Progress 40P mission with Spaceflight of Human Umbilical Vein Endothelial Cells (HUVECs): an Integrated Experiment (SPHINX), which consisted of 12 in-flight and 12 ground-based control modules and lasted 10 d. Postflight microarray analysis revealed 1023 significantly modulated genes, the majority of which are involved in cell adhesion, oxidative phosphorylation, stress responses, cell cycle, and apoptosis. Thioredoxin-interacting protein was the most up-regulated (33-fold), heat-shock proteins 70 and 90 the most down-regulated (5.6-fold). Ion channels (TPCN1, KCNG2, KCNJ14, KCNG1, KCNT1, TRPM1, CLCN4, CLCA2), mitochondrial oxidative phosphorylation, and focal adhesion were widely affected. Cytokine detection in the culture media indicated significant increased secretion of interleukin-1α and interleukin-1β. Nitric oxide was found not modulated. Our data suggest that in cultured HUVECs, microgravity affects the same molecular machinery responsible for sensing alterations of flow and generates a prooxidative environment that activates inflammatory responses, alters endothelial behavior, and promotes senescence. PMID:23913861

  3. Chinese herbal medicinal ingredients affect secretion of NO, IL-10, ICAM-1 and IL-2 by endothelial cells.

    PubMed

    Hu, Yiyi; He, Kongwang; Zhu, Haodan

    2015-06-01

    The aim of this study was to investigate the anti-endotoxin effects of sinomenine, fangchinoline, stachydrine, chuanxionggzine, oxymartrine and evodiamine alkaloids commonly found in Chinese herbal medicines. Porcine endothelial cells were challenged with 1 μg LPS/ml for 3 h and then treated with one of the six alkaloids at three concentrations (1, 5 or 10 μg/ml) for a further 21 h. The supernatants of the cultures were then collected and analyzed for levels of nitric oxide (NO), interleukin (IL)-10, intercellular cell adhesion molecule-1 (ICAM-1) and IL-2 using ELISA kits. The results revealed that sinomenine, stachydrine and chuanxionggzine inhibited production of NO; stachydrine and evodiamine inhibited secretion of IL-10; sinomenine and chuanxionggzine down-regulated ICAM-1 expression; oxymartrine and evodiamine decreased production of IL-2 by the LPS-stimulated endothelial cells. Overall, the data from these studies suggested to us that these six alkaloids might effectively reduce inflammatory responses in situ via changes in the formation of these key regulatory molecules/proteins. PMID:25986990

  4. High t-PA release by neonate brain microvascular endothelial cells under glutamate exposure affects neuronal fate.

    PubMed

    Henry, Vincent Jean; Lecointre, Maryline; Laudenbach, Vincent; Ali, Carine; Macrez, Richard; Jullienne, Amandine; Berezowski, Vincent; Carmeliet, Peter; Vivien, Denis; Marret, Stéphane; Gonzalez, Bruno José; Leroux, Philippe

    2013-02-01

    Glutamate excitotoxicity is a consolidated hypothesis in neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor mediated effects. In brain microvascular endothelial cell (nBMEC) cultures from neonates, t-PA content and release upon glutamate are higher than in adult (aBMECs) cultures. Owing to the variety of t-PA substrates and receptor targets, the study was aimed at determining the putative roles of endothelial t-PA in the neonatal brain parenchyma under glutamate challenge. Basal t-PA release was 4.4 fold higher in nBMECs vs aBMECs and glutamate was 20 fold more potent to allow Evans blue vascular permeability in neonate microvessels indicating that, under noxious glutamate (50 μM) exposure, high amounts of endothelial t-PA stores may be mobilized and may access the nervous parenchyma. Culture media from nBMECS or aBMECs challenged by excitotoxic glutamate were applied to neuron cultures at DIV 11. While media from adult cells did not evoke more LDH release in neuronal cultures that under glutamate alone, media from nBMECs enhanced 2.2 fold LDH release. This effect was not observed with media from t-PA(-/-) nBMECs and was inhibited by hr-PAI-1. In Cortical slices from 10 day-old mice, hrt-PA associated with glutamate evoked neuronal necrosis in deeper (more mature) layers, an effect reversed by NMDA receptor GluN1 amino-terminal domain antibody capable of inhibiting t-PA potentiation of the receptor. In superficial layers (less mature), hrt-PA alone inhibited apoptosis, an effect reversed by the EGF receptor antagonist AG1478. Applied to immature neurons in culture (DIV5), media from nBMEC rescued 85.1% of neurons from cell death induced by serum deprivation. In cortical slices, the anti-apoptotic effect of t-PA fitted with age dependent localization of less mature neurons. These data suggest that in the immature brain, propensity of vessels to release high amounts of t-PA may not only

  5. Inhibition of XO or NOX attenuates diethylstilbestrol-induced endothelial nitric oxide deficiency without affecting its effects on LNCaP cell invasion and apoptosis.

    PubMed

    Youn, Ji-Youn; Nguyen, Andrew; Cai, Hua

    2012-10-01

    Oestrogen protects cardiovascular health partially via an up-regulation of NO• (NO radical) production. Its synthetic analogue DES (diethylstilbestrol), used as a potent androgen deprivation therapy for patients with prostate cancer, is however associated with high incidence of thromboembolic events. Exposure of BAECs (bovine aortic endothelial cells) to pharmacologically relevant dosage (12.5 μmol/l, 24 h) of DES resulted in a marked reduction in endothelial NO• bioavailability determined by ESR (electron spin resonance), while 17β-oestradiol instead increased NO• production as expected. Intriguingly, endothelial O(2)•- (superoxide anion) production was up-regulated by DES in vitro and in vivo, which was, however, attenuated by the ER (oestrogen receptor) antagonist ICI 182780, the XO (xanthine oxidase) inhibitor oxypurinol or the NOX (NADPH oxidase) inhibitor NSC23766. These agents also restored NO• production. DES alone in a cell-free system did not produce any ESR-sound O(2)•- signal. Of note, eNOS (endothelial NO synthase) mRNA and protein remained unchanged in response to DES. These results suggest that receptor-dependent activation of XO or NOX, and subsequent production of O(2)•-, mediate DES-induced NO• deficiency. This could represent a previously unrecognized mechanism that is responsible for cardiovascular complications of DES administration. Importantly, DES-induced suppression of LNCaP cell invasion and apoptosis were not affected by XO or NOX inhibitor. Therefore combinatorial therapy of DES and XO/NOX inhibitor may prove to be an innovative and useful therapeutic option in eliminating cardiovascular complications of DES, while preserving its anti-cancer effects, benefiting patients with advanced cancer who do not respond well to any other treatments but DES.

  6. Endothelial Cell Response to Fusobacterium nucleatum.

    PubMed

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

    2016-07-01

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

  7. Endothelial-regenerating cells: an expanding universe.

    PubMed

    Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

    2010-03-01

    Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow-derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

  8. Exposure to GSM RF fields does not affect calcium homeostasis in human endothelial cells, rat pheocromocytoma cells or rat hippocampal neurons.

    PubMed

    O'Connor, Rodney P; Madison, Steve D; Leveque, Philippe; Roderick, H Llewelyn; Bootman, Martin D

    2010-07-27

    In the course of modern daily life, individuals are exposed to numerous sources of electromagnetic radiation that are not present in the natural environment. The strength of the electromagnetic fields from sources such as hairdryers, computer display units and other electrical devices is modest. However, in many home and office environments, individuals can experience perpetual exposure to an "electromagnetic smog", with occasional peaks of relatively high electromagnetic field intensity. This has led to concerns that such radiation can affect health. In particular, emissions from mobile phones or mobile phone masts have been invoked as a potential source of pathological electromagnetic radiation. Previous reports have suggested that cellular calcium (Ca2+) homeostasis is affected by the types of radiofrequency fields emitted by mobile phones. In the present study, we used a high-throughput imaging platform to monitor putative changes in cellular Ca2+ during exposure of cells to 900 MHz GSM fields of differing power (specific absorption rate 0.012-2 W/Kg), thus mimicking the type of radiation emitted by current mobile phone handsets. Data from cells experiencing the 900 Mhz GSM fields were compared with data obtained from paired experiments using continuous wave fields or no field. We employed three cell types (human endothelial cells, PC-12 neuroblastoma and primary hippocampal neurons) that have previously been suggested to be sensitive to radiofrequency fields. Experiments were designed to examine putative effects of radiofrequency fields on resting Ca2+, in addition to Ca2+ signals evoked by an InsP(3)-generating agonist. Furthermore, we examined putative effects of radiofrequency field exposure on Ca2+ store emptying and store-operated Ca2+ entry following application of the Ca2+ATPase inhibitor thapsigargin. Multiple parameters (e.g., peak amplitude, integrated Ca2+ signal, recovery rates) were analysed to explore potential impact of radiofrequency field

  9. Exposure to GSM RF fields does not affect calcium homeostasis in human endothelial cells, rat pheocromocytoma cells or rat hippocampal neurons.

    PubMed

    O'Connor, Rodney P; Madison, Steve D; Leveque, Philippe; Roderick, H Llewelyn; Bootman, Martin D

    2010-01-01

    In the course of modern daily life, individuals are exposed to numerous sources of electromagnetic radiation that are not present in the natural environment. The strength of the electromagnetic fields from sources such as hairdryers, computer display units and other electrical devices is modest. However, in many home and office environments, individuals can experience perpetual exposure to an "electromagnetic smog", with occasional peaks of relatively high electromagnetic field intensity. This has led to concerns that such radiation can affect health. In particular, emissions from mobile phones or mobile phone masts have been invoked as a potential source of pathological electromagnetic radiation. Previous reports have suggested that cellular calcium (Ca2+) homeostasis is affected by the types of radiofrequency fields emitted by mobile phones. In the present study, we used a high-throughput imaging platform to monitor putative changes in cellular Ca2+ during exposure of cells to 900 MHz GSM fields of differing power (specific absorption rate 0.012-2 W/Kg), thus mimicking the type of radiation emitted by current mobile phone handsets. Data from cells experiencing the 900 Mhz GSM fields were compared with data obtained from paired experiments using continuous wave fields or no field. We employed three cell types (human endothelial cells, PC-12 neuroblastoma and primary hippocampal neurons) that have previously been suggested to be sensitive to radiofrequency fields. Experiments were designed to examine putative effects of radiofrequency fields on resting Ca2+, in addition to Ca2+ signals evoked by an InsP(3)-generating agonist. Furthermore, we examined putative effects of radiofrequency field exposure on Ca2+ store emptying and store-operated Ca2+ entry following application of the Ca2+ATPase inhibitor thapsigargin. Multiple parameters (e.g., peak amplitude, integrated Ca2+ signal, recovery rates) were analysed to explore potential impact of radiofrequency field

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

    PubMed Central

    Kono, Ken; Hiruma, Hitomi; Kobayashi, Shingo; Sato, Yoji; Tanaka, Masaru; Sawada, Rumi; Niimi, Shingo

    2016-01-01

    Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials. PMID:27348615

  11. Retinal Endothelial Cell Apoptosis Stimulates Recruitment of Endothelial Progenitor Cells

    PubMed Central

    Bhatwadekar, Ashay D.; Glenn, Josephine V.; Curtis, Tim M.; Grant, Maria B.; Stitt, Alan W.; Gardiner, Tom A.

    2013-01-01

    Purpose Bone marrow–derived endothelial progenitor cells (EPCs) contribute to vascular repair although it is uncertain how local endothelial cell apoptosis influences their reparative function. This study was conducted to determine how the presence of apoptotic bodies at sites of endothelial damage may influence participation of EPCs in retinal microvascular repair. Methods Microlesions of apoptotic cell death were created in monolayers of retinal microvascular endothelial cells (RMECs) by using the photodynamic drug verteporfin. The adhesion of early-EPCs to these lesions was studied before detachment of the apoptotic cells or after their removal from the wound site. Apoptotic bodies were fed to normal RMECs and mRNA levels for adhesion molecules were analyzed. Results Endothelial lesions where apoptotic bodies were left attached at the wound site showed a fivefold enhancement in EPC recruitment (P < 0.05) compared with lesions where the apoptotic cells had been removed. In intact RMEC monolayers exposed to apoptotic bodies, expression of ICAM, VCAM, and E-selectin was upregulated by 5- to 15-fold (P < 0.05– 0.001). EPCs showed a characteristic chemotactic response (P < 0.05) to conditioned medium obtained from apoptotic bodies, whereas analysis of the medium showed significantly increased levels of VEGF, IL-8, IL-6, and TNF-α when compared to control medium; SDF-1 remained unchanged. Conclusions The data indicate that apoptotic bodies derived from retinal capillary endothelium mediate release of proangiogenic cytokines and chemokines and induce adhesion molecule expression in a manner that facilitates EPC recruitment. PMID:19474402

  12. ENDOTHELIAL CELLS IN ALLOGRAFT REJECTION

    PubMed Central

    Al-Lamki, Rafia S.; Bradley, John R.; Pober, Jordan S.

    2008-01-01

    In organ transplantation, blood borne cells and macromolecules (e.g. antibodies) of the host immune system are brought into direct contact with the endothelial cell (EC) lining of graft vessels. In this location, graft ECs play several roles in allograft rejection, including the initiation of rejection responses by presentation of alloantigen to circulating T cells; the development of inflammation and thrombosis; and as targets of injury and agents of repair. PMID:19034000

  13. Rapamycin inhibits re-endothelialization after percutaneous coronary intervention by impeding the proliferation and migration of endothelial cells and inducing apoptosis of endothelial progenitor cells.

    PubMed

    Liu, Hai-Tao; Li, Fei; Wang, Wen-Yong; Li, Xiao-Jing; Liu, Yi-Meng; Wang, Rui-An; Guo, Wen-Yi; Wang, Hai-Chang

    2010-01-01

    Endothelial-cell function is important in the healing of damaged endothelium after percutaneous coronary artery damage. In 3 different animal models, we sought to determine whether rapamycin (sirolimus) affects the proliferation and migration of endothelial cells and endothelial progenitor cells. First, after we implanted stents in dogs, we found that re-endothelialization was impeded more by drug-eluting stents than by bare-metal stents, 30 days after percutaneous coronary intervention. Second, in vitro in rats, we found that 1-100 ng/mL of rapamycin time- and dose-dependently inhibited proliferation over 72 hr (with effects evident as early as 24 hr) and also dose-dependently induced endothelial progenitor-cell apoptosis. Finally, in vivo in rats, we observed that vascular endothelial growth factor expression was decreased after 5 days of rapamycin treatment. We conclude that rapamycin impedes re-endothelialization after drug-eluting stent implantation by inhibiting the proliferation and migration of coronary endothelial cells, inducing endothelial progenitor-cell apoptosis, and decreasing vascular endothelial growth factor expression in the circulation. PMID:20401293

  14. Evolving functions of endothelial cells in inflammation.

    PubMed

    Pober, Jordan S; Sessa, William C

    2007-10-01

    Inflammation is usually analysed from the perspective of tissue-infiltrating leukocytes. Microvascular endothelial cells at a site of inflammation are both active participants in and regulators of inflammatory processes. The properties of endothelial cells change during the transition from acute to chronic inflammation and during the transition from innate to adaptive immunity. Mediators that act on endothelial cells also act on leukocytes and vice versa. Consequently, many anti-inflammatory therapies influence the behaviour of endothelial cells and vascular therapeutics influence inflammation. This Review describes the functions performed by endothelial cells at each stage of the inflammatory process, emphasizing the principal mediators and signalling pathways involved and the therapeutic implications. PMID:17893694

  15. Culture of human endothelial cells.

    PubMed

    Gallicchio, M A

    2001-01-01

    Endothelial cells line the luminal surface of all blood vessels in the body. The endothelial surface in adult humans is composed of approximately l-6×l0(13) cells and covers an area of 1-7 m(2). Endothelium serves many functions, including fluid and solute exchange through cell contraction, provision of an antithrombogenic surface through tissue plasminogen activator (tPA) and prostacyclin release, synthesis of angiogenic factors such as adenosine, allowance of leukocyte trafficking through adhesion molecule synthesis, presentation of antigens to the immune system, maintenance of vascular tone through nitric oxide and endothelin synthesis, and metabolism of circulating molecules through the release of enzymes such as lipoprotein lipase. PMID:21340938

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

    PubMed

    Stiles, Jessica; Amaya, Clarissa; Pham, Robert; Rowntree, Rebecca K; Lacaze, Mary; Mulne, Arlynn; Bischoff, Joyce; Kokta, Victor; Boucheron, Laura E; Mitchell, Dianne C; Bryan, Brad A

    2012-10-01

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

  17. Endothelial progenitor cell dysfunction in rheumatic disease.

    PubMed

    Westerweel, Peter E; Verhaar, Marianne C

    2009-06-01

    Rheumatic disease is characterized by inflammation and endothelial dysfunction, which contribute to accelerated atherosclerosis. Circulating endothelial progenitor cells (EPCs) can restore dysfunctional endothelium and thereby protect against atherosclerotic vascular disease. The number and function of EPCs are, however, affected in rheumatic diseases such as psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, and antineutrophil cytoplasmic autoantibody-associated vasculitis. rheumatic disease is often characterized by decreased numbers, and impaired function, of EPCs, although numbers of these cells might increase during the initial years of systemic sclerosis. Pioneering studies show that EPC dysfunction might be improved with pharmacological treatment. How best to restore EPC function, and whether achieving this aim can prevent long-term cardiovascular complications in rheumatic disease, remain to be established.

  18. Progenitor endothelial cell involvement in Alzheimer's disease

    SciTech Connect

    Budinger, Thomas F.

    2003-05-01

    There is compelling evidence that endothelial cells of the brain and periphery are dysfunctional in Alzheimer's Disease. There is evidence for a fundamental defect in, or abnormal aging of, endothelial progenitor cells in atherosclerosis. The possibility that endothelial cell defects are a primary cause for Alzheimer's Disease or other dementias can be researched by molecular and cell biology studies as well as cell trafficking studies using recently demonstrated molecular imaging methods. The evidence for abnormal endothelial function and the methods to explore this hypothesis are presented.

  19. Autocrine VEGF Isoforms Differentially Regulate Endothelial Cell Behavior

    PubMed Central

    Yamamoto, Hideki; Rundqvist, Helene; Branco, Cristina; Johnson, Randall S.

    2016-01-01

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

  20. Endothelial cells enhance migration of meniscus cells

    PubMed Central

    Yuan, Xiaoning; Eng, George M.; Arkonac, Derya E.; Chao, Pen-hsiu Grace; Vunjak-Novakovic, Gordana

    2014-01-01

    Objective To study the interactions between vascular endothelial cells and meniscal fibrochondrocytes from the inner avascular and outer vascular regions of the meniscus, and identify angiogenic factors that enhance cell migration and integrative repair. Methods Bovine meniscal fibrochondrocytes (bMFCs) from the inner and outer regions of meniscus were cultured for seven days with and without human umbilical vein endothelial cells (HUVECs) in a micropatterned three-dimensional hydrogel system for cell migration. Angiogenic factors secreted by HUVECs were probed for their role in paracrine mechanisms governing bMFC migration, and applied to a full-thickness defect model of meniscal repair in explants from the inner and outer regions over four weeks. Results Endothelial cells enhanced migration of inner and outer bMFCs in the micropatterned system via endothelin-1 (ET-1) signaling. Supplementation of ET-1 significantly enhanced integration strength of full-thickness defects in inner and outer explants, and cell migration at the macro-scale, compared to controls without ET-1 treatment. Conclusion We report for the first time that bMFCs from both the avascular and vascular regions respond to the presence of endothelial cells with increased migration. Paracrine signaling by endothelial cells regulates the bMFCs differentially by region, but we identify ET-1 as an angiogenic factor that stimulates migration of inner and outer cells at the micro-scale, and integrative repair of inner and outer explants at the macro-scale. These findings reveal the regional interactions between vasculature and MFCs, and suggest ET-1 as a potential new treatment modality for avascular meniscal injuries, in order to prevent the development of osteoarthritis. PMID:25307081

  1. ADP Signaling in Vascular Endothelial Cells

    PubMed Central

    Hess, Connie Ng; Kou, Ruqin; Johnson, Rosalyn P.; Li, Gordon K.; Michel, Thomas

    2009-01-01

    ADP responses underlie therapeutic approaches to many cardiovascular diseases, and ADP receptor antagonists are in widespread clinical use. The role of ADP in platelet biology has been extensively studied, yet ADP signaling pathways in endothelial cells remain incompletely understood. We found that ADP promoted phosphorylation of the endothelial isoform of nitric-oxide synthase (eNOS) at Ser1179 and Ser635 and dephosphorylation at Ser116 in cultured endothelial cells. Although eNOS activity was stimulated by both ADP and ATP, only ADP signaling was significantly inhibited by the P2Y1 receptor antagonist MRS 2179 or by knockdown of P2Y1 using small interfering RNA (siRNA). ADP activated the small GTPase Rac1 and promoted endothelial cell migration. siRNA-mediated knockdown of Rac1 blocked ADP-dependent eNOS Ser1179 and Ser635 phosphorylation, as well as eNOS activation. We analyzed pathways known to regulate eNOS, including phosphoinositide 3-kinase/Akt, ERK1/2, Src, and calcium/calmodulin-dependent kinase kinase-β (CaMKKβ) using the inhibitors wortmannin, PD98059, PP2, and STO-609, respectively. None of these inhibitors altered ADP-modulated eNOS phosphorylation. In contrast, siRNA-mediated knockdown of AMP-activated protein kinase (AMPK) inhibited ADP-dependent eNOS Ser635 phosphorylation and eNOS activity but did not affect eNOS Ser1179 phosphorylation. Importantly, the AMPK enzyme inhibitor compound C had no effect on ADP-stimulated eNOS activity, despite completely blocking AMPK activity. CaMKKβ knockdown suppressed ADP-stimulated eNOS activity, yet inhibition of CaMKKβ kinase activity using STO-609 failed to affect eNOS activation by ADP. These data suggest that the expression, but not the kinase activity, of AMPK and CaMKKβ is necessary for ADP signaling to eNOS. PMID:19783664

  2. 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

  3. Molecular mechanisms that control endothelial cell contacts.

    PubMed

    Vestweber, D

    2000-02-01

    Endothelial cell contacts control the permeability of the blood vessel wall. This allows the endothelium to form a barrier for solutes, macromolecules, and leukocytes between the vessel lumen and the interstitial space. Loss of this barrier function in pathophysiological situations can lead to extracellular oedema. The ability of leukocytes to enter tissue at sites of inflammation is dependent on molecular mechanisms that allow leukocytes to adhere to the endothelium and to migrate through the endothelial cell layer and the underlying basal lamina. It is a commonly accepted working hypothesis that inter-endothelial cell contacts are actively opened and closed during this process. Angiogenesis is another important process that requires well-controlled regulation of inter-endothelial cell contacts. The formation of new blood vessels by sprouting from pre-existing vessels depends on the loosening of established endothelial cell contacts and the migration of endothelial cells that form the outgrowing sprouts. This review focuses on the molecular composition of endothelial cell surface proteins and proteins of the cytoskeletal undercoat of the plasma membrane at sites of inter-endothelial cell contacts and discusses the current knowledge about the potential role of such molecules in the regulation of endothelial cell contacts. PMID:10685062

  4. Diesel exhaust particles and endothelial cells dysfunction: An update.

    PubMed

    Lawal, A O; Davids, L M; Marnewick, J L

    2016-04-01

    Epidemiological studies have shown a consistent positive correlation between exposure to particulate matter (PM) and increased mortality largely due to increased rates of cardiovascular morbidity and mortality. Diesel exhaust particles (DEPs) are major constituents of atmospheric PM and have been shown to cause disruption of the endothelial cell monolayer integrity, thereby affecting organ functions. Endothelial cells are very active metabolic components of biological tissue that performs a number of important physiological functions. Therefore, anything that compromises the integrity and functions of the endothelium will lead to organ dysfunction and disease. This review focuses on scientific evidence that link DEP exposure to endothelial cell dysfunction in various pathophysiological conditions affecting the cardiovascular system. The various mechanisms involved in the DEP-induced endothelial cell dysfunction are also addressed together with the preventive and therapeutic approaches to overcoming these challenges.

  5. Protective effects of vascular endothelial growth factor in cultured brain endothelial cells against hypoglycemia.

    PubMed

    Zhao, Fei; Deng, Jiangshan; Yu, Xiaoyan; Li, Dawei; Shi, Hong; Zhao, Yuwu

    2015-08-01

    Hypoglycemia is a common and serious problem among patients with type 1 diabetes receiving treatment with insulin. Clinical studies have demonstrated that hypoglycemic edema is involved in the initiation of hypoglycemic brain damage. However, the mechanisms of this edema are poorly understood. Vascular endothelial growth factor (VEGF), a potent regulator of blood vessel function, has been observed an important candidate hormone induced by hypoglycemia to protect neurons by restoring plasma glucose. Whether VEGF has a protective effect against hypoglycemia-induced damage in brain endothelial cells is still unknown. To investigate the effects of hypoglycemia on cerebral microvascular endothelial cells and assess the protective effect of exogenous VEGF on endothelial cells during hypoglycemia, confluent monolayers of the brain endothelial cell line bEnd.3 were treated with normal (5.5 mM glucose), hypoglycemic (0, 0.5, 1 mM glucose) medium or hypoglycemic medium in the presence of VEGF. The results clearly showed that hypoglycemia significantly downregulated the expression of claudin-5 in bEnd.3 cells, without affecting ZO-1 and occludin expression and distribution. Besides, transendothelial permeability significantly increased under hypoglycemic conditions compared to that under control conditions. Moreover, the hypoglycemic medium in presence of VEGF decreased endothelial permeability via the inhibition of claudin-5 degradation and improved hypoglycemia-induced cell toxicity. Furthermore, Glucose transporter-1 (Glut-1) and apoptosis regulator Bcl-2 expression were significantly upregulated. Taken together, hypoglycemia can significantly increase paraendocellular permeability by downregulating claudin-5 expression. We further showed that VEGF protected brain endothelial cells against hypoglycemia by enhancing glucose passage, reducing endothelial cell death, and ameliorating paraendocellular permeability.

  6. Variations in mass transfer to single endothelial cells.

    PubMed

    Van Doormaal, Mark A; Zhang, Ji; Wada, Shigeo; Shaw, James E; Won, Doyon; Cybulsky, Myron I; Yip, Chris M; Ethier, C Ross

    2009-06-01

    Mass transfer between flowing blood and arterial mural cells (including vascular endothelial cells) may play an important role in atherogenesis. Endothelial cells are known to have an apical surface topography that is not flat, and hence mass transfer patterns to individual endothelial cells are likely affected by the local cellular topography. The purpose of this paper is to investigate the relationship between vascular endothelial cell surface topography and cellular level mass transfer. Confluent porcine endothelial monolayers were cultured under both shear and static conditions and atomic force microscopy was used to measure endothelial cell topography. Using finite element methods and the measured cell topography, flow and concentration fields were calculated for a typical, small, blood-borne solute. A relative Sherwood number was defined as the difference between the computed Sherwood number and that predicted by the Leveque solution for mass transfer over a flat surface: this eliminates the effects of axial location on mass transfer efficiency. The average intracellular relative Sherwood number range was found to be dependent on cell height and not dependent on cell elongation due to shear stress in culture. The mass flux to individual cells reached a maximum at the highest point on the endothelial cell surface, typically corresponding to the nucleus of the cell. Therefore, for small receptor-mediated solutes, increased solute uptake efficiency can be achieved by concentrating receptors near the nucleus. The main conclusion of the work is that although the rate of mass transfer varies greatly over an individual cell, the average mass transfer rate to a cell is close to that predicted for a flat cell. In comparison to other hemodynamic factors, the topography of endothelial cells therefore seems to have little effect on mass transfer rates and is likely physiologically insignificant.

  7. Schwann cells promote endothelial cell migration

    PubMed Central

    Ramos, Tiago; Ahmed, Maqsood; Wieringa, Paul; Moroni, Lorenzo

    2015-01-01

    Directed cell migration is a crucial orchestrated process in embryonic development, wound healing, and immune response. The underlying substrate can provide physical and/or chemical cues that promote directed cell migration. Here, using electrospinning we developed substrates of aligned poly(lactic-co-glycolic acid) nanofibres to study the influence of glial cells on endothelial cells (ECs) in a 3-dimensional (3D) co-culture model. ECs build blood vessels and regulate their plasticity in coordination with neurons. Likewise, neurons construct nerves and regulate their circuits in coordination with ECs. In our model, the neuro-vascular cross-talk was assessed using a direct co-culture model of human umbilical vein endothelial cells (HUVECs) and rat Schwann cells (rSCs). The effect of rSCs on ECs behavior was demonstrated by earlier and higher velocity values and genetic expression profiles different of those of HUVECs when seeded alone. We observed 2 different gene expression trends in the co-culture models: (i) a later gene expression of angiogenic factors, such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and (ii) an higher gene expression of genes involved in actin filaments rearrangement, such as focal adhesion kinase (FAK), Mitogen-activated protein kinase-activated protein kinase 13 (MAPKAPK13), Vinculin (VCL), and Profilin (PROF). These results suggested that the higher ECs migration is mainly due to proteins involved in the actin filaments rearrangement and in the directed cell migration rather than the effect of angiogenic factors. This co-culture model provides an approach to enlighten the neurovascular interactions, with particular focus on endothelial cell migration. PMID:26491999

  8. Endothelial cells promote neural stem cell proliferation and differentiation associated with VEGF activated Notch and Pten signaling.

    PubMed

    Sun, Jinqiao; Zhou, Wenhao; Ma, Duan; Yang, Yi

    2010-09-01

    To investigate whether and how endothelial cells affect neurogenesis, we established a system to co-culture endothelial cells and brain slices of neonatal rat and observed how subventricular zone cells differentiate in the presence of endothelial cells. In the presence of endothelial cells, neural stem cells increased in number, as did differentiated neurons and glia. The augmentation of neurogenesis was reversed by diminishing vascular endothelial growth factor (VEGF) expression in endothelial cells with RNA interference (RNAi). Microarray analysis indicated that expression levels of 112 genes were significantly altered by co-culture and that expression of 81 of the 112 genes recovered to normal levels following RNAi of VEGF in endothelial cells. Pathway mapping showed an enrichment of genes in the Notch and Pten pathways. These data indicate that endothelial cells promote neural stem cell proliferation and differentiation associated with VEGF, possibly by activating the Notch and Pten pathways.

  9. Estetrol Modulates Endothelial Nitric Oxide Synthesis in Human Endothelial Cells

    PubMed Central

    Montt-Guevara, Maria Magdalena; Giretti, Maria Silvia; Russo, Eleonora; Giannini, Andrea; Mannella, Paolo; Genazzani, Andrea Riccardo; Genazzani, Alessandro David; Simoncini, Tommaso

    2015-01-01

    Estetrol (E4) is a natural human estrogen that is present at high concentrations during pregnancy. E4 has been reported to act as an endogenous estrogen receptor modulator, exerting estrogenic actions on the endometrium or the central nervous system but presenting antagonistic effects on the breast. Due to these characteristics, E4 is currently being developed for a number of clinical applications, including contraception and menopausal hormone therapy. Endothelial nitric oxide (NO) is a key player for vascular function and disease during pregnancy and throughout aging in women. Endothelial NO is an established target of estrogens that enhance its formation in human endothelial cells. We here addressed the effects of E4 on the activity and expression of the endothelial nitric oxide synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVEC). E4 stimulated the activation of eNOS and NO secretion in HUVEC. E4 was significantly less effective compared to E2, and a peculiar concentration-dependent effect was found, with higher amounts of E4 being less effective than lower concentrations. When E2 was combined with E4, an interesting pattern was noted. E4 antagonized NO synthesis induced by pregnancy-like E2 concentrations. However, E4 did not impede the modest induction of NO synthesis associated with postmenopausal-like E2 levels. These results support the hypothesis that E4 may be a regulator of NO synthesis in endothelial cells and raise questions on its peculiar signaling in this context. Our results may be useful to interpret the role of E4 during human pregnancy and possibly to help develop this interesting steroid for clinical use. PMID:26257704

  10. Estetrol Modulates Endothelial Nitric Oxide Synthesis in Human Endothelial Cells.

    PubMed

    Montt-Guevara, Maria Magdalena; Giretti, Maria Silvia; Russo, Eleonora; Giannini, Andrea; Mannella, Paolo; Genazzani, Andrea Riccardo; Genazzani, Alessandro David; Simoncini, Tommaso

    2015-01-01

    Estetrol (E4) is a natural human estrogen that is present at high concentrations during pregnancy. E4 has been reported to act as an endogenous estrogen receptor modulator, exerting estrogenic actions on the endometrium or the central nervous system but presenting antagonistic effects on the breast. Due to these characteristics, E4 is currently being developed for a number of clinical applications, including contraception and menopausal hormone therapy. Endothelial nitric oxide (NO) is a key player for vascular function and disease during pregnancy and throughout aging in women. Endothelial NO is an established target of estrogens that enhance its formation in human endothelial cells. We here addressed the effects of E4 on the activity and expression of the endothelial nitric oxide synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVEC). E4 stimulated the activation of eNOS and NO secretion in HUVEC. E4 was significantly less effective compared to E2, and a peculiar concentration-dependent effect was found, with higher amounts of E4 being less effective than lower concentrations. When E2 was combined with E4, an interesting pattern was noted. E4 antagonized NO synthesis induced by pregnancy-like E2 concentrations. However, E4 did not impede the modest induction of NO synthesis associated with postmenopausal-like E2 levels. These results support the hypothesis that E4 may be a regulator of NO synthesis in endothelial cells and raise questions on its peculiar signaling in this context. Our results may be useful to interpret the role of E4 during human pregnancy and possibly to help develop this interesting steroid for clinical use. PMID:26257704

  11. Scleroderma fibroblasts show increased responsiveness to endothelial cell-derived IL-1 and bFGF.

    PubMed

    Denton, C P; Xu, S; Black, C M; Pearson, J D

    1997-03-01

    Fibroblasts cultured from lesional skin in scleroderma (systemic sclerosis) demonstrate an activated phenotype that may be important in pathogenesis. Endothelial cell-derived cytokines can modulate fibroblast properties, and endothelial cell changes occur early in scleroderma. Thus, endothelial cell and fibroblast dysfunction may be linked through the paracrine activity of soluble endothelial cell products. We have explored endothelial cell-fibroblast interactions in vitro by investigating the modulation of scleroderma and control fibroblast properties by endothelial cell-conditioned medium (EC-CM). EC-CM caused a concentration-dependent stimulation of fibroblast DNA and protein synthesis and upregulation of cell surface ICAM-1 expression. Scleroderma fibroblasts showed consistently greater responses than control cells. Medium conditioned by mechanically wounded endothelial cells had a greater effect than that from resting endothelial cells. Pre-incubation of EC-CM with anti-bFGF significantly reduced the promotion of fibroblast thymidine incorporation but did not affect endothelial cell-induced leucine incorporation. Conversely, anti-IL-1 antibodies abrogated EC-CM-induced leucine incorporation and ICAM-1 expression but did not diminish thymidine incorporation. Recombinant bFGF or IL-1 modulated fibroblast properties similarly. These data demonstrate that endothelial cell-derived IL-1 and bFGF modulate fibroblast properties independently and that lesional scleroderma strains are more responsive than control fibroblasts to endothelial cell-induced modulation, which supports the hypothesis that altered endothelial cell-fibroblast communication may be involved in the pathogenesis of scleroderma.

  12. Endothelial cells in health and disease.

    PubMed

    Eckers, Anna; Haendeler, Judith

    2015-05-10

    According to the World Health Organization, from 2014, cardiovascular diseases (CVD) are the number one cause of death worldwide. One of the key players in maintaining proper cardiovascular function is the endothelium, the inner layer of all blood vessels. This monolayer of cells on one hand serves as a barrier between blood and the surrounding tissue and on the other hand regulates many aspects of vessel function. Therefore, it is not surprising that interventions reducing the risk for CVD improve endothelial function. There is a clear correlation between endothelial dysfunction, in which the endothelial homeostasis is disturbed, and the development and progression of many CVD. Thus, the development of diagnostic tools for early detection of disturbances in endothelial homeostasis or interventions aimed at improving endothelial function after insults require a comprehensive knowledge not only of the cellular reactions to the positive or negative stimuli but also of the molecular mechanisms relaying these responses. Thus, this Forum on "endothelial cells in health and disease" focuses on key molecules and processes intimately involved in endothelial cell function and covers areas from endothelial nitric oxide synthase-dependent processes, over the group of Phox-Bem1 domain proteins, cytochrome P450 epoxygenase-derived metabolites, and pre-mRNA splicing to microRNAs. Finally, one has to conclude that keeping endothelial homeostasis is the central key for a healthy long life of the human individual.

  13. Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

    SciTech Connect

    Vigl, Benjamin; Zgraggen, Claudia; Rehman, Nadia; Banziger-Tobler, Nadia E.; Detmar, Michael; Halin, Cornelia

    2009-01-15

    Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity.

  14. Circulating and tissue resident endothelial progenitor cells.

    PubMed

    Basile, David P; Yoder, Mervin C

    2014-01-01

    Progenitor cells for the endothelial lineage have been widely investigated for more than a decade, but continue to be controversial since no unique identifying marker has yet been identified. This review will begin with a discussion of the basic tenets originally proposed for proof that a cell displays properties of an endothelial progenitor cell. We then provide an overview of the methods for putative endothelial progenitor cell derivation, expansion, and enumeration. This discussion includes consideration of cells that are present in the circulation as well as cells resident in the vascular endothelial intima. Finally, we provide some suggested changes in nomenclature that would greatly clarify and demystify the cellular elements involved in vascular repair.

  15. Melatonin affects the dynamic steady-state equilibrium of estrogen sulfates in human umbilical vein endothelial cells by regulating the balance between estrogen sulfatase and sulfotransferase.

    PubMed

    González, Alicia; Martínez-Campa, Carlos; Alonso-González, Carolina; Cos, Samuel

    2015-12-01

    Melatonin is known to reduce the growth of endocrine-responsive breast cancers by interacting with estrogen signaling pathways. Estrogens play an important role in breast cancer, but also in various types of tissues, including vascular tissue. Estrogen sulfatase (STS) converts inactive estrogen sulfates into active estrogens, whereas estrogen sulfotransferase (EST) sulfonates estrogens to estrogen sulfates. Therefore, STS and EST are considered to be involved in the regulation of local estrogen levels in hormone‑dependent tumors and in non-pathologic tissues, such as those of the vascular system. Estrogens have a major impact on the vasculature, influencing vascular function, the expression of adhesion proteins, angiogenesis and the inflammatory state. In this study, we investigated the status of STS and EST in human umbilical vein endothelial cells (HUVECs) and the modulatory effects of melatonin. Both STS and EST were highly expressed in the HUVECs. The enzymatic activity correlated with the expression levels in these cells. Our findings also demonstrated that melatonin, at physiological concentrations, modulated the synthesis and transformation of biologically active estrogens in HUVECs through the inhibition of STS activity and expression, and the stimulation of EST activity and expression. Since melatonin decreased the STS levels and increased the EST levels, it modified the dynamic steady‑state equilibrium of estrogen sulfates by increasing the inactive estrogen levels and decreasing the active estrogen levels. Therefore, melatonin may modulate the known different biological actions of estrogens in endothelial cells, as well as in estrogen-dependent tumors and non-pathologic tissues. PMID:26458420

  16. Mitochondria, endothelial cell function, and vascular diseases

    PubMed Central

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

    2014-01-01

    Mitochondria are perhaps the most sophisticated and dynamic responsive sensing systems in eukaryotic cells. The role of mitochondria goes beyond their capacity to create molecular fuel and includes the generation of reactive oxygen species, the regulation of calcium, and the activation of cell death. In endothelial cells, mitochondria have a profound impact on cellular function under both healthy and diseased conditions. In this review, we summarize the basic functions of mitochondria in endothelial cells and discuss the roles of mitochondria in endothelial dysfunction and vascular diseases, including atherosclerosis, diabetic vascular dysfunction, pulmonary artery hypertension, and hypertension. Finally, the potential therapeutic strategies to improve mitochondrial function in endothelial cells and vascular diseases are also discussed, with a focus on mitochondrial-targeted antioxidants and calorie restriction. PMID:24834056

  17. Endothelial progenitor cells in cardiovascular diseases.

    PubMed

    Lee, Poay Sian Sabrina; Poh, Kian Keong

    2014-07-26

    Endothelial dysfunction has been associated with the development of atherosclerosis and cardiovascular diseases. Adult endothelial progenitor cells (EPCs) are derived from hematopoietic stem cells and are capable of forming new blood vessels through a process of vasculogenesis. There are studies which report correlations between circulating EPCs and cardiovascular risk factors. There are also studies on how pharmacotherapies may influence levels of circulating EPCs. In this review, we discuss the potential role of endothelial progenitor cells as both diagnostic and prognostic biomarkers. In addition, we look at the interaction between cardiovascular pharmacotherapies and endothelial progenitor cells. We also discuss how EPCs can be used directly and indirectly as a therapeutic agent. Finally, we evaluate the challenges facing EPC research and how these may be overcome.

  18. Glioma-associated endothelial cells show evidence of replicative senescence.

    PubMed

    Charalambous, Christiana; Virrey, Jenilyn; Kardosh, Adel; Jabbour, Mark N; Qazi-Abdullah, Lubna; Pen, Ligaya; Zidovetzki, Raphael; Schönthal, Axel H; Chen, Thomas C; Hofman, Florence M

    2007-04-01

    The innately programmed process of replicative senescence has been studied extensively with respect to cancer, but primarily from the perspective of tumor cells overcoming this stringent innate barrier and acquiring the capacity for unlimited proliferation. In this study, we focus on the potential role of replicative senescence affecting the non-transformed endothelial cells of the blood vessels within the tumor microenvironment. Based on the well-documented aberrant structural and functional features of blood vessels within solid tumors, we hypothesized that tumor-derived factors may lead to premature replicative senescence in tumor-associated brain endothelial cells (TuBEC). We show here that glioma tissue, but not normal brain tissue, contains cells that express the signature of replicative senescence, senescence-associated beta-galactosidase (SA-beta-gal), on CD31-positive endothelial cells. Primary cultures of human TuBEC stain for SA-beta-gal and exhibit characteristics of replicative senescence, including increased levels of the cell cycle inhibitors p21 and p27, increased resistance to cytotoxic drugs, increased growth factor production, and inability to proliferate. These data provide the first demonstration that tumor-derived brain endothelial cells may have reached an end-stage of differentiation known as replicative senescence and underscore the need for anti-angiogenic therapies to target this unique tumor-associated endothelial cell population.

  19. Glioma-associated endothelial cells show evidence of replicative senescence

    SciTech Connect

    Charalambous, Christiana; Virrey, Jenilyn; Kardosh, Adel; Jabbour, Mark N.; Qazi-Abdullah, Lubna; Pen, Ligaya; Zidovetzki, Raphael; Schoenthal, Axel H.; Chen, Thomas C.; Hofman, Florence M. . E-mail: hofman@usc.edu

    2007-04-01

    The innately programmed process of replicative senescence has been studied extensively with respect to cancer, but primarily from the perspective of tumor cells overcoming this stringent innate barrier and acquiring the capacity for unlimited proliferation. In this study, we focus on the potential role of replicative senescence affecting the non-transformed endothelial cells of the blood vessels within the tumor microenvironment. Based on the well-documented aberrant structural and functional features of blood vessels within solid tumors, we hypothesized that tumor-derived factors may lead to premature replicative senescence in tumor-associated brain endothelial cells (TuBEC). We show here that glioma tissue, but not normal brain tissue, contains cells that express the signature of replicative senescence, senescence-associated {beta}-galactosidase (SA-{beta}-gal), on CD31-positive endothelial cells. Primary cultures of human TuBEC stain for SA-{beta}-gal and exhibit characteristics of replicative senescence, including increased levels of the cell cycle inhibitors p21 and p27, increased resistance to cytotoxic drugs, increased growth factor production, and inability to proliferate. These data provide the first demonstration that tumor-derived brain endothelial cells may have reached an end-stage of differentiation known as replicative senescence and underscore the need for anti-angiogenic therapies to target this unique tumor-associated endothelial cell population.

  20. MicroRNAs in Hyperglycemia Induced Endothelial Cell Dysfunction

    PubMed Central

    Silambarasan, Maskomani; Tan, Jun Rong; Karolina, Dwi Setyowati; Armugam, Arunmozhiarasi; Kaur, Charanjit; Jeyaseelan, Kandiah

    2016-01-01

    Hyperglycemia is closely associated with prediabetes and Type 2 Diabetes Mellitus. Hyperglycemia increases the risk of vascular complications such as diabetic retinopathy, diabetic nephropathy, peripheral vascular disease and cerebro/cardiovascular diseases. Under hyperglycemic conditions, the endothelial cells become dysfunctional. In this study, we investigated the miRNA expression changes in human umbilical vein endothelial cells exposed to different glucose concentrations (5, 10, 25 and 40 mM glucose) and at various time intervals (6, 12, 24 and 48 h). miRNA microarray analyses showed that there is a correlation between hyperglycemia induced endothelial dysfunction and miRNA expression. In silico pathways analyses on the altered miRNA expression showed that the majority of the affected biological pathways appeared to be associated to endothelial cell dysfunction and apoptosis. We found the expression of ten miRNAs (miR-26a-5p, -26b-5p, 29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -140-5p, -192-5p, -221-3p and -320a) to increase gradually with increasing concentration of glucose. These miRNAs were also found to be involved in endothelial dysfunction. At least seven of them, miR-29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -221-3p, -320a and -192-5p, can be correlated to endothelial cell apoptosis. PMID:27070575

  1. MicroRNAs in Hyperglycemia Induced Endothelial Cell Dysfunction.

    PubMed

    Silambarasan, Maskomani; Tan, Jun Rong; Karolina, Dwi Setyowati; Armugam, Arunmozhiarasi; Kaur, Charanjit; Jeyaseelan, Kandiah

    2016-01-01

    Hyperglycemia is closely associated with prediabetes and Type 2 Diabetes Mellitus. Hyperglycemia increases the risk of vascular complications such as diabetic retinopathy, diabetic nephropathy, peripheral vascular disease and cerebro/cardiovascular diseases. Under hyperglycemic conditions, the endothelial cells become dysfunctional. In this study, we investigated the miRNA expression changes in human umbilical vein endothelial cells exposed to different glucose concentrations (5, 10, 25 and 40 mM glucose) and at various time intervals (6, 12, 24 and 48 h). miRNA microarray analyses showed that there is a correlation between hyperglycemia induced endothelial dysfunction and miRNA expression. In silico pathways analyses on the altered miRNA expression showed that the majority of the affected biological pathways appeared to be associated to endothelial cell dysfunction and apoptosis. We found the expression of ten miRNAs (miR-26a-5p, -26b-5p, 29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -140-5p, -192-5p, -221-3p and -320a) to increase gradually with increasing concentration of glucose. These miRNAs were also found to be involved in endothelial dysfunction. At least seven of them, miR-29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -221-3p, -320a and -192-5p, can be correlated to endothelial cell apoptosis. PMID:27070575

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

    PubMed

    Hempel, Casper; Hyttel, Poul; Kurtzhals, Jørgen A L

    2014-07-01

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

  3. Isolation and culture of pulmonary endothelial cells.

    PubMed

    Ryan, U S

    1984-06-01

    Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan. PMID:6090112

  4. LGN Directs Interphase Endothelial Cell Behavior via the Microtubule Network

    PubMed Central

    Wright, Catherine E.; Kushner, Erich J.; Du, Quansheng; Bautch, Victoria L.

    2015-01-01

    Angiogenic sprouts require coordination of endothelial cell (EC) behaviors as they extend and branch. Microtubules influence behaviors such as cell migration and cell-cell interactions via regulated growth and shrinkage. Here we investigated the role of the mitotic polarity protein LGN in EC behaviors and sprouting angiogenesis. Surprisingly, reduced levels of LGN did not affect oriented division of EC within a sprout, but knockdown perturbed overall sprouting. At the cell level, LGN knockdown compromised cell-cell adhesion and migration. EC with reduced LGN levels also showed enhanced growth and stabilization of microtubules that correlated with perturbed migration. These results fit a model whereby LGN influences interphase microtubule dynamics in endothelial cells to regulate migration, cell adhesion, and sprout extension, and reveal a novel non-mitotic role for LGN in sprouting angiogenesis. PMID:26398908

  5. Glassy Dynamics, Cell Mechanics and Endothelial Permeability

    PubMed Central

    Hardin, Corey; Rajendran, Kavitha; Manomohan, Greeshma; Tambe, Dhananjay T.; Butler, James P.; Fredberg, Jeffrey J.; Martinelli, Roberta; Carman, Christopher V.; Krishnan, Ramaswamy

    2013-01-01

    A key feature of all inflammatory processes is disruption of the vascular endothelial barrier. Such disruption is initiated in part through active contraction of the cytoskeleton of the endothelial cell (EC). Because contractile forces are propagated from cell to cell across a great many cell-cell junctions, this contractile process is strongly cooperative and highly nonlocal. We show here that the characteristic length scale of propagation is modulated by agonists and antagonists that impact permeability of the endothelial barrier. In the presence of agonists including thrombin, histamine, and H202, force correlation length increases, whereas in the presence of antagonists including sphingosine-1-phosphate, hepatocyte growth factor, and the rho kinase inhibitor, Y27632, force correlation length decreases. Intercellular force chains and force clusters are also evident, both of which are reminiscent of soft glassy materials approaching a glass transition. PMID:23638866

  6. Endothelial cells regulate neural crest and second heart field morphogenesis

    PubMed Central

    Milgrom-Hoffman, Michal; Michailovici, Inbal; Ferrara, Napoleone; Zelzer, Elazar; Tzahor, Eldad

    2014-01-01

    ABSTRACT Cardiac and craniofacial developmental programs are intricately linked during early embryogenesis, which is also reflected by a high frequency of birth defects affecting both regions. The molecular nature of the crosstalk between mesoderm and neural crest progenitors and the involvement of endothelial cells within the cardio–craniofacial field are largely unclear. Here we show in the mouse that genetic ablation of vascular endothelial growth factor receptor 2 (Flk1) in the mesoderm results in early embryonic lethality, severe deformation of the cardio–craniofacial field, lack of endothelial cells and a poorly formed vascular system. We provide evidence that endothelial cells are required for migration and survival of cranial neural crest cells and consequently for the deployment of second heart field progenitors into the cardiac outflow tract. Insights into the molecular mechanisms reveal marked reduction in Transforming growth factor beta 1 (Tgfb1) along with changes in the extracellular matrix (ECM) composition. Our collective findings in both mouse and avian models suggest that endothelial cells coordinate cardio–craniofacial morphogenesis, in part via a conserved signaling circuit regulating ECM remodeling by Tgfb1. PMID:24996922

  7. Rapid flow-induced responses in endothelial cells

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  8. Endothelial progenitor cells in diabetic foot syndrome.

    PubMed

    Drela, Ewelina; Stankowska, Katarzyna; Kulwas, Arleta; Rość, Danuta

    2012-01-01

    In the late 20th century endothelial progenitor cells (EPCs) were discovered and identified as cells capable of differentiating into endothelial cells. Antigens characteristic of endothelial cells and hematopoietic cells are located on their surface. EPCs can proliferate, adhere, migrate and have the specific ability to form vascular structure, and they have a wide range of roles: They participate in maintaining hemostasis, and play an important part in the processes of vasculogenesis and angiogenesis. They are sources of angiogenic factors, especially vascular endothelial growth factor (VEGF). EPCs exist in bone marrow, from which they are recruited into circulation in response to specific stimuli. Tissue ischemia is thought to be the strongest inductor of EPC mobilization. Local ischemia accompanies many pathological states, including diabetic foot syndrome (DFS). Impaired angiogenesis--in which EPCs participate--is typical of DFS. An analysis of the available literature indicates that in diabetic patients the number of EPCs declines and their functioning is impaired. Endothelial progenitor cells are crucial to vasculogenesis and angiogenesis during ischemic neovascularization. The pathomechanisms underlying impaired angiogenesis in patients with DFS is complicated, but the discovery of EPCs has shed new light on the pathogenesis of many diseases, including diabetes foot syndrome.

  9. 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.

  10. Endothelial progenitor cells in hematologic malignancies

    PubMed Central

    Saulle, Ernestina; Castelli, Germana; Pelosi, Elvira

    2016-01-01

    Studies carried out in the last years have improved the understanding of the cellular and molecular mechanisms controlling angiogenesis during adult life in normal and pathological conditions. Some of these studies have led to the identification of some progenitor cells that sustain angiogenesis through indirect, paracrine mechanisms (hematopoietic angiogenic cells) and through direct mechanisms, i.e., through their capacity to generate a progeny of phenotypically and functionally competent endothelial cells [endothelial colony forming cells (ECFCs)]. The contribution of these progenitors to angiogenetic processes under physiological and pathological conditions is intensively investigated. Angiogenetic mechanisms are stimulated in various hematological malignancies, including chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndromes and multiple myeloma, resulting in an increased angiogenesis that contributes to disease progression. In some of these conditions there is preliminary evidence that some endothelial cells could derive from the malignant clone, thus leading to the speculation that the leukemic cell derives from the malignant transformation of a hemangioblastic progenitor, i.e., of a cell capable of differentiation to the hematopoietic and to the endothelial cell lineages. Our understanding of the mechanisms underlying increased angiogenesis in these malignancies not only contributed to a better knowledge of the mechanisms responsible for tumor progression, but also offered the way for the discovery of new therapeutic targets. PMID:27583252

  11. Endothelial progenitor cells in hematologic malignancies.

    PubMed

    Testa, Ugo; Saulle, Ernestina; Castelli, Germana; Pelosi, Elvira

    2016-01-01

    Studies carried out in the last years have improved the understanding of the cellular and molecular mechanisms controlling angiogenesis during adult life in normal and pathological conditions. Some of these studies have led to the identification of some progenitor cells that sustain angiogenesis through indirect, paracrine mechanisms (hematopoietic angiogenic cells) and through direct mechanisms, i.e., through their capacity to generate a progeny of phenotypically and functionally competent endothelial cells [endothelial colony forming cells (ECFCs)]. The contribution of these progenitors to angiogenetic processes under physiological and pathological conditions is intensively investigated. Angiogenetic mechanisms are stimulated in various hematological malignancies, including chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndromes and multiple myeloma, resulting in an increased angiogenesis that contributes to disease progression. In some of these conditions there is preliminary evidence that some endothelial cells could derive from the malignant clone, thus leading to the speculation that the leukemic cell derives from the malignant transformation of a hemangioblastic progenitor, i.e., of a cell capable of differentiation to the hematopoietic and to the endothelial cell lineages. Our understanding of the mechanisms underlying increased angiogenesis in these malignancies not only contributed to a better knowledge of the mechanisms responsible for tumor progression, but also offered the way for the discovery of new therapeutic targets. PMID:27583252

  12. Endothelial cells modulate renin secretion from isolated mouse juxtaglomerular cells.

    PubMed Central

    Kurtz, A; Kaissling, B; Busse, R; Baier, W

    1991-01-01

    Utilizing cocultures of mouse renal juxtaglomerular cells with bovine microvascular endothelial cells, we have examined whether endothelial cells exert direct influence on renin secretion from renal juxtaglomerular cells. In the presence of endothelial cells both spontaneous and forskolin (10 microM) or isoproterenol (10 microM) stimulated renin release were markedly attenuated. The stimulatory effect of the calmodulin antagonist calmidazolium (10 microM) on renin secretion was not altered by endothelial cells, whereas the stimulatory effect of ethylisopropylamiloride (50 microM) an inhibitor of sodium-proton exchange was enhanced in the presence of endothelial cells. Indomethacin (10 microM) and NG-monomethyl-l-arginine (NMMA) (1 mM) used to inhibit cyclooxygenase activity and production of endothelium-derived relaxing factor (EDRF) decreased spontaneous renin release in the presence of endothelial cells only, but had no effect on forskolin stimulated renin secretion. Endothelin (1 microM) inhibited cAMP stimulated renin release both in the absence and in the presence of endothelial cells. ATP (10 microM) which acts on both endothelial and juxtaglomerular cells via purinergic P2 receptors inhibited cAMP stimulated renin release only in the absence but not in the presence of endothelial cells. This modulatory effect of endothelial cells was no altered by indomethacin nor by NMMA. Taken together, our findings provide first evidence for a local control function of the endothelium on cAMP stimulated renin secretion from renal juxtaglomerular cells, which could in part be mediated by endothelin. Images PMID:1717509

  13. Establishment of outgrowth endothelial cells from peripheral blood.

    PubMed

    Martin-Ramirez, Javier; Hofman, Menno; van den Biggelaar, Maartje; Hebbel, Robert P; Voorberg, Jan

    2012-09-01

    Blood outgrowth endothelial cells (BOECs) are important tools when investigating diagnostic and therapeutic approaches for vascular disease. In this protocol, mononuclear cells are isolated from peripheral blood and plated on type I collagen at ∼135,000 cells per cm(2) in endothelial cell differentiation medium. On average, 0.34 colonies of endothelial cells per milliliter of blood can be obtained. Colonies of endothelial cells become visible after 14-28 d. Upon confluence, these rapidly expanding colonies can be passaged and have been shown to propagate up to 10(18)-fold. Isolated BOECs are phenotypically similar to vascular endothelial cells, as revealed by their cobblestone morphology, the presence of endothelial cell-specific Weibel-Palade bodies and the expression of endothelial cell markers such as VE-cadherin. The protocol presented here also provides a particularly useful tool for the ex vivo assessment of endothelial cell function from patients with different vascular abnormalities. PMID:22918388

  14. Using cultured endothelial cells to study endothelial barrier dysfunction: Challenges and opportunities.

    PubMed

    Aman, Jurjan; Weijers, Ester M; van Nieuw Amerongen, Geerten P; Malik, Asrar B; van Hinsbergh, Victor W M

    2016-08-01

    Despite considerable progress in the understanding of endothelial barrier regulation and the identification of approaches that have the potential to improve endothelial barrier function, no drug- or stem cell-based therapy is presently available to reverse the widespread vascular leak that is observed in acute respiratory distress syndrome (ARDS) and sepsis. The translational gap suggests a need to develop experimental approaches and tools that better mimic the complex environment of the microcirculation in which the vascular leak develops. Recent studies have identified several elements of this microenvironment. Among these are composition and stiffness of the extracellular matrix, fluid shear stress, interaction of endothelial cells (ECs) with pericytes, oxygen tension, and the combination of toxic and mechanic injurious stimuli. Development of novel cell culture techniques that integrate these elements would allow in-depth analysis of EC biology that closely approaches the (patho)physiological conditions in situ. In parallel, techniques to isolate organ-specific ECs, to define EC heterogeneity in its full complexity, and to culture patient-derived ECs from inducible pluripotent stem cells or endothelial progenitor cells are likely to advance the understanding of ARDS and lead to development of therapeutics. This review 1) summarizes the advantages and pitfalls of EC cultures to study vascular leak in ARDS, 2) provides an overview of elements of the microvascular environment that can directly affect endothelial barrier function, and 3) discusses alternative methods to bridge the gap between basic research and clinical application with the intent of improving the translational value of present EC culture approaches. PMID:27343194

  15. Molecular response of liver sinusoidal endothelial cells on hydrogels.

    PubMed

    Bartneck, Matthias; Topuz, Fuat; Tag, Carmen Gabriele; Sauer-Lehnen, Sibille; Warzecha, Klaudia Theresa; Trautwein, Christian; Weiskirchen, Ralf; Tacke, Frank

    2015-06-01

    There is a high demand for the isolation of primary endothelial cells for biomaterial endotheliazation studies, tissue engineering, and artificial organ development. Further, biomarkers for monitoring the response of endothelial cells in biomaterials science are required. We systematically compared two strategies for isolating liver sinusoidal endothelial cells (LSEC) from mouse liver. We demonstrate that fluorescence-activated cell sorting results in a considerably higher purity (~97%) compared to magnetic-assisted cell sorting (~80%), but is associated with a lower yield and recovery rate. Cell repellent polyethylene glycol (PEG) substrates affected the morphology of primary LSEC in culture and significantly downregulated the intracellular adhesion molecule (ICAM) and upregulated the vascular cell adhesion molecule (VCAM). This molecular response could partially be reverted by further modification with arginylglycylaspartic acid (RGD). Thus, usage of PEGylated materials may reduce, while applying RGD may support endotheliazation of materials, and we could relate LSEC attachment to their expression of ICAM and VCAM mRNA, suggesting their usage as biomarkers for endothelialization.

  16. Enhancing endothelial progenitor cell for clinical use

    PubMed Central

    Ye, Lei; Poh, Kian-Keong

    2015-01-01

    Circulating endothelial progenitor cells (EPCs) have been demonstrated to correlate negatively with vascular endothelial dysfunction and cardiovascular risk factors. However, translation of basic research into the clinical practice has been limited by the lack of unambiguous and consistent definitions of EPCs and reduced EPC cell number and function in subjects requiring them for clinical use. This article critically reviews the definition of EPCs based on commonly used protocols, their value as a biomarker of cardiovascular risk factor in subjects with cardiovascular disease, and strategies to enhance EPCs for treatment of ischemic diseases. PMID:26240678

  17. Enhancing endothelial progenitor cell for clinical use.

    PubMed

    Ye, Lei; Poh, Kian-Keong

    2015-07-26

    Circulating endothelial progenitor cells (EPCs) have been demonstrated to correlate negatively with vascular endothelial dysfunction and cardiovascular risk factors. However, translation of basic research into the clinical practice has been limited by the lack of unambiguous and consistent definitions of EPCs and reduced EPC cell number and function in subjects requiring them for clinical use. This article critically reviews the definition of EPCs based on commonly used protocols, their value as a biomarker of cardiovascular risk factor in subjects with cardiovascular disease, and strategies to enhance EPCs for treatment of ischemic diseases.

  18. Transport of lipoprotein lipase across endothelial cells

    SciTech Connect

    Saxena, U.; Klein, M.G.; Goldberg, I.J. )

    1991-03-15

    Lipoprotein lipase (LPL), synthesized in muscle and fat, hydrolyzes plasma triglycerides primarily while bound to luminal endothelial cell surfaces. To obtain information about the movement of LPL from the basal to the luminal endothelial cell surface, the authors studied the transport of purified bovine milk LPL across bovine aortic endothelial cell monolayers. {sup 125}I-labeled LPL ({sup 125}I-LPL) added to the basal surface of the monolayers was detected on the apical side of the cells in two compartments: (1) in the medium of the upper chamber, and (2) bound to the apical cell surface. The amount of {sup 125}I-LPL on the cell surface, but not in the medium, reached saturation with time and LPL dose. Catalytically active LPL was transported to the apical surface but very little LPL activity appeared in the medium. Heparinase treatment of the basal cell surface and addition of dextran sulfate to the lower chamber decreased the amount of {sup 125}I-LPL appearing on the apical surface. Similarly, the presence of increasing molar ratios of oleic acid/bovine serum albumin at the basal surface decreased the transport of active LPL across the monolayer. Thus, a saturable transport system, which requires haparan sulfate proteoglycans and is inhibited by high concentrations of free fatty acids on the basal side of the cells, appears to exist for passage of enzymatically active LPL across endothelial cells. They postulate that regulation of LPL transport to the endothelial luminal surface modulates the physiologically active pool of LPL in vivo.

  19. Are endothelial cell bioeffects from acoustic droplet vaporization proximity dependent?

    NASA Astrophysics Data System (ADS)

    Seda, Robinson; Li, David; Fowlkes, J. Brian; Bull, Joseph

    2013-11-01

    Acoustic droplet vaporization (ADV) produces gas microbubbles that provide a means of selective occlusion in gas embolotherapy. Vaporization and subsequent occlusion occur inside blood vessels supplying the targeted tissue, such as tumors. Theoretical and computational studies showed that ADV within a vessel can impart high fluid mechanical stresses on the vessel wall. Previous in vitro studies have demonstrated that vaporization at an endothelial layer may affect cell attachment and viability. The current study is aimed at investigating the role of vaporization distance away from the endothelial layer. HUVECs were cultured in OptiCell™ chambers until reaching confluence. Dodecafluoropentane microdroplets were added, attaining a 10:1 droplet to cell ratio. A single ultrasound pulse (7.5 MHz) consisting of 16 cycles (~ 2 μs) and a 5 MPa peak rarefactional pressure was used to produce ADV while varying the vaporization distance from the endothelial layer (0 μm, 500 μm, 1000 μm). Results indicated that cell attachment and viability was significantly different if the distance was 0 μm (at the endothelial layer). Other distances were not significantly different from the control. ADV will significantly affect the endothelium if droplets are in direct contact with the cells. Droplet concentration and flow conditions inside blood vessels may play an important role. This work was supported by NIH grant R01EB006476.

  20. Circulating endothelial cells in cardiovascular disease.

    PubMed

    Boos, Christopher J; Lip, Gregory Y H; Blann, Andrew D

    2006-10-17

    Quantification of circulating endothelial cells (CECs) in peripheral blood is developing as a novel and reproducible method of assessing endothelial damage/dysfunction. The CECs are thought to be mature cells that have detached from the intimal monolayer in response to endothelial injury and are a different cell population to endothelial progenitor cells (EPCs). The EPCs are nonleukocytes derived from the bone marrow that are believed to have proliferative potential and may be important in vascular regeneration. Currently accepted methods of CEC quantification include the use of immunomagnetic bead separation (with cell counting under fluorescence microscopy) and flow cytometry. Several recent studies have shown increased numbers of CECs in cardiovascular disease and its risk factors, such as unstable angina, acute myocardial infarction, stroke, diabetes mellitus, and critical limb ischemia, but no change in stable intermittent claudication, essential hypertension, or atrial fibrillation. Furthermore, CEC quantification at 48 h after acute myocardial infarction has been shown to be an accurate predictor of major adverse coronary events and death at both 1 month and 1 year. This article presents an overview of the pathophysiology of CECs in the setting of cardiovascular disease and a brief comparison with EPCs. PMID:17045885

  1. Pathogenic hantaviruses direct the adherence of quiescent platelets to infected endothelial cells.

    PubMed

    Gavrilovskaya, Irina N; Gorbunova, Elena E; Mackow, Erich R

    2010-05-01

    Hantavirus infections are noted for their ability to infect endothelial cells, cause acute thrombocytopenia, and trigger 2 vascular-permeability-based diseases. However, hantavirus infections are not lytic, and the mechanisms by which hantaviruses cause capillary permeability and thrombocytopenia are only partially understood. The role of beta(3) integrins in hemostasis and the inactivation of beta(3) integrin receptors by pathogenic hantaviruses suggest the involvement of hantaviruses in altered platelet and endothelial cell functions that regulate permeability. Here, we determined that pathogenic hantaviruses bind to quiescent platelets via a beta(3) integrin-dependent mechanism. This suggests that platelets may contribute to hantavirus dissemination within infected patients and provides a means by which hantavirus binding to beta(3) integrin receptors prevents platelet activation. The ability of hantaviruses to bind platelets further suggested that cell-associated hantaviruses might recruit platelets to the endothelial cell surface. Our findings indicate that Andes virus (ANDV)- or Hantaan virus (HTNV)-infected endothelial cells specifically direct the adherence of calcein-labeled platelets. In contrast, cells comparably infected with nonpathogenic Tula virus (TULV) failed to recruit platelets to the endothelial cell surface. Platelet adherence was dependent on endothelial cell beta(3) integrins and neutralized by the addition of the anti-beta(3) Fab fragment, c7E3, or specific ANDV- or HTNV-neutralizing antibodies. These findings indicate that pathogenic hantaviruses displayed on the surface of infected endothelial cells bind platelets and that a platelet layer covers the surface of infected endothelial cells. This fundamentally changes the appearance of endothelial cells and has the potential to alter cellular immune responses, platelet activation, and endothelial cell functions that affect vascular permeability. Hantavirus-directed platelet quiescence and

  2. Flow over glycocalyx covered endothelial cells.

    NASA Astrophysics Data System (ADS)

    Waters, Sarah L.; Liu, Shu Q.; Grotberg, James B.

    1997-11-01

    Blood vessels are lined with a monolayer of endothelial cells that interact with the blood flow. Two structural features of the endothelial membrane may influence the fluid dynamics over the endothelium: 1) the endothelial membrane bumpiness; and 2) the porous glycocalyx layer covering the cell surface. The bumpiness of the endothelium may induce regions of recirculation, and the glycocalyx may effect the flow pattern at the cell surface. An analytical study is presented for pressure driven blood flow in the microcirculation. The vessel is modeled as a rigid, impermeable, symmetric two--dimensional channel, which has sinusoidally wavy walls. The vessel has two regions: 1) the glycocalyx layer which is modeled as a uniformly thick poroelastic deformable wall layer using biphasic mixture theory; and 2) the free lumen where the Navier--Stokes equations of motion apply. Analytical results are obtained by making the long wavelength approximation. The model predicts the fluid flow and hence the shear stress exerted by the flow on the individual endothelial cells and at the glycocalyx--lumen interface. Implications of the results for biological events such as molecular transport and signal transduction are considered.

  3. Platelet endothelial cell adhesion molecule-1 and mechanotransduction in vascular endothelial cells.

    PubMed

    Fujiwara, K

    2006-04-01

    Endothelial cells are known to respond to mechanical forces such as fluid shear stress and cyclic stretch, but elucidating the mechanism for mechanosensing has been difficult. Experimental data indicate that there are probably several sensing mechanisms. We have recently proposed a novel mechanoresponse mechanism that involves platelet endothelial cell adhesion molecule-1 (PECAM-1). When endothelial cells are stimulated by fluid shear stress, PECAM-1 is tyrosine phosphorylated and activates the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signalling cascade. The same signalling events occurred when we applied pulling force directly on PECAM-1 on the endothelial cell surface using magnetic beads coated with antibodies against the external domain of PECAM-1. These results appear to indicate that PECAM-1 is a mechanotransduction molecule. To our knowledge, this is the first mammalian molecule that is shown to respond to mechanical force directly exerted to it. PMID:16594905

  4. Endothelial progenitor cells--an evolving story.

    PubMed

    Pearson, Jeremy D

    2010-05-01

    The first description of endothelial progenitor cells (EPC) in 1997 led rapidly to substantial changes in our understanding of angiogenesis, and within 5 years to the first clinical studies in humans using bone marrow derived EPC to enhance coronary neovascularisation and cardiac function after myocardial ischemia. However, to improve the success of this therapy a clearer understanding of the biology of EPC is needed. This article summarises recent data indicating that most EPC are not, in fact, endothelial progenitors but can be better described as angiogenic monocytes, and explores the implications this has for their future therapeutic use.

  5. Adhesion behavior of endothelial progenitor cells to endothelial cells in simple shear flow

    NASA Astrophysics Data System (ADS)

    Gong, Xiao-Bo; Li, Yu-Qing; Gao, Quan-Chao; Cheng, Bin-Bin; Shen, Bao-Rong; Yan, Zhi-Qiang; Jiang, Zong-Lai

    2011-12-01

    The adhesion of endothelial progenitor cells (EPCs) on endothelial cells (ECs) is one of the critical physiological processes for the regenesis of vascular vessels and the prevention of serious cardiovascular diseases. Here, the rolling and adhesion behavior of EPCs on ECs was studied numerically. A two-dimensional numerical model was developed based on the immersed boundary method for simulating the rolling and adhesion of cells in a channel flow. The binding force arising from the catch bond of a receptor and ligand pair was modeled with stochastic Monte Carlo method and Hookean spring model. The effect of tumor necrosis factor alpha (TNF- α) on the expression of the number of adhesion molecules in ECs was analyzed experimentally. A flow chamber system with CCD camera was set up to observe the top view of the rolling of EPCs on the substrate cultivated with ECs. Numerical results prove that the adhesion of EPC on ECs is closely related to membrane stiffness of the cell and shear rate of the flow. It also suggests that the adhesion force between EPC and EC by P-selectin glycoprotein ligand-1 only is not strong enough to bond the cell onto vessel walls unless contributions of other catch bond are considered. Experimental results demonstrate that TNF- α enhanced the expressions of VCAM, ICAM, P-selectin and E-selectin in ECs, which supports the numerical results that the rolling velocity of EPC on TNF- α treated EC substrate decreases obviously compared with its velocity on the untreated one. It is found that because the adhesion is affected by both the rolling velocity and the deformability of the cell, an optimal stiffness of EPC may exist at a given shear rate of flow for achieving maximum adhesion rates.

  6. Characterization of Bioeffects on Endothelial Cells under Acoustic Droplet Vaporization.

    PubMed

    Seda, Robinson; Li, David S; Fowlkes, J Brian; Bull, Joseph L

    2015-12-01

    Gas embolotherapy is achieved by locally vaporizing microdroplets through acoustic droplet vaporization, which results in bubbles that are large enough to occlude blood flow directed to tumors. Endothelial cells, lining blood vessels, can be affected by these vaporization events, resulting in cell injury and cell death. An idealized monolayer of endothelial cells was subjected to acoustic droplet vaporization using a 3.5-MHz transducer and dodecafluoropentane droplets. Treatments included insonation pressures that varied from 2 to 8 MPa (rarefactional) and pulse lengths that varied from 4 to 16 input cycles. The bubble cloud generated was directly dependent on pressure, but not on pulse length. Cellular damage increased with increasing bubble cloud size, but was limited to the bubble cloud area. These results suggest that vaporization near the endothelium may impact the vessel wall, an effect that could be either deleterious or beneficial depending on the intended overall therapeutic application.

  7. Adipose-derived stem cells (ASCs) as a source of endothelial cells in the reconstruction of endothelialized skin equivalents.

    PubMed

    Auxenfans, C; Lequeux, C; Perrusel, E; Mojallal, A; Kinikoglu, B; Damour, O

    2012-07-01

    Tissue-engineered autologous skin is a potential alternative to autograft for burn coverage, but produces poor clinical responses such as unsatisfactory graft intake due to insufficient vascularization. Endothelialized skin equivalents comprising human umbilical vein endothelial cells (HUVECs) survive significantly longer due to inosculation with the capillaries of the host, but these cells are allogeneic by definition. The aim of this study was to reconstruct an autologous endothelialized skin equivalent by incorporating progenitor or pre-differentiated endothelial cells derived from adipose tissue, easily accessible source for autologous transplantation. Human adipose tissue-derived stem cells were isolated from lipoaspirates and amplified to obtain endothelial progenitor cells, which were subsequently differentiated into endothelial cells. These cells were then seeded along with human fibroblasts into a porous collagen-glycosaminoglycan-chitosan scaffold to obtain an endothelialized dermal equivalent. Then, human keratinocytes give rise to a endothelialized skin equivalent. Immunohistochemistry and transmission electron microscopy results demonstrate the presence of capillary-like tubular structures in skin equivalents comprising pre-differentiated endothelial cells, but not endothelial progenitor cells. The former expressed both EN4 and von Willebrand factor, and Weibel-Palade bodies were detected in their cytoplasm. This study demonstrates that adipose tissue is an excellent source of autologous endothelial cells to reconstruct endothelialized tissue equivalents, and that pre-differentiation of stem cells is necessary to obtain vasculature in such models. PMID:21755603

  8. Reduced Ang2 expression in aging endothelial cells.

    PubMed

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

    2016-06-01

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. PMID:27137842

  9. Isolation of Murine Embryonic Hemogenic Endothelial Cells

    PubMed Central

    Marcelo, Kathrina L.; Hirschi, Karen K.

    2016-01-01

    The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level. PMID:27341393

  10. Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells

    PubMed Central

    Dushpanova, Anar; Agostini, Silvia; Ciofini, Enrica; Cabiati, Manuela; Casieri, Valentina; Matteucci, Marco; Del Ry, Silvia; Clerico, Aldo; Berti, Sergio; Lionetti, Vincenzo

    2016-01-01

    Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2−) levels, known triggers of ET-1 expression. Moreover, no increase in O2− or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2− production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction. PMID:27443965

  11. 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

  12. Brain microvascular endothelial cell transplantation ameliorates ischemic white matter damage.

    PubMed

    Puentes, Sandra; Kurachi, Masashi; Shibasaki, Koji; Naruse, Masae; Yoshimoto, Yuhei; Mikuni, Masahiko; Imai, Hideaki; Ishizaki, Yasuki

    2012-08-21

    Ischemic insults affecting the internal capsule result in sensory-motor disabilities which adversely affect the patient's life. Cerebral endothelial cells have been reported to exert a protective effect against brain damage, so the transplantation of healthy endothelial cells might have a beneficial effect on the outcome of ischemic brain damage. In this study, endothelin-1 (ET-1) was injected into the rat internal capsule to induce lacunar infarction. Seven days after ET-1 injection, microvascular endothelial cells (MVECs) were transplanted into the internal capsule. Meningeal cells or 0.2% bovine serum albumin-Hank's balanced salt solution were injected as controls. Two weeks later, the footprint test and histochemical analysis were performed. We found that MVEC transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (P<0.01) and induced remyelination (P<0.01) compared with the control groups. Also the inflammatory response was repressed by MVEC transplantation, judging from fewer ED-1-positive activated microglial cells in the MVEC-transplanted group than in the other groups. Elucidation of the mechanisms by which MVECs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia.

  13. Brain microvascular endothelial cell transplantation ameliorates ischemic white matter damage.

    PubMed

    Puentes, Sandra; Kurachi, Masashi; Shibasaki, Koji; Naruse, Masae; Yoshimoto, Yuhei; Mikuni, Masahiko; Imai, Hideaki; Ishizaki, Yasuki

    2012-08-21

    Ischemic insults affecting the internal capsule result in sensory-motor disabilities which adversely affect the patient's life. Cerebral endothelial cells have been reported to exert a protective effect against brain damage, so the transplantation of healthy endothelial cells might have a beneficial effect on the outcome of ischemic brain damage. In this study, endothelin-1 (ET-1) was injected into the rat internal capsule to induce lacunar infarction. Seven days after ET-1 injection, microvascular endothelial cells (MVECs) were transplanted into the internal capsule. Meningeal cells or 0.2% bovine serum albumin-Hank's balanced salt solution were injected as controls. Two weeks later, the footprint test and histochemical analysis were performed. We found that MVEC transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (P<0.01) and induced remyelination (P<0.01) compared with the control groups. Also the inflammatory response was repressed by MVEC transplantation, judging from fewer ED-1-positive activated microglial cells in the MVEC-transplanted group than in the other groups. Elucidation of the mechanisms by which MVECs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia. PMID:22771710

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

    PubMed

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

    2015-12-01

    Efficacy of therapeutic angiogenesis needs a comprehensive understanding of endothelial cell (EC) function and biological factors and cells that interplay with ECs. Stem cells are considered the key components of pro- and anti-angiogenic milieu in a wide variety of physiopathological states, and interactions of EC-stem cells have been the subject of controversy in recent years. In this study, the potential effects of three tissue-specific adult stem cells, namely rat marrow-derived mesenchymal stem cells (rBMSCs), rat adipose-derived stem cells (rADSCs) and rat muscle-derived satellite cells (rSCs), on the endothelial activation of key angiogenic signaling molecules, including VEGF, Ang-2, VEGFR-2, Tie-2, and Tie2-pho, were investigated. Human umbilical vein endothelial cells (HUVECs) and rat lung microvascular endothelial cells (RLMECs) were cocultured with the stem cells or incubated with the stem cell-derived conditioned media on Matrigel. Following HUVEC-stem cell coculture, CD31-positive ECs were flow sorted and subjected to western blotting to analyze potential changes in the expression of the pro-angiogenic signaling molecules. Elongation and co-alignment of the stem cells were seen along the EC tubes in the EC-stem cell cocultures on Matrigel, with cell-to-cell dye communication in the EC-rBMSC cocultures. Moreover, rBMSCs and rADSCs significantly improved endothelial tubulogenesis in both juxtacrine and paracrine manners. These two latter stem cells dynamically up-regulated VEGF, Ang-2, VREGR-2, and Tie-2 but down-regulated Tie2-pho and the Tie2-pho/Tie-2 ratio in HUVECs. Induction of pro-angiogenic signaling in ECs by marrow- and adipose-derived MSCs further indicates the significance of stem cell milieu in angiogenesis dynamics. PMID:26068799

  15. The actin cytoskeleton in endothelial cell phenotypes

    PubMed Central

    Prasain, Nutan; Stevens, Troy

    2009-01-01

    Endothelium forms a semi-permeable barrier that separates blood from the underlying tissue. Barrier function is largely determined by cell-cell and cell-matrix adhesions that define the limits of cell borders. Yet, such cell-cell and cell-matrix tethering is critically reliant upon the nature of adherence within the cell itself. Indeed, the actin cytoskeleton fulfills this essential function, to provide a strong, dynamic intracellular scaffold that organizes integral membrane proteins with the cell’s interior, and responds to environmental cues to orchestrate appropriate cell shape. The actin cytoskeleton is comprised of three distinct, but interrelated structures, including actin cross-linking of spectrin within the membrane skeleton, the cortical actin rim, and actomyosin-based stress fibers. This review addresses each of these actin-based structures, and discusses cellular signals that control the disposition of actin in different endothelial cell phenotypes. PMID:19028505

  16. Conserved arginine residues in the carboxyl terminus of the equine arteritis virus E protein may play a role in heparin binding but may not affect viral infectivity in equine endothelial cells.

    PubMed

    Lu, Zhengchun; Sarkar, Sanjay; Zhang, Jianqiang; Balasuriya, Udeni B R

    2016-04-01

    Equine arteritis virus (EAV), the causative agent of equine viral arteritis, has relatively broad cell tropism in vitro. In horses, EAV primarily replicates in macrophages and endothelial cells of small blood vessels. Until now, neither the cellular receptor(s) nor the mechanism(s) of virus attachment and entry have been determined for this virus. In this study, we investigated the effect of heparin on EAV infection in equine endothelial cells (EECs). Heparin, but not other glycosaminoglycans, could reduce EAV infection up to 93 %. Sequence analysis of the EAV E minor envelope protein revealed a conserved amino acid sequence (52 RSLVARCSRGARYR 65) at the carboxy terminus of the E protein, which was predicted to be the heparin-binding domain. The basic arginine (R) amino acid residues were subsequently mutated to glycine by site-directed mutagenesis of ORF2a in an E protein expression vector and an infectious cDNA clone of EAV. Two single mutations in E (R52G and R57G) did not affect the heparin-binding capability, whereas the E double mutation (R52,60G) completely eliminated the interaction between the E protein and heparin. Although the mutant R52,60G EAV did not bind heparin, the mutations did not completely abolish infectivity, indicating that heparin is not the only critical factor for EAV infection. This also suggested that other viral envelope protein(s) might be involved in attachment through heparin or other cell-surface molecules, and this warrants further investigation.

  17. Phagocytosis of platelets enhances endothelial cell survival under serum deprivation.

    PubMed

    Jiang, Ping; Ren, Ya-Li; Lan, Yong; Li, Jia-Liang; Luo, Jun; Li, Jian; Cai, Jian-Ping

    2015-07-01

    Platelets are key players in fundamental processes of vascular biology, such as angiogenesis, tissue regeneration, and tumor metastasis. However, the underlying mechanisms remain unclear. In this study, some tumor vascular endothelial cells were positively stained by antiplatelet antibodies. Further investigation revealed that platelets were taken up by endothelial cells in vitro and in vivo. Human umbilical vascular endothelial cells were rendered apoptotic under conditions of serum deprivation. However, endothelial apoptosis was suppressed and cell viability was enhanced when platelets were added to the cultures. Endothelial survival was paralleled by an upregulation of phosphorylated Akt and p70 S6K. In conclusion, this study demonstrated that platelets can be phagocytosed by endothelial cells, and the phagocytosed platelets could suppress endothelial apoptosis and promote cell viability level. The mechanism underlying this process involves the activation of Akt signaling.

  18. Phagocytosis of platelets enhances endothelial cell survival under serum deprivation

    PubMed Central

    Ren, Ya-Li; Lan, Yong; Li, Jia-Liang; Luo, Jun; Li, Jian; Cai, Jian-Ping

    2015-01-01

    Platelets are key players in fundamental processes of vascular biology, such as angiogenesis, tissue regeneration, and tumor metastasis. However, the underlying mechanisms remain unclear. In this study, some tumor vascular endothelial cells were positively stained by antiplatelet antibodies. Further investigation revealed that platelets were taken up by endothelial cells in vitro and in vivo. Human umbilical vascular endothelial cells were rendered apoptotic under conditions of serum deprivation. However, endothelial apoptosis was suppressed and cell viability was enhanced when platelets were added to the cultures. Endothelial survival was paralleled by an upregulation of phosphorylated Akt and p70 S6K. In conclusion, this study demonstrated that platelets can be phagocytosed by endothelial cells, and the phagocytosed platelets could suppress endothelial apoptosis and promote cell viability level. The mechanism underlying this process involves the activation of Akt signaling. PMID:25577801

  19. Role of endothelial cells in bovine mammary gland health and disease.

    PubMed

    Ryman, Valerie E; Packiriswamy, Nandakumar; Sordillo, Lorraine M

    2015-12-01

    The bovine mammary gland is a dynamic and complex organ composed of various cell types that work together for the purpose of milk synthesis and secretion. A layer of endothelial cells establishes the blood-milk barrier, which exists to facilitate the exchange of solutes and macromolecules necessary for optimal milk production. During bacterial challenge, however, endothelial cells divert some of their lactation function to protect the underlying tissue from damage by initiating inflammation. At the onset of inflammation, endothelial cells tightly regulate the movement of plasma components and leukocytes into affected tissue. Unfortunately, endothelial dysfunction as a result of exacerbated or sustained inflammation can negatively affect both barrier integrity and the health of surrounding extravascular tissue. The objective of this review is to highlight the role of endothelial cells in supporting milk production and regulating optimal inflammatory responses. The consequences of endothelial dysfunction and sustained inflammation on milk synthesis and secretion are discussed. Given the important role of endothelial cells in orchestrating the inflammatory response, a better understanding of endothelial function during mastitis may support development of targeted therapies to protect bovine mammary tissue and mammary endothelium.

  20. Circulating endothelial cells: a new biomarker of endothelial dysfunction in hematological diseases.

    PubMed

    Gendron, Nicolas; Smadja, David M

    2016-08-01

    The endothelium and its integrity are in the center of numerous cardiovascular, pulmonary and tumoral diseases. Several studies identified different circulating cellular sub-populations, which allow a noninvasive exploration of endothelial dysfunction. Furthermore, angiogenesis plays a major role in the biology of benign and malignant hematologic diseases. Among these biomarkers, circulating endothelial cells could be considered as a marker of endothelial injury and/or endothelial activation as well as vascular remodeling, whereas circulating endothelial progenitor cells would be only involved in the vascular regeneration. In the future, the quantification of circulating endothelial cells in many diseases could be a noninvasive biomarker used in diagnosis, prognostic and therapeutic follow-up of lung vasculopathy and/or residual disease of hematological malignancies.

  1. Protein tyrosine phosphatase regulation of endothelial cell apoptosis and differentiation.

    PubMed

    Yang, C; Chang, J; Gorospe, M; Passaniti, A

    1996-02-01

    Apoptosis, or programmed cell death, occurs during development and may also be an important factor in many diseases. However, little is known about the signal transduction pathways regulating apoptosis. In these studies, loss of endothelial cell-substrate attachment and apoptosis after removal of growth factors was associated with dephosphorylation of tyrosine residues at the cell periphery. Dephosphorylation of total cellular proteins accompanied apoptosis and was reduced by orthovanadate, an inhibitor of protein tyrosine phosphatases. Orthovanadate blocked the fragmentation of nuclear DNA, inhibited DNA laddering, and suppressed the expression of TRPM-2, an apoptosis-associated gene. The tyrosine phosphorylation levels of FAK125, erk1 (mitogen-activated kinase kinase), and cdc-2 were reduced during apoptosis. FAK125 dephosphorylation was inhibited by orthovanadate, but premature activation (tyrosine dephosphorylation) of cdc-2 was not. Orthovanadate was as effective as basic fibroblast growth factor in activating erk1 without increasing cell proliferation and in preventing the apoptosis of endothelial cells after treatment with tumor necrosis factor alpha. Endothelial cell differentiation on extracellular matrix (Matrigel) was also stimulated by orthovanadate in the absence of basic fibroblast growth factor without affecting growth arrest and inhibition of DNA synthesis. Expression of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1) was down-regulated during the early stages of differentiation, remained low for at least 6 hours as differentiation proceeded, and increased upon completion of differentiation. Cells that failed to down-regulate p21 mRNA on Matrigel in the absence of angiogenic factors underwent apoptosis. These results suggest that protein tyrosine phosphatases are actively involved in signal transduction during apoptosis and may regulate p21 expression to inhibit endothelial cell differentiation.

  2. Morphological study of endothelial cells during freezing

    NASA Astrophysics Data System (ADS)

    Zhang, A.; Xu, L. X.; Sandison, G. A.; Cheng, S.

    2006-12-01

    Microvascular injury is recognized as a major tissue damage mechanism of ablative cryosurgery. Endothelial cells lining the vessel wall are thought to be the initial target of freezing. However, details of this injury mechanism are not yet completely understood. In this study, ECMatrix™ 625 was used to mimic the tumour environment and to allow the endothelial cells cultured in vitro to form the tube-like structure of the vasculature. The influence of water dehydration on the integrity of this structure was investigated. It was found that the initial cell shape change was mainly controlled by water dehydration, dependent on the cooling rate, resulting in the shrinkage of cells in the direction normal to the free surface. As the cooling was prolonged and temperature was lowered, further cell shape change could be induced by the chilling effects on intracellular proteins, and focal adhesions to the basement membrane. Quantitative analysis showed that the freezing induced dehydration greatly enhanced the cell surface stresses, especially in the axial direction. This could be one of the major causes of the final breaking of the cell junction and cell detachment.

  3. Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells.

    PubMed

    Gioia, Magda; Vindigni, Giulia; Testa, Barbara; Raniolo, Sofia; Fasciglione, Giovanni Francesco; Coletta, Massimiliano; Biocca, Silvia

    2015-01-01

    The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MβCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding. PMID:26495844

  4. Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells

    PubMed Central

    Testa, Barbara; Raniolo, Sofia; Fasciglione, Giovanni Francesco; Coletta, Massimiliano; Biocca, Silvia

    2015-01-01

    The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MβCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding. PMID:26495844

  5. Endothelial cell permeability to water and antipyrine

    SciTech Connect

    Garrick, R.A.

    1986-03-05

    The endothelium provides a structural barrier between plasma constituents and the tissues. The permeability characteristics of the the endothelial cells regulate the transcellular movement of materials across this barrier while other movement is paracellular. In this study the permeability of the endothelial cells to tritiated water (/sup 3/HHO) and /sup 14/C-labeled antipyrine (AP) was investigated. The cells were isolated non-enzymatically from calf pulmonary artery and were maintained in culture and used between the seventh and fifteenth passage. The cells were removed from the T-flasks with a rubber policeman, titurated with a 22g needle and centrifuged. The cells were mixed with an extracellular marker, drawn into polyethylene tubing and packed by centrifugation for use in the linear diffusion technique. All measurements were made at 37 C. The diffusion coefficients for /sup 3/HHO through the packed cells (D), the intracellular material (D/sub 2/), and the extracellular material (D/sub 1/) were 0.682, 0.932 and 2.45 x 10/sup -5/ cm/sup 2/ s/sup -1/ and for AP were 0.273, 0.355 and 1.13 x 10/sup -5/ cm/sup 2/ s/sup -1/ respectively. The permeability coefficient calculated by the series-parallel pathway model for /sup 3/HHO was higher than that for AP and for both /sup 3/HHO and AP were lower than those calculated for isolated lung cells and erythrocytes.

  6. Vascular endothelial growth factor B, a novel growth factor for endothelial cells.

    PubMed Central

    Olofsson, B; Pajusola, K; Kaipainen, A; von Euler, G; Joukov, V; Saksela, O; Orpana, A; Pettersson, R F; Alitalo, K; Eriksson, U

    1996-01-01

    We have isolated and characterized a novel growth factor for endothelial cells, vascular endothelial growth factor B (VEGF-B), with structural similarities to vascular endothelial growth factor (VEGF) and placenta growth factor. VEGF-B was particularly abundant in heart and skeletal muscle and was coexpressed with VEGF in these and other tissues. VEGF-B formed cell-surface-associated disulfide-linked homodimers and heterodimerized with VEGF when coexpressed. Conditioned medium from transfected 293EBNA cells expressing VEGF-B stimulated DNA synthesis in endothelial cells. Our results suggest that VEGF-B has a role in angiogenesis and endothelial cell growth, particularly in muscle. Images Fig. 3 Fig. 4 Fig. 5 PMID:8637916

  7. KRIT1 Protein Depletion Modifies Endothelial Cell Behavior via Increased Vascular Endothelial Growth Factor (VEGF) Signaling*

    PubMed Central

    DiStefano, Peter V.; Kuebel, Julia M.; Sarelius, Ingrid H.; Glading, Angela J.

    2014-01-01

    Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1+/− mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1. PMID:25320085

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

    SciTech Connect

    Vallon, Mario; Rohde, Franziska; Janssen, Klaus-Peter; Essler, Markus

    2010-02-01

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

  9. Endothelial cell heterogeneity: antioxidant profiles determine vulnerability to oxidant injury.

    PubMed

    Vercellotti, G M; Dobson, M; Schorer, A E; Moldow, C F

    1988-02-01

    Human umbilical vein endothelial cells were more sensitive to hydrogen peroxide lysis than cow pulmonary artery endothelial cells. Conversely, activated neutrophils which utilize hydrogen peroxide-mediated cell cytotoxicity cell mechanisms were more toxic to the cow pulmonary artery cells. This discordance was not related to neutrophil adhesion to either cell type or cell passage number. The antioxidant profiles of the endothelial cells revealed that cow pulmonary artery cells were rich in catalase to consume bolus hydrogen peroxide presented to them, while human umbilical vein endothelial cells utilize glutathione peroxidase-linked mechanisms to detoxify a slower more sustained release of hydrogen peroxide generated by neutrophils. Endothelial cells from different species and sites may utilize diversified antioxidant protective mechanisms. PMID:3340627

  10. Transcriptional Regulation oa Endothelial Cell And Vascular Development

    PubMed Central

    Park, Changwon; Kim, Tae Min; Malik, Asrar B.

    2013-01-01

    The establishment and maintenance of the vascular system is critical for embryonic development and postnatal life. Defects in endothelial cell development and vessel formation and function lead to embryonic lethality and are important in the etiology of vascular diseases. Here we review the underlying molecular mechanisms of endothelial cell differentiation, plasticity, and the development of the vasculature. This review focuses on the interplay among transcription factors and signaling molecules that specify the differentiation of vascular endothelial cells. We also discuss recent progress on reprogramming of somatic cells towards distinct endothelial cell lineages and its promise in regenerative vascular medicine. PMID:23661712

  11. Production of soluble Neprilysin by endothelial cells.

    PubMed

    Kuruppu, Sanjaya; Rajapakse, Niwanthi W; Minond, Dmitriy; Smith, A Ian

    2014-04-01

    A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35±0.70 and 6.54±0.41 μmols of substrate cleaved over 3h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC50 values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37±1.43 and 38.30±4.70, respectively as a % of control; P<0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.

  12. Imbalance of mitochondrial-nuclear cross talk in isocyanate mediated pulmonary endothelial cell dysfunction.

    PubMed

    Panwar, Hariom; Jain, Deepika; Khan, Saba; Pathak, Neelam; Raghuram, Gorantla V; Bhargava, Arpit; Banerjee, Smita; Mishra, Pradyumna K

    2013-01-01

    Mechanistic investigations coupled with epidemiology, case-control, cohort and observational studies have increasingly linked isocyanate exposure (both chronic and acute) with pulmonary morbidity and mortality. Though ascribed for impairment in endothelial cell function, molecular mechanisms of these significant adverse pulmonary outcomes remains poorly understood. As preliminary studies conducted in past have failed to demonstrate a cause-effect relationship between isocyanate toxicity and compromised pulmonary endothelial cell function, we hypothesized that direct exposure to isocyanate may disrupt endothelial structural lining, resulting in cellular damage. Based on this premise, we comprehensively evaluated the molecular repercussions of methyl isocyanate (MIC) exposure on human pulmonary arterial endothelial cells (HPAE-26). We examined MIC-induced mitochondrial oxidative stress, pro-inflammatory cytokine response, oxidative DNA damage response and apoptotic index. Our results demonstrate that exposure to MIC, augment mitochondrial reactive oxygen species production, depletion in antioxidant defense enzymes, elevated pro-inflammatory cytokine response and induced endothelial cell apoptosis via affecting the balance of mitochondrial-nuclear cross talk. We herein delineate the first and direct molecular cascade of isocyanate-induced pulmonary endothelial cell dysfunction. The results of our study might portray a connective link between associated respiratory morbidities with isocyanate exposure, and indeed facilitate to discern the exposure-phenotype relationship in observed deficits of pulmonary endothelial cell function. Further, understanding of inter- and intra-cellular signaling pathways involved in isocyanate-induced endothelial damage would not only aid in biomarker identification but also provide potential new avenues to target specific therapeutic interventions.

  13. Effects of ultrasound upon endothelial cell ultrastructure

    NASA Astrophysics Data System (ADS)

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

    2012-11-01

    A number of new brain applications for therapeutic ultrasound are emerging including drug delivery through BBB opening, enhancement of angiogenesis, sonothrombolysis and neuromodulation. Safety remains important as alterations in the cytoskeleton and tight junctions of endothelial cells have been described. In this study we characterize the in vitro effects of ultrasound on cell morphology using a new human brain cell line (hCMEC/D3). Changes in ultrastructure were analyzed with antibodies against tubulin, actin and catenin. Transport was analyzed by measuring transferrin uptake. No significant changes were seen after continuous wave ultrasound treatment of hCMEC/D3 cells grown in Opticell{trade mark, serif} chambers. We could not observe disassembled actin stress fibers or variations in the microtubule network. However, severe damage occurred in cells cultured in petri dishes.

  14. Production of soluble Neprilysin by endothelial cells

    SciTech Connect

    Kuruppu, Sanjaya; Rajapakse, Niwanthi W.; Minond, Dmitriy; Smith, A. Ian

    2014-04-04

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

  15. Endothelial Cell Dynamics during Anastomosis in vitro

    PubMed Central

    Diaz-Santana, Anthony; Shan, Mengrou; Stroock, Abraham D.

    2015-01-01

    Vascular anastomosis –the fusion of vessels from two distinct branches of the vascular system – represents a critical step in vascular growth under both healthy and pathological conditions, in vivo, and presents an important target for engineering of vascularized tissues, in vitro. Recent works in animal models have advanced our understanding of the molecular and cellular players in vascular anastomosis, but questions remain related to cellular dynamics and control of this process, in vitro. In this study, we exploited a three-dimensional (3-D) culture platform to examine the dynamics of endothelial cell (EC) during and after vascular anastomosis by allowing angiogenesis and vasculogenesis to proceed in parallel. We show that anastomosis occurs between sprouts formed by angiogenesis from an endothelium and tubes formed by vasculogenesis in the bulk of a 3-D matrix. This fusion leads to highly connected vessels that span from the surface of the matrix into the bulk in a manner that depends on cell density and identity. Further, we observe and analyze intermixing of endothelial cells of distinct origin (surface versus bulk) within the vessels structures that are formed; we provide evidence that the cells migrate along pre-existing vessels segments as part of this intermixing process. We conclude that anastomosis can occur between vessels emerging by angiogenesis and vasculogenesis and that this process may play an important role in contexts such as wound healing. PMID:25790315

  16. Regulation of allergic lung inflammation by endothelial cell transglutaminase 2.

    PubMed

    Soveg, Frank; Abdala-Valencia, Hiam; Campbell, Jackson; Morales-Nebreda, Luisa; Mutlu, Gökhan M; Cook-Mills, Joan M

    2015-09-15

    Tissue transglutaminase 2 (TG2) is an enzyme with multiple functions, including catalysis of serotonin conjugation to proteins (serotonylation). Previous research indicates that TG2 expression is upregulated in human asthma and in the lung endothelium of ovalbumin (OVA)-challenged mice. It is not known whether endothelial cell TG2 is required for allergic inflammation. Therefore, to determine whether endothelial cell TG2 regulates allergic inflammation, mice with an endothelial cell-specific deletion of TG2 were generated, and these mice were sensitized and challenged in the airways with OVA. Deletion of TG2 in endothelial cells blocked OVA-induced serotonylation in lung endothelial cells, but not lung epithelial cells. Interestingly, deletion of endothelial TG2 reduced allergen-induced increases in respiratory system resistance, number of eosinophils in the bronchoalveolar lavage, and number of eosinophils in the lung tissue. Endothelial cell deletion of TG2 did not alter expression of adhesion molecules, cytokines, or chemokines that regulate leukocyte recruitment, consistent with other studies, demonstrating that deletion of endothelial cell signals does not alter lung cytokines and chemokines during allergic inflammation. Taken together, the data indicate that endothelial cell TG2 is required for allergic inflammation by regulating the recruitment of eosinophils into OVA-challenged lungs. In summary, TG2 functions as a critical signal for allergic lung responses. These data identify potential novel targets for intervention in allergy/asthma.

  17. The relationship between oxidised LDL, endothelial progenitor cells and coronary endothelial function in patients with CHD

    PubMed Central

    Watt, Jonathan; Kennedy, Simon; Ahmed, Nadeem; Hayhurst, James; McClure, John D; Berry, Colin; Wadsworth, Roger M; Oldroyd, Keith G

    2016-01-01

    Objective The balance between coronary endothelial dysfunction and repair is influenced by many protective and deleterious factors circulating in the blood. We studied the relationship between oxidised low-density lipoprotein (oxLDL), circulating endothelial progenitor cells (EPCs) and coronary endothelial function in patients with stable coronary heart disease (CHD). Methods 33 patients with stable CHD were studied. Plasma oxLDL was measured using ELISA, coronary endothelial function was assessed using intracoronary acetylcholine infusion and EPCs were quantified using flow cytometry for CD34+/KDR+ cells. Results Plasma oxLDL correlated positively with the number of EPCs in the blood (r=0.46, p=0.02). There was a positive correlation between the number of circulating EPCs and coronary endothelial function (r=0.42, p=0.04). There was no significant correlation between oxLDL and coronary endothelial function. Conclusions Plasma levels of oxLDL are associated with increased circulating EPCs in the blood of patients with CHD, which may reflect a host-repair response to endothelial injury. Patients with stable CHD had a high prevalence of coronary endothelial dysfunction, which was associated with lower numbers of circulating EPCs, suggesting a mechanistic link between endothelial dysfunction and the pathogenesis of atherosclerosis. PMID:26848395

  18. Adherence of Candida to cultured vascular endothelial cells: mechanisms of attachment and endothelial cell penetration.

    PubMed

    Rotrosen, D; Edwards, J E; Gibson, T R; Moore, J C; Cohen, A H; Green, I

    1985-12-01

    To elucidate the pathogenesis of hematogenous Candida infections, we developed an in vitro model of Candida adherence to and penetration of human endothelial cells. We enhanced or inhibited adherence in order to probe mechanisms of attachment. Adherence of Candida albicans showed a linear relation to Candida inoculum (range, 10(2)-10(5) cfu, r = .99, P less than .01) and exceeded that of less virulent Candida species and that of Saccharomyces cerevisiae (P less than .01). Candida immune serum blocked attachment (greater than 95% inhibition; P less than .001), however, this activity was abolished by immunoprecipitation of immune serum with C. albicans mannan (P less than .001) and was unaffected by immunoprecipitation with S. cerevisiae mannan or by adsorption with particulate chitin. Adherence was diminished by exposing C. albicans to heat (greater than 99% inhibition; P less than .01), UV light (98% inhibition; P less than .01), or sodium periodate (greater than 72% inhibition; P less than .01). An extract from heat-exposed C. albicans blocked adherence (greater than 51% inhibition; P less than .001). Transmission electron microscopy demonstrated that viable or killed Candida organisms were attached to endothelial cells, were enveloped by membrane processes from the endothelial cell surface, and were incorporated into the endothelial cells within phagosomes. Cytochalasin B blocked incorporation without blocking surface attachment. PMID:3905987

  19. Regulation of endothelial proliferation by the renin-angiotensin system in human umbilical vein endothelial cells.

    PubMed

    Herr, D; Rodewald, M; Fraser, H M; Hack, G; Konrad, R; Kreienberg, R; Wulff, C

    2008-07-01

    This study was performed in order to evaluate the role of angiotensin II in physiological angiogenesis. Human umbilical vein endothelial cells (HUVEC) were stained for angiotensin II type 1 receptor (AGTR1) immunocytochemically and for gene expression of renin-angiotensin system (RAS) components. The regulation of the angiogenesis-associated genes vascular endothelial growth factor (VEGF) and angiopoietins (ANGPT1 and ANGPT2) were studied using quantitative RT-PCR. Furthermore, we examined the effect of angiotensin II on the proliferation of HUVEC using Ki-67 as well as BrdU immunocytochemistry and investigated whether the administration of the AGTR1 blocker candesartan or the VEGF antagonist FLT1-Fc could suppress the observed angiotensin II-dependent proangiogenic effect. AGTR1 was expressed in HUVEC and the administration of angiotensin II significantly increased the gene expression of VEGF and decreased the gene expression of ANGPT1. Since the expression of ANGPT2 was not affected significantly the ratio of ANGPT1/ANGPT2 was decreased. In addition, a significantly increased endothelial cell proliferation was observed after stimulation with angiotensin II, which was suppressed by the simultaneous administration of candesartan or the VEGF antagonist FLT1-Fc. These results indicate the potential capacity of angiotensin II in influencing angiogenesis by the regulation of angiogenesis-associated genes via AGTR1. Since VEGF blockade opposed the effect of angiotensin II on cell proliferation, it is hypothesised that VEGF mediates the angiotensin II-dependent effect in concert with the changes in angiopoietin expression. This is the first report of the RAS on the regulation of angiogenesis-associated genes in physiology.

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

    PubMed Central

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

    2016-01-01

    The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL), a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF) signaling and angiogenesis in endothelial cells (ECs). We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day) recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis. PMID:26863518

  1. Fibrinogen Induces Alterations of Endothelial Cell Tight Junction Proteins

    PubMed Central

    PATIBANDLA, PHANI K.; TYAGI, NEETU; DEAN, WILLIAM L.; TYAGI, SURESH C.; ROBERTS, ANDREW M.; LOMINADZE, DAVID

    2009-01-01

    We previously showed that an elevated content of fibrinogen (Fg) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Fg binding to endothelial cells (ECs) alters expression of actin-associated endothelial tight junction proteins (TJP). Rat cardiac microvascular ECs were grown in gold plated chambers of an electrical cell-substrate impedance system, 8-well chambered, or in 12-well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with mitogen-activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126), Fg (4 mg/ml) with anti-ICAM-1 antibody or BQ788 (endothelin type B receptor blocker), endothelin-1, endothelin-1 with BQ788, or medium alone for 24 h. Fg induced a dose-dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Western blot analysis and RT-PCR data showed that the higher dose of Fg decreased the contents of TJPs, occludin, zona occluden-1 (ZO-1), and zona occluden-2 (ZO-2) in ECs. Fg-induced decreases in contents of the TJPs were blocked by PD98059, U0126, or anti-ICAM-1 antibody. While BQ788 inhibited endothelin-1-induced decrease in TEER, it did not affect Fg-induced decrease in TEER. These data suggest that Fg increases EC layer permeability via the MEK kinase signaling pathway by affecting occludin, ZO-1, and ZO-2, TJPs, which are bound to actin filaments. Therefore, increased binding of Fg to its major EC receptor, ICAM-1, during cardiovascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs. PMID:19507189

  2. Endothelial Cell Implantation and Survival within Experimental Gliomas

    NASA Astrophysics Data System (ADS)

    Lal, Bachchu; Indurti, Ravi R.; Couraud, Pierre-Olivier; Goldstein, Gary W.; Laterra, John

    1994-10-01

    The delivery of therapeutic genes to primary brain neoplasms opens new opportunities for treating these frequently fatal tumors. Efficient gene delivery to tissues remains an important obstacle to therapy, and this problem has unique characteristics in brain tumors due to the blood-brain and blood-tumor barriers. The presence of endothelial mitogens and vessel proliferation within solid tumors suggests that genetically modified endothelial cells might efficiently transplant to brain tumors. Rat brain endothelial cells immortalized with the adenovirus E1A gene and further modified to express the β-galactosidase reporter were examined for their ability to survive implantation to experimental rat gliomas. Rats received 9L, F98, or C6 glioma cells in combination with endothelial cells intracranially to caudate/putamen or subcutaneously to flank. Implanted endothelial cells were identified by β-galactosidase histochemistry or by polymerase chain reaction in all tumors up to 35 days postimplantation, the latest time examined. Implanted endothelial cells appeared to cooperate in tumor vessel formation and expressed the brain-specific endothelial glucose transporter type 1 as identified by immunohistochemistry. The proliferation of implanted endothelial cells was supported by their increased number within tumors between postimplantation days 14 and 21 (P = 0.015) and by their expression of the proliferation antigen Ki67. These findings establish that genetically modified endothelial cells can be stably engrafted to growing gliomas and suggest that endothelial cell implantation may provide a means of delivering therapeutic genes to brain neoplasms and other solid tumors. In addition, endothelial implantation to brain may be useful for defining mechanisms of brain-specific endothelial differentiation.

  3. Differentiation state determines neural effects on microvascular endothelial cells

    SciTech Connect

    Muffley, Lara A.; Pan, Shin-Chen; Smith, Andria N.; Ga, Maricar; Hocking, Anne M.; Gibran, Nicole S.

    2012-10-01

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

  4. Immunological functions of liver sinusoidal endothelial cells

    PubMed Central

    Knolle, Percy A; Wohlleber, Dirk

    2016-01-01

    Liver sinusoidal endothelial cells (LSECs) line the liver sinusoids and separate passenger leukocytes in the sinusoidal lumen from hepatocytes. LSECs further act as a platform for adhesion of various liver-resident immune cell populations such as Kupffer cells, innate lymphoid cells or liver dendritic cells. In addition to having an extraordinary scavenger function, LSECs possess potent immune functions, serving as sentinel cells to detect microbial infection through pattern recognition receptor activation and as antigen (cross)-presenting cells. LSECs cross-prime naive CD8 T cells, causing their rapid differentiation into memory T cells that relocate to secondary lymphoid tissues and provide protection when they re-encounter the antigen during microbial infection. Cross-presentation of viral antigens by LSECs derived from infected hepatocytes triggers local activation of effector CD8 T cells and thereby assures hepatic immune surveillance. The immune function of LSECs complements conventional immune-activating mechanisms to accommodate optimal immune surveillance against infectious microorganisms while preserving the integrity of the liver as a metabolic organ. PMID:27041636

  5. 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.

  6. Asiaticoside Inhibits TNF-α-Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells.

    PubMed

    Fong, Lai Yen; Ng, Chin Theng; Zakaria, Zainul Amiruddin; Baharuldin, Mohamad Taufik Hidayat; Arifah, Abdul Kadir; Hakim, Muhammad Nazrul; Zuraini, Ahmad

    2015-10-01

    The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.

  7. Modulation of endothelial cell phenotype by physical activity: impact on obesity-related endothelial dysfunction.

    PubMed

    Bender, Shawn B; Laughlin, M Harold

    2015-07-01

    Increased levels of physical activity are associated with reduced cardiovascular disease (CVD) risk and mortality in obesity and diabetes. Available evidence suggests that local factors, including local hemodynamics, account for a significant portion of this CVD protection, and numerous studies have interrogated the therapeutic benefit of physical activity/exercise training in CVD. Less well established is whether basal differences in endothelial cell phenotype between/among vasculatures related to muscle recruitment patterns during activity may account for reports of nonuniform development of endothelial dysfunction in obesity. This is the focus of this review. We highlight recent work exploring the vulnerability of two distinct vasculatures with established differences in endothelial cell phenotype. Specifically, based largely on dramatic differences in underlying hemodynamics, arteries perfusing soleus muscle (slow-twitch muscle fibers) and those perfusing gastrocnemius muscle (fast-twitch muscle fibers) in the rat exhibit an exercise training-like versus an untrained endothelial cell phenotype, respectively. In the context of obesity, therefore, arteries to soleus muscle exhibit protection from endothelial dysfunction compared with vulnerable arteries to gastrocnemius muscle. This disparate vulnerability is consistent with numerous animal and human studies, demonstrating increased skeletal muscle blood flow heterogeneity in obesity coincident with reduced muscle function and exercise intolerance. Mechanistically, we highlight emerging areas of inquiry exploring novel aspects of hemodynamic-sensitive signaling in endothelial cells and the time course of physical activity-associated endothelial adaptations. Lastly, further exploration needs to consider the impact of endothelial heterogeneity on the development of endothelial dysfunction because endothelial dysfunction independently predicts CVD events.

  8. Cell biology of diabetic nephropathy: Roles of endothelial cells, tubulointerstitial cells and podocytes

    PubMed Central

    Maezawa, Yoshiro; Takemoto, Minoru; Yokote, Koutaro

    2015-01-01

    Diabetic nephropathy is the major cause of end-stage renal failure throughout the world in both developed and developing countries. Diabetes affects all cell types of the kidney, including endothelial cells, tubulointerstitial cells, podocytes and mesangial cells. During the past decade, the importance of podocyte injury in the formation and progression of diabetic nephropathy has been established and emphasized. However, recent findings provide additional perspectives on pathogenesis of diabetic nephropathy. Glomerular endothelial damage is already present in the normoalbuminuric stage of the disease when podocyte injury starts. Genetic targeting of mice that cause endothelial injury leads to accelerated diabetic nephropathy. Tubulointerstitial damage, previously considered to be a secondary effect of glomerular protein leakage, was shown to have a primary significance in the progression of diabetic nephropathy. Emerging evidence suggests that the glomerular filtration barrier and tubulointerstitial compartment is a composite, dynamic entity where any injury of one cell type spreads to other cell types, and leads to the dysfunction of the whole apparatus. Accumulation of novel knowledge would provide a better understanding of the pathogenesis of diabetic nephropathy, and might lead to a development of a new therapeutic strategy for the disease. PMID:25621126

  9. 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.

  10. Effects of surface viscoelasticity on cellular responses of endothelial cells

    PubMed Central

    Hosseini, Motahare-Sadat; Katbab, Ali Asghar

    2014-01-01

    Background: One area of nanoscience deals with nanoscopic interactions between nanostructured materials and biological systems. To elucidate the effects of the substrate surface morphology and viscoelasticity on cell proliferation, fractal analysis was performed on endothelial cells cultured on nanocomposite samples based on silicone rubber (SR) and various concentrations of organomodified nanoclay (OC). Methods: The nanoclay/SR ratio was tailored to enhance cell behavior via changes in sample substrate surface roughness and viscoelasticity. Results: Surface roughness of the cured SR filled with negatively-charged nanosilicate layers had a greater effect than elasticity on cell growth. The surface roughness of SR nanocomposite samples increased with increasing the OC content, leading to enhanced cell growth and extracellular matrix (ECM) remodeling. This was consistent with the decrease in SR segmental motions and damping factor as the primary viscoelastic parameters by the nanosilicate layers with increasing clay concentrations. Conclusions: The inclusion of clay nanolayers affected the growth and behavior of endothelial cells on microtextured SR. PMID:26989733

  11. Development of Endothelial-Specific Single Inducible Lentiviral Vectors for Genetic Engineering of Endothelial Progenitor Cells.

    PubMed

    Yang, Guanghua; Kramer, M Gabriela; Fernandez-Ruiz, Veronica; Kawa, Milosz P; Huang, Xin; Liu, Zhongmin; Prieto, Jesus; Qian, Cheng

    2015-11-27

    Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties.

  12. Anti-endothelial cell antibodies in patients with Wegener's granulomatosis and micropolyarteritis.

    PubMed Central

    Ferraro, G; Meroni, P L; Tincani, A; Sinico, A; Barcellini, W; Radice, A; Gregorini, G; Froldi, M; Borghi, M O; Balestrieri, G

    1990-01-01

    Anti-endothelial cell antibodies (AECA) have been detected by cell surface radioimmunoassay in nine out of 15 patients with micropolyarteritis (MPA) and in two out of five patients with Wegener's granulomatosis. AECA mostly belonged to the IgG isotype and were present in the active phase of the diseases. These antibodies were not detectable in 10 sera from patients with essential mixed cryoglobulinaemia, suggesting that they were not a mere epiphenomenon consequent to the inflammatory vascular injury. The binding activity was not related to ABH antigens or to HLA class I antigens displayed by resting human endothelial cells in culture and was not influenced by removing immune complexes. Absorption of the anti-neutrophil cytoplasmic antibodies (ANCA), present in MPA and Wegener's granulomatosis sera, did not affect the endothelial binding. AECA-positive sera did not display lytic activity against endothelial cells, neither alone nor after addition of fresh complement or normal human peripheral blood mononuclear cells. Although AECA are not cytolytic for endothelial cell monolayers in vitro, the reactivity against intact endothelial cells suggests their possible involvement in in vivo pathological processes affecting vascular structures in small vessel primary vasculitides. PMID:2302834

  13. 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

  14. Tracing behavior of endothelial cells promotes vascular network formation.

    PubMed

    Yasuda, Noriko; Sekine, Hidekazu; Bise, Ryoma; Okano, Teruo; Shimizu, Tatsuya

    2016-05-01

    The in vitro formation of network structures derived from endothelial cells in grafts before transplantation contributes to earlier engraftment. In a previous study, endothelial cells migrated to form a net-shaped structure in co-culture. However, the specific network formation behavior of endothelial cells during migration remains unclear. In this study, we demonstrated the tracing behavior and cell cycle of endothelial cells using Fucci-labeled (Fluorescent Ubiquitination-based Cell Cycle Indicator) endothelial cells. Here, we observed the co-culture of Fucci-labeled human umbilical vein endothelial cells (HUVECs) together with normal human dermal fibroblasts (NHDFs) using time-lapse imaging and analyzed by multicellular concurrent tracking. In the G0/G1 period, HUVECs migrate faster than in the S/G2/M period, because G0/G1 is the mobile phase and S/G2/M is the proliferation phase in the cell cycle. When HUVECs are co-cultured, they tend to move randomly until they find existing tracks that they then follow to form clusters. Extracellular matrix (ECM) staining showed that collagen IV, laminin and thrombospondin deposited in accordance with endothelial cell networks. Therefore the HUVECs may migrate on the secreted ECM and exhibit tracing behavior, where the HUVECs migrate toward each other. These results suggested that ECM and a cell phase contributed to form a network by accelerating cell migration.

  15. Endothelial progenitor cells: a new player in lupus?

    PubMed

    Haque, Sahena; Alexander, M Yvonne; Bruce, Ian N

    2012-01-01

    Patients with systemic lupus erythematosus (SLE) have a greatly increased risk of cardiovascular disease. There is growing interest in the link between vascular damage and lupus-specific inflammatory factors. Impaired endothelial repair could account for the endothelial dysfunction in this patient group. This review describes the contribution that endothelial progenitor cells could play in the pathogenesis of premature vascular damage in this disease. The methods of isolation, detection, and characterization of endothelial progenitor cells, together with their potential role in repair of the endothelium and as a therapeutic target in SLE, are discussed. PMID:22356717

  16. Transient disruptions of aortic endothelial cell plasma membranes.

    PubMed Central

    Yu, Q. C.; McNeil, P. L.

    1992-01-01

    Cells of gut, skin, and muscle frequently suffer transient survivable plasma membrane disruptions ("wounds") under physiological conditions, but it is not known whether endothelial cells of the aorta, which are constantly exposed to hemodynamically generated mechanical forces, similarly are injured in vivo. We have used serum albumin as a molecular probe for identifying endothelial cells of the rat aorta that incurred and survived transient plasma membrane wounds in vivo. Such wounded endothelial cells were in fact observed in the aortas of all rats examined. However, the percentage of wounded cells in the total aortic endothelial population varied remarkably between individuals ranging from 1.4% to 17.9% with a mean of 6.5% (+/- 4.6% SD). Wounded endothelial cells were heterogeneously distributed, being found in distinct clusters often in the shape of streaks aligned with the long axis of the vessel, or in the shape of partial or complete rims surrounding bifurcation openings, such as the ostia of the intercostal arteries. Physical exercise (running) did not increase the frequency of aortic endothelial cell membrane wounding, nor did spontaneous hypertension. Surprisingly, 80% of mitotic endothelial cell figures were identified as wounded. This article identified a previously unrecognized form of endothelial cell injury, survivable disruptions of the plasma membrane, and shows that injury to the endothelial cells of the normal aorta is far more commonplace than previously suspected. Plasma membrane wounding of endothelial cells could be linked to the initiation of atherosclerosis. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 11 Figure 6 Figure 8 PMID:1466399

  17. Transcriptional targeting of tumor endothelial cells for gene therapy

    PubMed Central

    Dong, Zhihong; Nör, Jacques E.

    2009-01-01

    It is well known that angiogenesis plays a critical role in the pathobiology of tumors. Recent clinical trials have shown that inhibition of angiogenesis can be an effective therapeutic strategy for patients with cancer. However, one of the outstanding issues in anti-angiogenic treatment for cancer is the development of toxicities related to off-target effects of drugs. Transcriptional targeting of tumor endothelial cells involves the use of specific promoters for selective expression of therapeutic genes in the endothelial cells lining the blood vessels of tumors. Recently, several genes that are expressed specifically in tumor-associated endothelial cells have been identified and characterized. These discoveries have enhanced the prospectus of transcriptionaly targeting tumor endothelial cells for cancer gene therapy. In this manuscript, we review the promoters, vectors, and therapeutic genes that have been used for transcriptional targeting of tumor endothelial cells, and discuss the prospects of such approaches for cancer gene therapy. PMID:19393703

  18. Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases

    PubMed Central

    Wang, Yingying; Boerma, Marjan; Zhou, Daohong

    2016-01-01

    Exposure to ionizing radiation induces not only apoptosis but also senescence. While the role of endothelial cell apoptosis in mediating radiation-induced acute tissue injury has been extensively studied, little is known about the role of endothelial cell senescence in the pathogenesis of radiation-induced late effects. Senescent endothelial cells exhibit decreased production of nitric oxide and expression of thrombomodulin, increased expression of adhesion molecules, elevated production of reactive oxygen species and inflammatory cytokines and an inability to proliferate and form capillary-like structures in vitro. These findings suggest that endothelial cell senescence can lead to endothelial dysfunction by dysregulation of vasodilation and hemostasis, induction of oxidative stress and inflammation and inhibition of angiogenesis, which can potentially contribute to radiation-induced late effects such as cardiovascular diseases (CVDs). In this article, we discuss the mechanisms by which radiation induces endothelial cell senescence, the roles of endothelial cell senescence in radiation-induced CVDs and potential strategies to prevent, mitigate and treat radiation-induced CVDs by targeting senescent endothelial cells. PMID:27387862

  19. 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

  20. Cilostazol suppresses angiotensin II-induced apoptosis in endothelial cells.

    PubMed

    Shi, Miao-Qian; Su, Fei-Fei; Xu, Xuan; Liu, Xiong-Tao; Wang, Hong-Tao; Zhang, Wei; Li, Xue; Lian, Cheng; Zheng, Qiang-Sun; Feng, Zhi-Chun

    2016-03-01

    Patients with essential hypertension undergo endothelial dysfunction, particularly in the conduit arteries. Cilostazol, a type III phosphodiesterase inhibitor, serves a role in the inhibition of platelet aggregation and it is widely used in the treatment of peripheral vascular diseases. Previous studies have suggested that cilostazol suppresses endothelial dysfunction; however, it remains unknown whether cilostazol protects the endothelial function in essential hypertension. The aim of the present study was to investigate whether, and how, cilostazol suppresses angiotensin II (angII)‑induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) and Sprague Dawley rats were exposed to angII and treated with cilostazol. Endothelial cell apoptosis and function, nitric oxide and superoxide production, phosphorylation (p) of Akt, and caspase‑3 protein expression levels were investigated. AngII exposure resulted in the apoptosis of endothelial cells in vitro and in vivo. In vitro, cilostazol significantly suppressed the angII‑induced apoptosis of HUVECs; however, this effect was reduced in the presence of LY294002, a phosphoinositide 3 kinase (PI3K) inhibitor. Furthermore, cilostazol suppressed the angII‑induced p‑Akt downregulation and cleaved caspase‑3 upregulation. These effects were also alleviated by LY294002. In vivo, cilostazol suppressed the angII‑induced endothelial cell apoptosis and dysfunction. Cilostazol was also demonstrated to partially reduced the angII‑induced increase in superoxide production. The results of the present study suggested that cilostazol suppresses endothelial apoptosis and dysfunction by modulating the PI3K/Akt pathway.

  1. Cilostazol suppresses angiotensin II-induced apoptosis in endothelial cells

    PubMed Central

    SHI, MIAO-QIAN; SU, FEI-FEI; XU, XUAN; LIU, XIONG-TAO; WANG, HONG-TAO; ZHANG, WEI; LI, XUE; LIAN, CHENG; ZHENG, QIANG-SUN; FENG, ZHI-CHUN

    2016-01-01

    Patients with essential hypertension undergo endothelial dysfunction, particularly in the conduit arteries. Cilostazol, a type III phosphodiesterase inhibitor, serves a role in the inhibition of platelet aggregation and it is widely used in the treatment of peripheral vascular diseases. Previous studies have suggested that cilostazol suppresses endothelial dysfunction; however, it remains unknown whether cilostazol protects the endothelial function in essential hypertension. The aim of the present study was to investigate whether, and how, cilostazol suppresses angiotensin II (angII)-induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) and Sprague Dawley rats were exposed to angII and treated with cilostazol. Endothelial cell apoptosis and function, nitric oxide and superoxide production, phosphorylation (p) of Akt, and caspase-3 protein expression levels were investigated. AngII exposure resulted in the apoptosis of endothelial cells in vitro and in vivo. In vitro, cilostazol significantly suppressed the angII-induced apoptosis of HUVECs; however, this effect was reduced in the presence of LY294002, a phosphoinositide 3 kinase (PI3K) inhibitor. Furthermore, cilostazol suppressed the angII-induced p-Akt downregulation and cleaved caspase-3 upregulation. These effects were also alleviated by LY294002. In vivo, cilostazol suppressed the angII-induced endothelial cell apoptosis and dysfunction. Cilostazol was also demonstrated to partially reduced the angII-induced increase in superoxide production. The results of the present study suggested that cilostazol suppresses endothelial apoptosis and dysfunction by modulating the PI3K/Akt pathway. PMID:26862035

  2. Activation of small ruminant aortic endothelial cells after in vitro infection by caprine arthritis encephalitis virus.

    PubMed

    Jan, C L; Greenland, T; Gounel, F; Balleydier, S; Mornex, J F

    2000-12-01

    Small ruminants infected by the lentiviruses caprine arthritis-encephalitis virus (CAEV), originally isolated from a goat, or maedi-visna virus, originally from sheep, typically develop an organising lymphoid infiltration of affected tissues. This could reflect modulation of the migration pattern of lymphocytes in infected animals. Possible active contribution by vascular endothelial cells was investigated using an in vitro model. Low-passage cultured ovine aortic endothelium proved susceptible to productive infection by CAEV without significant cytotoxicity. Infected endothelial cells maintained expression of endothelial markers, increased MHC class I antigen expression and initiated expression of the adhesion molecule VCAM -1 and, at a late stage, MHC class II antigens. Infected endothelial cells showed a two-fold increase in binding capacity for sheep peripheral blood leucocytes over uninfected controls. Such events could contribute to the tissue distribution of lymphoid cells and local immune responses in lentiviral infections of small ruminants. PMID:11124093

  3. Implantation of Vascular Grafts Lined with Genetically Modified Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Wilson, James M.; Birinyi, Louis K.; Salomon, Robert N.; Libby, Peter; Callow, Allan D.; Mulligan, Richard C.

    1989-06-01

    The possibility of using the vascular endothelial cell as a target for gene replacement therapy was explored. Recombinant retroviruses were used to transduce the lacZ gene into endothelial cells harvested from mongrel dogs. Prosthetic vascular grafts seeded with the genetically modified cells were implanted as carotid interposition grafts into the dogs from which the original cells were harvested. Analysis of the graft 5 weeks after implantation revealed genetically modified endothelial cells lining the luminal surface of the graft. This technology could be used in the treatment of atherosclerosis disease and the design of new drug delivery systems.

  4. Effect of metformin on insulin-resistant endothelial cell function

    PubMed Central

    CHEN, HAIYAN; LI, JIE; YANG, OU; KONG, JIAN; LIN, GUANGZHU

    2015-01-01

    The aim of the present study was to investigate the effect of metformin on the function of insulin-resistant (IR) endothelial cells. A model of IR endothelial cells was established by incubating cells with 30 mM glucose, 1 μM dexamethasone and various concentrations of insulin. The nitric oxide (NO) content of the endothelial cells was determined by measuring the rate of nitroreductase production; the endothelin (ET) concentration was examined by enzyme-linked immunosorbent assay; and the expression levels of endothelial nitric oxide synthase (eNOS) were detected using western blotting. The optimal conditions for inducing insulin resistance in endothelial cells were a combination treatment of 10−4 mmol/l insulin, 30 mM glucose and 1 μM dexamethasone for 48 h. Notably, metformin administration significantly increased the NO content and reduced the ET-1 concentration in the IR cells compared with the non-treated control cells (P<0.05); furthermore, metformin significantly increased the intracellular eNOS protein expression in IR endothelial cells compared with the non-treated control cells (P<0.05), with an optimal metformin concentration of 10−3 mmol/l. Thus, the present study identified that metformin improves the function of IR endothelial cells, possibly through promoting eNOS protein expression and increasing the NO content. PMID:25663871

  5. Endothelial Cell Density to Predict Endothelial Graft Failure After Penetrating Keratoplasty

    PubMed Central

    Lass, Jonathan H.; Sugar, Alan; Benetz, Beth Ann; Beck, Roy W.; Dontchev, Mariya; Gal, Robin L.; Kollman, Craig; Gross, Robert; Heck, Ellen; Holland, Edward J.; Mannis, Mark J.; Raber, Irving; Stark, Walter; Stulting, R. Doyle

    2010-01-01

    Objective To determine whether preoperative and/or postoperative central endothelial cell density (ECD) and its rate of decline postoperatively are predictive of graft failure caused by endothelial decompensation following penetrating keratoplasty to treat a moderate-risk condition, principally, Fuchs dystrophy or pseudophakic corneal edema. Methods In a subset of Cornea Donor Study participants, a central reading center determined preoperative and postoperative ECD from available specular images for 17 grafts that failed because of endothelial decompensation and 483 grafts that did not fail. Results Preoperative ECD was not predictive of graft failure caused by endothelial decompensation (P = .91). However, the 6-month ECD was predictive of subsequent failure (P < .001). Among those that had not failed within the first 6 months, the 5-year cumulative incidence (±95% confidence interval) of failure was 13% (±12%) for the 33 participants with a 6-month ECD of less than 1700 cells/mm2 vs 2%(±3%) for the 137 participants with a 6-monthECDof 2500 cells/mm2 or higher. After 5 years’ follow-up, 40 of 277 participants (14%) with a clear graft had an ECD below 500 cells/mm2. Conclusions Preoperative ECD is unrelated to graft failure from endothelial decompensation, whereas there is a strong correlation of ECD at 6 months with graft failure from endothelial decompensation. A graft can remain clear after 5 years even when the ECD is below 500 cells/mm2. PMID:20065219

  6. [Dynamics of the cell cycle in human endothelial cell culture infected with influenza virus].

    PubMed

    Prochukhanova, A R; Lyublinskaya, O G; Azarenok, A A; Nazarova, A V; Zenin, V V; Zhilinskaya, I N

    2015-01-01

    Cell cycle in a culture of endothelial cells EAhy 926 infected with influenza virus was investigated. Cytometric analysis of culture, synchronized using contact inhibition, has shown that the exposure to the influenza virus in cells EAhy 926 lengthened S-phase of the cell cycle. This result has been tested and proven on culture EAhy 926 treated with nocodazole. Compared with lung carcinoma cells A549, in which influenza virus provokes the arrest of G0/G1 phase of the cycle, elongation of S-phase of cycle at a similar infection of endothelial culture EAhy 926 indicates that the influenza virus differently affects the dynamics of the cell cycle according to the origin of the infected culture.

  7. Apoptosis of Endothelial Cells by 13-HPODE Contributes to Impairment of Endothelial Barrier Integrity

    PubMed Central

    Ryman, Valerie E.; Packiriswamy, Nandakumar

    2016-01-01

    Inflammation is an essential host response during bacterial infections such as bovine mastitis. Endothelial cells are critical for an appropriate inflammatory response and loss of vascular barrier integrity is implicated in the pathogenesis of Streptococcus uberis-induced mastitis. Previous studies suggested that accumulation of linoleic acid (LA) oxygenation products derived from 15-lipoxygenase-1 (15-LOX-1) metabolism could regulate vascular functions. The initial LA derivative from the 15-LOX-1 pathway, 13-hydroperoxyoctadecadienoic acid (HPODE), can induce endothelial death, whereas the reduced hydroxyl product, 13-hydroxyoctadecadienoic acid (HODE), is abundantly produced during vascular activation. However, the relative contribution of specific LA-derived metabolites on impairment of mammary endothelial integrity is unknown. Our hypothesis was that S. uberis-induced LA-derived 15-LOX-1 oxygenation products impair mammary endothelial barrier integrity by apoptosis. Exposure of bovine mammary endothelial cells (BMEC) to S. uberis did not increase 15-LOX-1 LA metabolism. However, S. uberis challenge of bovine monocytes demonstrated that monocytes may be a significant source of both 13-HPODE and 13-HODE during mastitis. Exposure of BMEC to 13-HPODE, but not 13-HODE, significantly reduced endothelial barrier integrity and increased apoptosis. Changing oxidant status by coexposure to an antioxidant during 13-HPODE treatment prevented adverse effects of 13-HPODE, including amelioration of apoptosis. A better understanding of how the oxidant status of the vascular microenvironment impacts endothelial barrier properties could lead to more efficacious treatments for S. uberis mastitis.

  8. Human monocyte-endothelial cell interaction in vitro.

    PubMed

    Pawlowski, N A; Abraham, E L; Pontier, S; Scott, W A; Cohn, Z A

    1985-12-01

    We have examined the interaction of freshly isolated human blood monocytes with cultured human umbilical vein endothelial cells in vitro. Purified monocytes incubated with confluent primary or passaged endothelial cells (EC) for 90 min at 37 degrees C bound at maximal densities of 6.5-7.0 X 10(3)/mm2 (8 or 9 per EC) without causing disruption of the monolayer. Monocyte-EC binding proceeded in the presence of plasma proteins or optimal phagocytic doses of opsonized zymosan particles. The avidity of attachment was not diminished by alternative monocyte isolation techniques. Monocyte attachment to EC was dependent upon the presence of divalent cations (magnesium greater than calcium) and was inhibited at 4 degrees C. Monocytes selectively bound to EC when incubated with monolayers composed of smooth muscle cells and EC. Neither EC monolayer confluence nor a variety of EC culture conditions affected the high levels of monocyte binding. In contrast, human neutrophils (less than 1 per EC) and lymphocytes (less than 2-3.5 per EC) bound at lower maximal densities under the same conditions, while platelet reactivity remained minimal. The distinctively higher affinity of human blood monocytes relative to other circulating white cells for binding to cultured human EC may have relevance to their function in vivo.

  9. Apoptosis in capillary endothelial cells in ageing skeletal muscle

    PubMed Central

    Wang, Huijuan; Listrat, Anne; Meunier, Bruno; Gueugneau, Marine; Coudy-Gandilhon, Cécile; Combaret, Lydie; Taillandier, Daniel; Polge, Cécile; Attaix, Didier; Lethias, Claire; Lee, Kijoon; Goh, Kheng Lim; Béchet, Daniel

    2014-01-01

    The age-related loss of skeletal muscle mass and function (sarcopenia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonuclei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostaining for dystrophin, paired box protein-7 (Pax7) or laminin-2α, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the age-related rise in apoptotic nuclei was essentially due to stromal cells. Myofibre-associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7+ satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age-dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing. PMID:24245531

  10. Immunohistochemical localization of endothelial cell markers in solitary fibrous tumor.

    PubMed

    Sawada, Namie; Ishiwata, Toshiyuki; Naito, Zenya; Maeda, Shotaro; Sugisaki, Yuichi; Asano, Goro

    2002-12-01

    Solitary fibrous tumor (SFT) is an uncommon tumor first reported in the pleura, but recently described in other tissues. CD34, which is expressed in hematopoietic stem cells, endothelial progenitor cells and vascular endothelial cells, is observed in most SFT and some investigators believe that its expression is a definitive marker of this tumor. In the present study, the expression of vascular endothelial cell markers, such as vascular endothelial growth factor receptor (VEGFR)-1 (flt-1), VEGFR-2 (flk-1/KDR), Tie-2 and c-Met, was examined in SFT to clarify the relationship between SFT and endothelial cells. By immunohistochemical staining of tumor cells from 26 patients, VEGFR-1 was detected in 24 (92%), VEGFR-2 in five (19%), Tie-2 in 14 (54%), and c-Met, a specific receptor of hepatocyte growth factor (HGF) in 23 patients (88%). Furthermore, VEGFR-3 (flt-4) immunoreactivity was detected in eight of 26 patients (31%). In contrast, VEGF, VEGF-C and HGF, which are ligands for the receptors, were not localized in the SFT cells. These findings indicate that most SFT may closely relate to vascular or lymphatic endothelial cells and the endothelial growth factors may contribute to the growth of SFT in a paracrine manner.

  11. Dermal stem cells can differentiate down an endothelial lineage.

    PubMed

    Bell, Emma; Richardson, Gavin D; Jahoda, Colin A; Gledhill, Karl; Phillips, Helen M; Henderson, Deborah; Owens, W Andrew; Hole, Nicholas

    2012-11-01

    In this study, we have demonstrated that cells of neural crest origin located in the dermal papilla (DP) exhibit endothelial marker expression and a functional activity. When grown in endothelial growth media, DP primary cultures upregulate expression of vascular endothelial growth factor receptor 1 (FLT1) mRNA and downregulate expression of the dermal stem cell marker α-smooth muscle actin. DP cells have demonstrated functional characteristics of endothelial cells, including the ability to form capillary-like structures on Matrigel, increase uptake of low-density lipoprotein and upregulate ICAM1 (CD54) in response to tumour necrosis factor alpha (TNF-α) stimulation. We confirmed that these observations were not due to contaminating endothelial cells, by using DP clones. We have also used the WNT1cre/ROSA26R and WNT1cre/YFP lineage-tracing mouse models to identify a population of neural crest-derived cells in DP cultures that express the endothelial marker PECAM (CD31); these cells also form capillary-like structures on Matrigel. Importantly, cells of neural crest origin that express markers of endothelial and mesenchymal lineages exist within the dermal sheath of the vibrissae follicle.

  12. Endothelial Cell Morphology and Migration are Altered by Changes in Gravitational Fields

    NASA Technical Reports Server (NTRS)

    Melhado, Caroline; Sanford, Gary; Harris-Hooker, Sandra

    1997-01-01

    Endothelial cell migration is important to vascular wall regeneration following injury or stress. However, the mechanism(s) governing this response is not well understood. The microgravity environment of space may complicate the response of these cells to injury. To date, there are no reports in this area. We examined how bovine aortic (BAEC) and pulmonary (BPEC) endothelial cells respond to denudation injury under hypergravity (HGrav) and simulated microgravity (MGrav), using image analysis. In 10% FBS, the migration of confluent BAEC and BPEC into the denuded area was not affected by HGrav or MGrav. However, in low FBS (0.5%), signficantly retarded migration under MGrav, and increased migration under HGrav was found. MGrav also decreased the migration of postconfluent BPEC while HGrav showed no difference. Both MGrav and HGrav strongly decreased the migration of postconfluent BAEC. Also, both cell lines showed significant morphological changes by scanning electron microscopy. These studies indicate that endothelial cell function is affected by changes in gravity.

  13. Synthesis of heparan and chondroitin sulfate proteoglycans by human endothelial cells is differentially affected by herpes simplex virus type I (HSV-1)

    SciTech Connect

    Kaner, R.J.; Iozzo, R.V.; Ziaie, Z.; Kefalides, N.A.

    1987-05-01

    Effects of HSV-1 infection on proteoglycan (PG) synthesis by human EC were studied as a model of EC injury. Confluent cultures of early passage umbilical vein EC were infected with HSV-1 at multiplicities of infection (MOI) from 0.001 to 1.0. In uninfected cultures, over 90% of the total (/sup 35/S)sulfate-labeled macromolecules were divided into two major peaks on ion-exchange chromatography. One contained heparan sulfate (HSPG) and the other chondroitin/dermatan sulfate (CS/DSPG) proteoglycan. HSV-1 infection produced a dose-dependent inhibition of total PG synthesis of up to 75%. There were widely divergent effects on individual PG species. Inhibition of CS/DSPG synthesis was much more marked than that of the HSPG. At a MOI of 1.0, CS/DSPG was present at 13% of control values, compared to 30% for HSPG. There was about one log difference in the dose of virus required to produce 50% inhibition of cell/matrix HSPG relative to CS/DSPG. HSV-1 did not inhibit the generation of an undersulfated HSPG, which contained shorter glycosaminoglycan chains than the predominant species. The authors conclude that HSV-1 infection of human EC produces complex differential effects on proteoglycan synthesis.

  14. Ozone inhibits endothelial cell cyclooxygenase activity through formation of hydrogen peroxide

    SciTech Connect

    Madden, M.C.; Eling, T.E.; Friedman, M.

    1987-09-01

    We have previously demonstrated that a 2H exposure of cultured pulmonary endothelial cells to ozone (0.0-1.0 ppm) in-vitro resulted in a concentration-dependent reduction of endothelial prostacyclin production (90% decrease at the 1.0 ppm level). Ozone-exposed endothelial cells, incubated with 20 uM arachidonate, also demonstrated a significant inhibition of prostacyclin synthesis. To further examine the mechanisms of the inhibition of prostacyclin synthesis, bovine pulmonary endothelial cells were exposed to 1.0 ppm ozone for 2H. A significant decrease in prostacyclin synthesis was found within 5 min of exposure (77 +/- 36% of air-exposed control values, p less than 0.05). Endothelial prostacyclin synthesis returned to baseline levels by 12H after ozone exposure, a time point which was similar to the recovery time of unexposed endothelium treated with 0.5 uM acetylsalicylic acid. Incubation of endothelial cells, previously exposed to 1.0 ppm ozone for 2 hours, with 4 uM PGH2 resulted in restoration of essentially normal prostacyclin synthesis. When endothelial cells were co-incubated with catalase (5 U/ml) during ozone exposure, no inhibition of prostacyclin synthesis was observed. Co-incubation with either heat-inactivated catalase or superoxide dismutase (10 U/ml) did not affect the ozone-induced inhibition of prostacyclin synthesis. These data suggest that H/sub 2/O/sub 2/ is a major toxic species produced in endothelial cells during ozone exposure and responsible for the inhibition of endothelial cyclooxygenase activity.

  15. Phenotypic Expression of ADAMTS13 in Glomerular Endothelial Cells

    PubMed Central

    Tati, Ramesh; Kristoffersson, Ann-Charlotte; Ståhl, Anne-lie; Mörgelin, Matthias; Motto, David; Satchell, Simon; Mathieson, Peter; Manea-Hedström, Minola; Karpman, Diana

    2011-01-01

    Background ADAMTS13 is the physiological von Willebrand factor (VWF)-cleaving protease. The aim of this study was to examine ADAMTS13 expression in kidneys from ADAMTS13 wild-type (Adamts13+/+) and deficient (Adamts13−/−) mice and to investigate the expression pattern and bioactivity in human glomerular endothelial cells. Methodology/Principal Findings Immunohistochemistry was performed on kidney sections from ADAMTS13 wild-type and ADAMTS13-deficient mice. Phenotypic differences were examined by ultramorphology. ADAMTS13 expression in human glomerular endothelial cells and dermal microvascular endothelial cells was investigated by real-time PCR, flow cytometry, immunofluorescence and immunoblotting. VWF cleavage was demonstrated by multimer structure analysis and immunoblotting. ADAMTS13 was demonstrated in glomerular endothelial cells in Adamts13+/+ mice but no staining was visible in tissue from Adamts13−/− mice. Thickening of glomerular capillaries with platelet deposition on the vessel wall was detected in Adamts13−/− mice. ADAMTS13 mRNA and protein were detected in both human endothelial cells and the protease was secreted. ADAMTS13 activity was demonstrated in glomerular endothelial cells as cleavage of VWF. Conclusions/Significance Glomerular endothelial cells express and secrete ADAMTS13. The proteolytic activity could have a protective effect preventing deposition of platelets along capillary lumina under the conditions of high shear stress present in glomerular capillaries. PMID:21720563

  16. The Novel Methods for Analysis of Exosomes Released from Endothelial Cells and Endothelial Progenitor Cells

    PubMed Central

    Wang, Jinju; Guo, Runmin; Yang, Yi; Jacobs, Bradley; Chen, Suhong; Iwuchukwu, Ifeanyi; Gaines, Kenneth J.; Chen, Yanfang; Simman, Richard; Lv, Guiyuan; Wu, Keng; Bihl, Ji C.

    2016-01-01

    Exosomes (EXs) are cell-derived vesicles that mediate cell-cell communication and could serve as biomarkers. Here we described novel methods for purification and phenotyping of EXs released from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads and fluorescence quantum dots (Q-dots®) techniques. EXs from the culture medium of ECs and EPCs were isolated and detected with cell-specific antibody conjugated microbeads and second antibody conjugated Q-dots by using nanoparticle tracking analysis (NTA) system. The sensitivities of the cell origin markers for ECs (CD105, CD144) and EPCs (CD34, KDR) were evaluated. The sensitivity and specificity were determined by using positive and negative markers for EXs (CD63), platelets (CD41), erythrocytes (CD235a), and microvesicles (Annexin V). Moreover, the methods were further validated in particle-free plasma and patient samples. Results showed that anti-CD105/anti-CD144 and anti-CD34/anti-KDR had the highest sensitivity and specificity for isolating and detecting EC-EXs and EPC-EXs, respectively. The methods had the overall recovery rate of over 70% and were able to detect the dynamical changes of circulating EC-EXs and EPC-EXs in acute ischemic stroke. In conclusion, we have developed sensitive and specific microbeads/Q-dots fluorescence NTA methods for EC-EX and EPC-EX isolation and detection, which will facilitate the functional study and biomarker discovery. PMID:27118976

  17. The Novel Methods for Analysis of Exosomes Released from Endothelial Cells and Endothelial Progenitor Cells.

    PubMed

    Wang, Jinju; Guo, Runmin; Yang, Yi; Jacobs, Bradley; Chen, Suhong; Iwuchukwu, Ifeanyi; Gaines, Kenneth J; Chen, Yanfang; Simman, Richard; Lv, Guiyuan; Wu, Keng; Bihl, Ji C

    2016-01-01

    Exosomes (EXs) are cell-derived vesicles that mediate cell-cell communication and could serve as biomarkers. Here we described novel methods for purification and phenotyping of EXs released from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads and fluorescence quantum dots (Q-dots®) techniques. EXs from the culture medium of ECs and EPCs were isolated and detected with cell-specific antibody conjugated microbeads and second antibody conjugated Q-dots by using nanoparticle tracking analysis (NTA) system. The sensitivities of the cell origin markers for ECs (CD105, CD144) and EPCs (CD34, KDR) were evaluated. The sensitivity and specificity were determined by using positive and negative markers for EXs (CD63), platelets (CD41), erythrocytes (CD235a), and microvesicles (Annexin V). Moreover, the methods were further validated in particle-free plasma and patient samples. Results showed that anti-CD105/anti-CD144 and anti-CD34/anti-KDR had the highest sensitivity and specificity for isolating and detecting EC-EXs and EPC-EXs, respectively. The methods had the overall recovery rate of over 70% and were able to detect the dynamical changes of circulating EC-EXs and EPC-EXs in acute ischemic stroke. In conclusion, we have developed sensitive and specific microbeads/Q-dots fluorescence NTA methods for EC-EX and EPC-EX isolation and detection, which will facilitate the functional study and biomarker discovery.

  18. Glioma-associated endothelial cells are chemoresistant to temozolomide.

    PubMed

    Virrey, Jenilyn J; Golden, Encouse B; Sivakumar, Walavan; Wang, Weijun; Pen, Ligaya; Schönthal, Axel H; Hofman, Florence M; Chen, Thomas C

    2009-10-01

    Temozolomide is considered the standard of care and drug of choice for the treatment of initially diagnosed malignant gliomas. Although well tolerated, temozolomide still has limited clinical efficacy. Following drug treatment, patient prognosis still remains poor; tumor recurrence is almost universal. We hypothesized that this lack of effectiveness with temozolomide is because this drug does not target the glioma microenvironment, which is highly vascular in malignant gliomas. To test this hypothesis we analyzed the effects of temozolomide on the tumor vasculature in vitro and in vivo. We found that this drug did not affect the viability or proliferation rate of endothelial cells isolated from human glioma specimens, although temozolomide was highly cytotoxic to the glioma cell lines U87MG and U251. Furthermore, temozolomide did not inhibit the migration of these glioma-associated endothelial cells, a key mechanism responsible for tumor angiogenesis. In in vivo studies, using the intracranial glioma mouse model, temozolomide did not cause a pronounced effect on microvessel density. Our findings show that temozolomide has no apparent effect on the glioma vascular microenvironment. Thus combination therapy with anti-vascular agents may enhance temozolomide effectiveness as glioma therapeutic protocol.

  19. Activated Brain Endothelial Cells Cross-Present Malaria Antigen

    PubMed Central

    Howland, Shanshan W.; Poh, Chek Meng; Rénia, Laurent

    2015-01-01

    In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention. PMID:26046849

  20. PTEN overexpression attenuates angiogenic processes of endothelial cells by blockade of endothelin-1/endothelin B receptor signaling.

    PubMed

    Kuo, Hsiao-Mei; Lin, Chun-Yao; Lam, Hing-Chung; Lin, Pey-Ru; Chan, Hoi-Hung; Tseng, Jui-Cheng; Sun, Cheuk-Kwan; Hsu, Te-Fa; Wu, Chia-Ching; Yang, Chao-Yuh; Hsu, Ching-Mei; Tai, Ming-Hong

    2012-04-01

    Arteriovenous (AV) graft is frequently used as vascular access in hemodialysis patients. However, clotting or thrombosis of AV grafts often occurs and requires surgical removal. At present, the molecular pathogenesis underlying thrombosis of AV graft is not clear. The PTEN/Akt signaling has been implicated in the pathogenesis of vascular diseases. In this study, elevated PTEN expression and concomitant Akt inactivation was observed in endothelium of atherosclerotic brachial arteries from hemodialysis patients. To investigate whether PTEN upregulation affects endothelial function, adenovirus-mediated PTEN (Ad-PTEN) overexpression was performed in aorta rings and cultured endothelial cells. It was found that PTEN overexpression potently inhibited the microvessel sprouting in aorta rings and the angiogenic activities of endothelial cells including migration and tube formation. On the contrary, PTEN knockdown by RNA interference promoted the endothelial migration and reversed the Ad-PTEN-induced inhibition of endothelial migration. Expression analysis showed that PTEN overexpression attenuated the expression of endothelin-1 (ET-1) and endothelin B receptor (ETBR) in endothelial cells at transcriptional levels. However, exogenous ET-1 supply only partially reversed the PTEN-induced inhibition of migration and tube formation. This was delineated due to that PTEN overexpression also perturbed endothelial nitric oxide synthase (eNOS) activation and vascular endothelial growth factor (VEGF) release. In summary, PTEN upregulation induces endothelial dysfunction by attenuating the availability and signaling of multiple angiogenic pathways in endothelial cells, thereby may contribute to thrombosis of AV graft.

  1. Endothelial cell markers reflecting endothelial cell dysfunction in patients with mixed connective tissue disease

    PubMed Central

    2010-01-01

    Introduction The aim of the present study was to investigate the association between cardiovascular risk factors and endothelial dysfunction in patients with mixed connective tissue disease (MCTD) and to determine which biomarkers are associated with atherosclerotic complications, such as cardiovascular disease. Methods Fifty MCTD patients and 38 healthy age-matched and sex-matched controls were enrolled in this study. In order to describe endothelial dysfunction, we assessed flow-mediated dilation (FMD), nitrate-mediated dilation (NMD) and carotid artery intima-media thickness (IMT). We investigated FMD of the brachial artery after reactive hyperemia and NMD after sublingual nitroglycerin administration, while the IMT of the common carotid artery was determined by ultrasound. Anti-U1 ribonucleoprotein (anti-U1RNP) antibodies, anti-cardiolipin (anti-CL) antibodies, anti-endothelial cell antibody (AECA) and endothelial cell markers, such as soluble thrombomodulin (TM) and von Willebrand factor antigen (vWFAg), were assessed. Results The endothelium-dependent vasodilation (FMD) was significantly impaired in patients with MCTD, as compared with controls (%FMD: 4.7 ± 4.2% vs. 8.7 ± 5.0%; P < 0.001), while the percentage NMD did not differ (%NMD: 14.3 ± 6.6% vs. 17.1 ± 6.7%; P = 0.073). Mean carotid IMT values were higher in patients than in controls (IMT: MCTD, 0.64 ± 0.13 mm vs. controls, 0.53 ± 0.14 mm; P < 0.001). FMD negatively correlated with disease duration, the levels of apolipoprotein A1, the paraoxonase-1 activity, and systolic blood pressure in MCTD patients. The percentage FMD was significantly lower in MCTD patients with cardiovascular diseases (CVD), than in those without CVD (%FMD: 3.5 ± 2.9 vs. 5.8 ± 4.8, P < 0.0002), while percentage NMD did not differ between patients with and without CVDs. Serum levels of autoantibodies (anti-U1RNP, AECA and anti-CL) were significantly higher in MCTD patients and differed between MCTD patients with and

  2. Phagocytosis by glomerular endothelial cells in infection-related glomerulopathy.

    PubMed

    van Velthuysen, M L; Mayen, A E; Prins, F A; de Heer, E; Bruijn, J A; Fleuren, G J

    1994-01-01

    Glomerulonephritis in BALB/c mice following infection with Trypanosoma brucei is characterized by albuminuria and glomerular deposition of immunoglobulins. Electron-dense deposits are present in the mesangium, as well as subendothelially and subepithelially along the glomerular capillary wall. In this study the nature of intracytoplasmic, electron-dense, round structures observed in glomerular endothelial cells was investigated by immunoelectron-microscopy and enzyme histochemistry. The presence of these structures was related in time with the development of proteinuria. Mice from the C57BL10 strain, which upon infection develop glomerular immune complexes without proteinuria, were examined as well. The results demonstrated that the first endothelial changes, occurring 3-4 weeks after infection, were swelling of endothelial cells containing intracytoplasmic, electron-dense, round structures. These changes were seen prior to the onset of proteinuria, and were not present in glomeruli of mice that did not develop proteinuria. The endothelial granules were shown to contain immunoglobulins and typical lysosomal enzymes, providing evidence for phagocytosis by the glomerular endothelial cells. Liver endothelial cells did not show comparable changes. Thus, local phagocytosis by glomerular endothelial cells is shown to be a specific event in the development of glomerular disease. PMID:7800204

  3. Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions

    PubMed Central

    1996-01-01

    Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE- cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE- cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen- reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE

  4. Crosstalk between cancer cells and blood endothelial and lymphatic endothelial cells in tumour and organ microenvironment

    PubMed Central

    Lee, Esak; Pandey, Niranjan B.; Popel, Aleksander S.

    2015-01-01

    Tumour and organ microenvironments are crucial for cancer progression and metastasis. Crosstalk between multiple non-malignant cell types in the microenvironments and cancer cells promotes tumour growth and metastasis. Blood and lymphatic endothelial cells (BEC and LEC) are two of the components in the microenvironments. Tumour blood vessels (BV), comprising BEC, serve as conduits for blood supply into the tumour, and are important for tumour growth as well as haematogenous tumour dissemination. Lymphatic vessels (LV), comprising LEC, which are relatively leaky compared with BV, are essential for lymphogenous tumour dissemination. In addition to describing the conventional roles of the BV and LV, we also discuss newly emerging roles of these endothelial cells: their crosstalk with cancer cells via molecules secreted by the BEC and LEC (also called angiocrine and lymphangiocrine factors). This review suggests that BEC and LEC in various microenvironments can be orchestrators of tumour progression and proposes new mechanism-based strategies to discover new therapies to supplement conventional anti-angiogenic and anti-lymphangiogenic therapies. PMID:25634527

  5. Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

    SciTech Connect

    Askari, Ara A.; Thomson, Scott; Edin, Matthew L.; Lih, Fred B.; Zeldin, Darryl C.; Bishop-Bailey, David

    2014-04-04

    Highlights: • We examined epoxygenase product formation and regulation in endothelial cells. • The epoxygenase CYP2J2 is an LPS (TLR-4) inducible enzyme in endothelial cells. • The endothelial cell line EA.Hy926 synthesises epoxygenase products. • Inhibition of endothelial epoxygenases increases TNFα secretion. • Soluble epoxide hydrolase inhibitors reduce inflammation-induced TNFα and NFκB. - Abstract: The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity.

  6. Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation.

    PubMed

    Cary, Lynnette H; Noutai, Daniel; Salber, Rudolph E; Williams, Margaret S; Ngudiankama, Barbara F; Whitnall, Mark H

    2014-06-01

    caspase 3. Irradiation of Jurkat cells caused a G2/M arrest and increased adherence to HUVEC. When co-cultured with HUVEC, irradiated Jurkat cells exhibited G0/G1 arrest and increased apoptosis. The data indicate that gene expression and cell function of endothelial cells and hematopoietic cells are influenced by radiation and by interactions between the two cell types. These phenomena may affect the success of therapies for ARS and cancer. PMID:24828109

  7. The natural antioxidants, pomegranate extract and soy isoflavones, favourably modulate canine endothelial cell function.

    PubMed

    Baumgartner-Parzer, Sabina M; Waldenberger, Ferdinand Rudolf; Freudenthaler, Angelika; Ginouvès-Guerdoux, Amandine; McGahie, David; Gatto, Hugues

    2012-01-01

    Cardiovascular disease, preceded by vascular endothelial dysfunction, is a prominent cause of death in dogs. L-carnitine and taurine, well known for their antioxidative capacity, beneficially affect cardiovascular disease as well as certain dog cardiomyopathies. It is well established that vascular endothelial dysfunction precedes cardiovascular disease and that "vasoprotective factors" (NO and antioxidants) prevent apoptosis, whereas "risk factors" such as oxidized LDL, hyperglycemia, and free fatty acids trigger it in cultured human vascular endothelial cells. Whereas human vascular cell in vitro models are widely established and used for the characterisation of potential vasoprotective substances, such models are not available for canine endothelial cells. In the present study we therefore developed an in vitro model, which allows the testing of the effects of different substances on proliferation and apoptosis in canine aortic endothelial cells. This model was used to test L-carnitine, taurine, pomegranate extract, and Soy Isoflavones in comparison to reference substances (glutathione and pioglitazone) previously shown to modulate human endothelial cell function. L-carnitine and taurine neither exhibited antiproliferative nor antiapoptotic activities in the context of this study. However extracts from pomegranate and soy isoflavones dramatically reduced proliferation and apoptosis in a dose dependent fashion, being in line with a vasoprotective activity in dogs.

  8. The Impact of Microgravity and Hypergravity on Endothelial Cells

    PubMed Central

    Maier, Jeanette A. M.

    2015-01-01

    The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature. PMID:25654101

  9. The impact of microgravity and hypergravity on endothelial cells.

    PubMed

    Maier, Jeanette A M; Cialdai, Francesca; Monici, Monica; Morbidelli, Lucia

    2015-01-01

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

  10. Fibronectin coating of oxygenator membranes enhances endothelial cell attachment

    PubMed Central

    2013-01-01

    Background Extracorporeal membrane oxygenation (ECMO) can replace the lungs’ gas exchange capacity in refractory lung failure. However, its limited hemocompatibility, the activation of the coagulation and complement system as well as plasma leakage and protein deposition hamper mid- to long-term use and have constrained the development of an implantable lung assist device. In a tissue engineering approach, lining the blood contact surfaces of the ECMO device with endothelial cells might overcome these limitations. As a first step towards this aim, we hypothesized that coating the oxygenator’s gas exchange membrane with proteins might positively influence the attachment and proliferation of arterial endothelial cells. Methods Sheets of polypropylene (PP), polyoxymethylpentene (TPX) and polydimethylsiloxane (PDMS), typical material used for oxygenator gas exchange membranes, were coated with collagen, fibrinogen, gelatin or fibronectin. Tissue culture treated well plates served as controls. Endothelial cell attachment and proliferation were analyzed for a period of 4 days by microscopic examination and computer assisted cell counting. Results Endothelial cell seeding efficiency is within range of tissue culture treated controls for fibronectin treated surfaces only. Uncoated membranes as well as all other coatings lead to lower cell attachment. A confluent endothelial cell layer develops on fibronectin coated PDMS and the control surface only. Conclusions Fibronectin increases endothelial cells’ seeding efficiency on different oxygenator membrane material. PDMS coated with fibronectin shows sustained cell attachment for a period of four days in static culture conditions. PMID:23356939

  11. Endothelial cell tumor growth is Ape/ref-1 dependent.

    PubMed

    Biswas, Ayan; Khanna, Savita; Roy, Sashwati; Pan, Xueliang; Sen, Chandan K; Gordillo, Gayle M

    2015-09-01

    Tumor-forming endothelial cells have highly elevated levels of Nox-4 that release H2O2 into the nucleus, which is generally not compatible with cell survival. We sought to identify compensatory mechanisms that enable tumor-forming endothelial cells to survive and proliferate under these conditions. Ape-1/ref-1 (Apex-1) is a multifunctional protein that promotes DNA binding of redox-sensitive transcription factors, such as AP-1, and repairs oxidative DNA damage. A validated mouse endothelial cell (EOMA) tumor model was used to demonstrate that Nox-4-derived H2O2 causes DNA oxidation that induces Apex-1 expression. Apex-1 functions as a chaperone to keep transcription factors in a reduced state. In EOMA cells Apex-1 enables AP-1 binding to the monocyte chemoattractant protein-1 (mcp-1) promoter and expression of that protein is required for endothelial cell tumor formation. Intraperitoneal injection of the small molecule inhibitor E3330, which specifically targets Apex-1 redox-sensitive functions, resulted in a 50% decrease in tumor volume compared with mice injected with vehicle control (n = 6 per group), indicating that endothelial cell tumor proliferation is dependent on Apex-1 expression. These are the first reported results to establish Nox-4 induction of Apex-1 as a mechanism promoting endothelial cell tumor formation.

  12. Stereoselectivity of ectonucleotidases on vascular endothelial cells.

    PubMed Central

    Cusack, N J; Pearson, J D; Gordon, J L

    1983-01-01

    We have investigated the stereoselectivity of ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5'-nucleotidase, EC 3.1.3.5) on pig aortic endothelial cells using two classes of nucleotide analogue. In experiments with nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety, the rate of catabolism of 100 microM-L-ATP was one-fifth that of D-ATP, the rate of catabolism of 100 microM-L-ADP was one-fifteenth that of D-ADP and there was no detectable catabolism of 100 microM-L-AMP. Each of the L-enantiomers inhibited, apparently competitively, the catabolism of the corresponding D-enantiomer; Ki values were approx. 0.6 mM, 1.0 mM and 3.9 mM for L-ATP, L-ADP and L-AMP respectively. Experiments with adenosine 5'-[beta, gamma-imido]triphosphate and with D- and L-enantiomers of adenosine 5'-[beta, gamma-methylene]triphosphate revealed modest ectopyrophosphatase activity, undetectable in experiments with natural nucleotides, which was also stereoselective. Use of phosphorothioate nucleotide analogues demonstrated that ATP catabolism was virtually stereospecific with respect to the geometry of the thiol group substituted on the beta-phosphate: the Rp isomer was degraded, whereas there was little or no breakdown of the Sp isomer. ADP catabolism was also stereospecific with respect to the geometry of the thiol group substituted on the alpha-phosphate: the Sp isomer but not the Rp isomer was degraded. The geometry of thiol-group substitution on the alpha-phosphate had no effect on ATP catabolism to ADP. There was no detectable catabolism of analogues with thiol-group substitution on the terminal phosphate. Each of the phosphorothioate analogues that was catabolized broke down at a rate similar to that of the natural nucleotide from which it was derived. These results demonstrate that the ectonucleotidases on pig aortic endothelial cells exhibit a high degree of stereoselectivity

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

    PubMed

    Tran, Jason; Magenau, Astrid; Rodriguez, Macarena; Rentero, Carles; Royo, Teresa; Enrich, Carlos; Thomas, Shane R; Grewal, Thomas; Gaus, Katharina

    2016-01-01

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

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

    PubMed

    Tran, Jason; Magenau, Astrid; Rodriguez, Macarena; Rentero, Carles; Royo, Teresa; Enrich, Carlos; Thomas, Shane R; Grewal, Thomas; Gaus, Katharina

    2016-01-01

    Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells. PMID:26977592

  15. Substituted oxines inhibit endothelial cell proliferation and angiogenesis†

    PubMed Central

    Bhat, Shridhar; Shim, Joong Sup; Zhang, Feiran; Chong, Curtis Robert; Liu, Jun O.

    2013-01-01

    Two substituted oxines, nitroxoline (5) and 5-chloroquinolin-8-yl phenylcarbamate (22), were identified as hits in a high-throughput screen aimed at finding new anti-angiogenic agents. In a previous study, we have elucidated the molecular mechanism of antiproliferative activity of nitroxoline in endothelial cells, which comprises of a dual inhibition of type 2 human methionine aminopeptidase (MetAP2) and sirtuin 1 (SIRT1). Structure–activity relationship study (SAR) of nitroxoline offered many surprises where minor modifications yielded oxine derivatives with increased potency against human umbilical vein endothelial cells (HUVEC), but with entirely different as yet unknown mechanisms. For example, 5-nitrosoquinolin-8-ol (33) inhibited HUVEC growth with sub-micromolar IC50, but did not affect MetAP2 or MetAP1, and it only showed weak inhibition against SIRT1. Other sub-micromolar inhibitors were derivatives of 5-aminoquinolin-8-ol (34) and 8-sulfonamidoquinoline (32). A sulfamate derivative of nitroxoline (48) was found to be more potent than nitroxoline with the retention of activities against MetAP2 and SIRT1. The bioactivity of the second hit, micromolar HUVEC and MetAP2 inhibitor carbamate 22 was improved further with an SAR study culminating in carbamate 24 which is a nanomolar inhibitor of HUVEC and MetAP2. PMID:22391578

  16. Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function.

    PubMed Central

    Tiruppathi, C; Malik, A B; Del Vecchio, P J; Keese, C R; Giaever, I

    1992-01-01

    We have developed an electrical method to study endothelial cell shape changes in real time in order to examine the mechanisms of alterations in the endothelial barrier function. Endothelial shape changes were quantified by using a monolayer of endothelial cells grown on a small (10(-3) cm2) evaporated gold electrode and measuring the changes in electrical impedance. Bovine pulmonary microvessel endothelial cells and bovine pulmonary artery endothelial cells were used to study the effects of alpha-thrombin on cell-shape dynamics by the impedance measurement. alpha-Thrombin produced a dose-dependent decrease in impedance that occurred within 0.5 min in both cell types, indicative of retraction of endothelial cells and widening of interendothelial junctions because of "rounding up" of the cells. The alpha-thrombin-induced decrease in impedance persisted for approximately 2 hr, after which the value recovered to basal levels. Pretreatment of endothelial cells with the protein kinase C inhibitor, calphostin C, or with 8-bromoadenosine 3',5'-cyclic monophosphate prevented the decreased impedance, suggesting that the endothelial cell change is modulated by activation of second-messenger pathways. The alpha-thrombin-induced decrease in impedance was in agreement with the previously observed increases in transendothelial albumin permeability and evidence of formation of intercellular gaps after alpha-thrombin challenge. The impedance measurement may be a valuable in vitro method for the assessment of mechanisms of decreased endothelial barrier function occurring with inflammatory mediators. Since the rapidly occurring changes in endothelial cell shape in response to mediators such as thrombin are mediated activation of second-messenger pathways, the ability to monitor endothelial cell dynamics in real time may provide insights into the signal-transduction events mediating the increased endothelial permeability. PMID:1518814

  17. Platelet endothelial cell adhesion molecule-1 modulates endothelial cell motility through the small G-protein Rho.

    PubMed

    Gratzinger, Dita; Canosa, Sandra; Engelhardt, Britta; Madri, Joseph A

    2003-08-01

    Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoglobulin family vascular adhesion molecule, is involved in endothelial cell migration and angiogenesis (1, 2). We found that endothelial cells lacking PECAM-1 exhibit increased single cell motility and extension formation but poor wound healing migration, reminiscent of cells in which Rho activity has been suppressed by overexpressing a GTPase-activating protein (3). The ability of PECAM-1 to restore wound healing migration to PECAM-1-deficient cells was independent of its extracellular domain or signaling via its immunoreceptor tyrosine-based inhibitory motif. PECAM-1-deficient endothelial cells had a selective defect in RhoGTP loading, and inhibition of Rho activity mimicked the PECAM-1-deficient phenotype of increased chemokinetic single cell motility at the expense of coordinated wound healing migration. The wound healing advantage of PECAM-1-positive endothelial cells was not only Rho mediated but pertussis toxin inhibitable, characteristic of migration mediated by heterotrimeric G-protein-linked seven-transmembrane receptor signaling such as signaling in response to the serum sphingolipid sphingosine-1-phosphate (S1P) (4, 5). Indeed, we found that the wound healing defect of PECAM-1 null endothelial cells is minimized in sphingolipid-depleted media; moreover, PECAM-1 null endothelial cells fail to increase their migration in response to S1P. We have also found that PECAM-1 localizes to rafts and that in its absence heterotrimeric G-protein components are differentially recruited to rafts, providing a potential mechanism for PECAM-1-mediated coordination of S1P signaling. PECAM-1 may thus support the effective S1P/RhoGTP signaling required for wound healing endothelial migration by allowing for the spatially directed, coordinated activation of Galpha signaling pathways. PMID:12890700

  18. Changes of myoid and endothelial cells in the peritubular wall during contraction of the seminiferous tubule.

    PubMed

    Losinno, Antonella D; Sorrivas, Viviana; Ezquer, Marcelo; Ezquer, Fernando; López, Luis A; Morales, Alfonsina

    2016-08-01

    The wall of the seminiferous tubule in rodents consists of an inner layer of myoid cells covered by an outer layer of endothelial cells. Myoid cells are a type of smooth muscle cell containing α-actin filaments arranged in two independent layers that contract when stimulated by endothelin-1. The irregular surface relief of the tubular wall is often considered a hallmark of contraction induced by a variety of stimuli. We examine morphological changes of the rat seminiferous tubule wall during contraction by a combination of light, confocal, transmission and scanning electron microscopy. During ET-1-induced contraction, myoid cells changed from a flat to a conical shape, but their actin filaments remained in independent layers. As a consequence of myoid cell contraction, the basement membrane became wavy, orientation of collagen fibers in the extracellular matrix was altered and the endothelial cell layer became folded. To observe the basement of the myoid cell cone, the endothelial cell monolayer was removed by collagenase digestion prior to SEM study. In contracted tubules, it is possible to distinguish cell relief: myoid cells have large folds on the external surface oriented parallel to the tubular axis, whereas endothelial cells have numerous cytoplasmic projections facing the interstitium. The myoid cell cytoskeleton is unusual in that the actin filaments are arranged in two orthogonal layers, which adopt differing shapes during contraction with myoid cells becoming cone-shaped. This arrangement impacts on other components of the seminiferous tubule wall and affects the propulsion of the tubular contents to the rete testis.

  19. Cellular and molecular biology of aging endothelial cells.

    PubMed

    Donato, Anthony J; Morgan, R Garrett; Walker, Ashley E; Lesniewski, Lisa A

    2015-12-01

    Cardiovascular disease (CVD) is the leading cause of death in the United States and aging is a major risk factor for CVD development. One of the major age-related arterial phenotypes thought to be responsible for the development of CVD in older adults is endothelial dysfunction. Endothelial function is modulated by traditional CVD risk factors in young adults, but advancing age is independently associated with the development of vascular endothelial dysfunction. This endothelial dysfunction results from a reduction in nitric oxide bioavailability downstream of endothelial oxidative stress and inflammation that can be further modulated by traditional CVD risk factors in older adults. Greater endothelial oxidative stress with aging is a result of augmented production from the intracellular enzymes NADPH oxidase and uncoupled eNOS, as well as from mitochondrial respiration in the absence of appropriate increases in antioxidant defenses as regulated by relevant transcription factors, such as FOXO. Interestingly, it appears that NFkB, a critical inflammatory transcription factor, is sensitive to this age-related endothelial redox change and its activation induces transcription of pro-inflammatory cytokines that can further suppress endothelial function, thus creating a vicious feed-forward cycle. This review will discuss the two macro-mechanistic processes, oxidative stress and inflammation, that contribute to endothelial dysfunction with advancing age as well as the cellular and molecular events that lead to the vicious cycle of inflammation and oxidative stress in the aged endothelium. Other potential mediators of this pro-inflammatory endothelial phenotype are increases in immune or senescent cells in the vasculature. Of note, genomic instability, telomere dysfunction or DNA damage has been shown to trigger cell senescence via the p53/p21 pathway and result in increased inflammatory signaling in arteries from older adults. This review will discuss the current state

  20. Toxicity of oxygen radicals in cultured pulmonary endothelial cells

    SciTech Connect

    Autor, A.P.; Bonham, A.C.; Thies, R.L.

    1984-01-01

    Superoxide dismutase and catalase, which catalytically remove superoxide radicals and hydrogen perioxide, respectively, each separately protected cultured pulmonary artery endothelial cells from loss of membrane integrity after exposure to oxygen radicals generated either cellularly (polymorphonuclear leukocytes) or chemically (dihydroxyfumarate). Nicotinamide, a precursor of nicotinamide-adenine dinucleotide (NAD) and an inhibitor of ADP-ribose synthetase, also protected cultured endothelial cells from loss of membrane integrity in a concentration-dependent manner after exposure to dihydroxyfumarate.

  1. 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

  2. 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.

  3. Putative Key Role of Inositol Messengers in Endothelial Cells in Preeclampsia

    PubMed Central

    Kunjara, Sirilaksana; McLean, Patricia; Rademacher, Laurens; Rademacher, Thomas W.; Fascilla, Fabiana; Bettocchi, Stefano

    2016-01-01

    Immunological alterations, endothelial dysfunction, and insulin resistance characterize preeclampsia. Endothelial cells hold the key role in the pathogenesis of this disease. The signaling pathways mediating these biological abnormalities converge on PKB/Akt, an intracellular kinase regulating cell survival, proliferation, and metabolism. Inositol second messengers are involved in metabolic and cell signaling pathways and are highly expressed during preeclampsia. Intracellular action of these molecules is deeply affected by zinc, manganese, and calcium. To evaluate the pathophysiological significance, we present the response of the intracellular pathways of inositol phosphoglycans involved in cellular metabolism and propose a link with the disease. PMID:27738431

  4. 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

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

    SciTech Connect

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

    2007-12-01

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

  6. Traction Forces of Endothelial Cells under Slow Shear Flow

    PubMed Central

    Perrault, Cecile M.; Brugues, Agusti; Bazellieres, Elsa; Ricco, Pierre; Lacroix, Damien; Trepat, Xavier

    2015-01-01

    Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeostasis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow regime in which endothelial cell mechanics is acutely responsive to shear stress. PMID:26488643

  7. Response of endothelial cells to decellularized extracellular matrix deposited by bone marrow mesenchymal stem cells

    PubMed Central

    Xu, Yue; Yan, Mengdie; Gong, Yihong; Chen, Lei; Zhao, Feng; Zhang, Zhaoqiang

    2014-01-01

    Objective: Evaluate the behavior and function of human umbilical vein endothelial cells (HUVECs) on decellularized extracellular matrix (ECM) deposited by bone marrow mesenchymal stem cells (BMSCs). Methods: Prepared through chemical approach, decellularized ECM was characterized by use of immunofluorescence staining. The morphology, attachment, proliferation and migration of HUVECs cultured on six-well tissue culture plastic (TCP) and decellularized ECM were investigated. Results: Decellularized ECM was successfully prepared without three-dimensional architecture disruption. This biological scaffold is similar to nature vascular ECM, preserved various matrix proteins such as type I collagen, type III collagen and fibronection. HUVECs on decellularized ECM showed well attachment and regular arrangement. Decellularized ECM could also significantly enhance the migration and proliferation potential of HUVECs in contrast to TCP. Conclusion: Deposited by BMSCs, ECM can affect the behavior of endothelial cell and could be used as a promising material in tissue engineering. PMID:25663998

  8. Oxidative stress modulates nucleobase transport in microvascular endothelial cells.

    PubMed

    Bone, Derek B J; Antic, Milica; Vilas, Gonzalo; Hammond, James R

    2014-09-01

    Purine nucleosides and nucleobases play key roles in the physiological response to vascular ischemia/reperfusion events. The intra- and extracellular concentrations of these compounds are controlled, in part, by equilibrative nucleoside transporter subtype 1 (ENT1; SLC29A1) and by equilibrative nucleobase transporter subtype 1 (ENBT1). These transporters are expressed at the membranes of numerous cell types including microvascular endothelial cells. We studied the impact of reactive oxygen species on the function of ENT1 and ENBT1 in primary (CMVEC) and immortalized (HMEC-1) human microvascular endothelial cells. Both cell types displayed similar transporter expression profiles, with the majority (>90%) of 2-chloro[(3)H]adenosine (nucleoside) uptake mediated by ENT1 and [(3)H]hypoxanthine (nucleobase) uptake mediated by ENBT1. An in vitro mineral oil-overlay model of ischemia/reperfusion had no effect on ENT1 function, but significantly reduced ENBT1 Vmax in both cell types. This decrease in transport function was mimicked by the intracellular superoxide generator menadione and could be reversed by the superoxide dismutase mimetic MnTMPyP. In contrast, neither the extracellular peroxide donor TBHP nor the extracellular peroxynitrite donor 3-morpholinosydnonimine (SIN-1) affected ENBT1-mediated [(3)H]hypoxanthine uptake. SIN-1 did, however, enhance ENT1-mediated 2-chloro[(3)H]adenosine uptake. Our data establish HMEC-1 as an appropriate model for study of purine transport in CMVEC. Additionally, these data suggest that the generation of intracellular superoxide in ischemia/reperfusion leads to the down-regulation of ENBT1 function. Modification of purine transport by oxidant stress may contribute to ischemia/reperfusion induced vascular damage and should be considered in the development of therapeutic strategies.

  9. Impact of simulated microgravity on microvascular endothelial cell apoptosis.

    PubMed

    Kang, Chun-Yan; Zou, Lin; Yuan, Ming; Wang, Yang; Li, Tian-Zhi; Zhang, Ye; Wang, Jun-Feng; Li, Yan; Deng, Xiao-Wei; Liu, Chang-Ting

    2011-09-01

    Cardiovascular deconditioning is known to occur in astronauts exposed to microgravity. Endothelial dysfunction at microcirculatory sites might contribute to cardiovascular deconditioning induced by weightlessness. Recent studies have reported changes in the morphology and gene expression of endothelial cells exposed to conditions of simulated microgravity. The present study was aimed at examining the effects of microgravity on the apoptosis of microvascular endothelial cells and the mechanism underlying these effects. We simulated a microgravity environment and found that microgravity induced microvascular endothelial cell apoptosis and that this effect was correlated with the downregulation of the PI3K/Akt pathway, increased expression of NF-κB, and depolymerization of F-actin. These findings may provide important insights into the origin of the adverse physiological changes occurring due to exposure to microgravity conditions. PMID:21287193

  10. Impact of simulated microgravity on microvascular endothelial cell apoptosis.

    PubMed

    Kang, Chun-Yan; Zou, Lin; Yuan, Ming; Wang, Yang; Li, Tian-Zhi; Zhang, Ye; Wang, Jun-Feng; Li, Yan; Deng, Xiao-Wei; Liu, Chang-Ting

    2011-09-01

    Cardiovascular deconditioning is known to occur in astronauts exposed to microgravity. Endothelial dysfunction at microcirculatory sites might contribute to cardiovascular deconditioning induced by weightlessness. Recent studies have reported changes in the morphology and gene expression of endothelial cells exposed to conditions of simulated microgravity. The present study was aimed at examining the effects of microgravity on the apoptosis of microvascular endothelial cells and the mechanism underlying these effects. We simulated a microgravity environment and found that microgravity induced microvascular endothelial cell apoptosis and that this effect was correlated with the downregulation of the PI3K/Akt pathway, increased expression of NF-κB, and depolymerization of F-actin. These findings may provide important insights into the origin of the adverse physiological changes occurring due to exposure to microgravity conditions.

  11. Neuropilin2 expressed in gastric cancer endothelial cells increases the proliferation and migration of endothelial cells in response to VEGF

    SciTech Connect

    Kim, Woo Ho; Lee, Sun Hee; Jung, Myung Hwan; Seo, Ji Heun; Kim, Jin; Kim, Min A; Lee, You Mie

    2009-08-01

    The structure and characteristics of the tumor vasculature are known to be different from those of normal vessels. Neuropilin2 (Nrp2), which is expressed in non-endothelial cell types, such as neuronal or cancer cells, functions as a receptor for both semaphorin and vascular endothelial growth factor (VEGF). After isolating tumor and normal endothelial cells from advanced gastric cancer tissue and normal gastric mucosa tissues, respectively, we identified genes that were differentially expressed in gastric tumor endothelial (TEC) and normal endothelial cells (NEC) using DNA oligomer chips. Using reverse transcriptase-PCR, we confirmed the chip results by showing that Nrp2 gene expression is significantly up-regulated in TEC. Genes that were found to be up-regulated in TEC were also observed to be up-regulated in human umbilical vein endothelial cells (HUVECs) that were co-cultured with gastric cancer cells. In addition, HUVECs co-cultured with gastric cancer cells showed an increased reactivity to VEGF-induced proliferation and migration. Moreover, overexpression of Nrp2 in HUVECs significantly enhanced the proliferation and migration induced by VEGF. Observation of an immunohistochemical analysis of various human tumor tissue arrays revealed that Nrp2 is highly expressed in the tumor vessel lining and to a lesser extent in normal tissue microvessels. From these results, we suggest that Nrp2 may function to increase the response to VEGF, which is more significant in TEC than in NEC given the differential expression, leading to gastric TEC with aggressive angiogenesis phenotypes.

  12. Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells.

    PubMed

    Coso, Sanja; Zeng, Yiping; Sooraj, Dhanya; Williams, Elizabeth D

    2011-10-15

    Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.

  13. The effect of bisphosphonates on the endothelial differentiation of mesenchymal stem cells.

    PubMed

    Sharma, Dileep; Hamlet, Stephen Mark; Petcu, Eugen Bogdan; Ivanovski, Saso

    2016-02-09

    The contribution of the local stem cell niche to providing an adequate vascular framework during healing cannot be overemphasized. Bisphosphonates (BPs) are known to have a direct effect on the local vasculature, but their effect on progenitor cell differentiation is unknown. This in vitro study evaluated the effect(s) of various BPs on the differentiation of human placental mesenchymal stem cells (pMSCs) along the endothelial lineage and their subsequent functional and morphogenic capabilities. pMSC multipotency was confirmed by successful differentiation into cells of both the osteogenic and endothelial lineages, as demonstrated by positive Alizarin Red S staining and Ac-LDL uptake. pMSC differentiation in the presence of non-cytotoxic BP concentrations showed that nitrogen containing BPs had a significant inhibitory effect on cell migration and endothelial marker gene expression, as well as compromised endothelial differentiation as demonstrated using von Willebrand factor immunofluorescence staining and tube formation assay. This in vitro study demonstrated that at non-cytotoxic levels, nitrogen-containing BPs inhibit differentiation of pMSCs into cells of an endothelial lineage and affect the downstream functional capability of these cells supporting a multi-modal effect of BPs on angiogenesis as pathogenic mechanism contributing to bone healing disorders such as bisphosphonate related osteonecrosis of the jaws (BRONJ).

  14. The effect of bisphosphonates on the endothelial differentiation of mesenchymal stem cells

    PubMed Central

    Sharma, Dileep; Hamlet, Stephen Mark; Petcu, Eugen Bogdan; Ivanovski, Saso

    2016-01-01

    The contribution of the local stem cell niche to providing an adequate vascular framework during healing cannot be overemphasized. Bisphosphonates (BPs) are known to have a direct effect on the local vasculature, but their effect on progenitor cell differentiation is unknown. This in vitro study evaluated the effect(s) of various BPs on the differentiation of human placental mesenchymal stem cells (pMSCs) along the endothelial lineage and their subsequent functional and morphogenic capabilities. pMSC multipotency was confirmed by successful differentiation into cells of both the osteogenic and endothelial lineages, as demonstrated by positive Alizarin Red S staining and Ac-LDL uptake. pMSC differentiation in the presence of non-cytotoxic BP concentrations showed that nitrogen containing BPs had a significant inhibitory effect on cell migration and endothelial marker gene expression, as well as compromised endothelial differentiation as demonstrated using von Willebrand factor immunofluorescence staining and tube formation assay. This in vitro study demonstrated that at non-cytotoxic levels, nitrogen-containing BPs inhibit differentiation of pMSCs into cells of an endothelial lineage and affect the downstream functional capability of these cells supporting a multi-modal effect of BPs on angiogenesis as pathogenic mechanism contributing to bone healing disorders such as bisphosphonate related osteonecrosis of the jaws (BRONJ). PMID:26857282

  15. Lack of vimentin impairs endothelial differentiation of embryonic stem cells

    PubMed Central

    Boraas, Liana C.; Ahsan, Tabassum

    2016-01-01

    The cytoskeletal filament vimentin is inherent to the endothelial phenotype and is critical for the proper function of endothelial cells in adult mice. It is unclear, however, if the presence of vimentin is necessary during differentiation to the endothelial phenotype. Here we evaluated gene and protein expression of differentiating wild type embryonic stem cells (WT ESCs) and vimentin knockout embryonic stem cells (VIM −/− ESCs) using embryoid bodies (EBs) formed from both cell types. Over seven days of differentiation VIM −/− EBs had altered morphology compared to WT EBs, with a rippled outer surface and a smaller size due to decreased proliferation. Gene expression of pluripotency markers decreased similarly for EBs of both cell types; however, VIM −/− EBs had impaired differentiation towards the endothelial phenotype. This was quantified with decreased expression of markers along the specification pathway, specifically the early mesodermal marker Brachy-T, the lateral plate mesodermal marker FLK1, and the endothelial-specific markers TIE2, PECAM, and VE-CADHERIN. Taken together, these results indicate that the absence of vimentin impairs spontaneous differentiation of ESCs to the endothelial phenotype in vitro. PMID:27480130

  16. Lack of vimentin impairs endothelial differentiation of embryonic stem cells.

    PubMed

    Boraas, Liana C; Ahsan, Tabassum

    2016-01-01

    The cytoskeletal filament vimentin is inherent to the endothelial phenotype and is critical for the proper function of endothelial cells in adult mice. It is unclear, however, if the presence of vimentin is necessary during differentiation to the endothelial phenotype. Here we evaluated gene and protein expression of differentiating wild type embryonic stem cells (WT ESCs) and vimentin knockout embryonic stem cells (VIM -/- ESCs) using embryoid bodies (EBs) formed from both cell types. Over seven days of differentiation VIM -/- EBs had altered morphology compared to WT EBs, with a rippled outer surface and a smaller size due to decreased proliferation. Gene expression of pluripotency markers decreased similarly for EBs of both cell types; however, VIM -/- EBs had impaired differentiation towards the endothelial phenotype. This was quantified with decreased expression of markers along the specification pathway, specifically the early mesodermal marker Brachy-T, the lateral plate mesodermal marker FLK1, and the endothelial-specific markers TIE2, PECAM, and VE-CADHERIN. Taken together, these results indicate that the absence of vimentin impairs spontaneous differentiation of ESCs to the endothelial phenotype in vitro. PMID:27480130

  17. Cytotoxic effects of four antibiotics on endothelial cells.

    PubMed

    Lanbeck, P; Paulsen, O

    1995-12-01

    Intravenous administration of antibiotics often causes local pain and thrombophlebitis at the site of injection. An in vitro model that could predict these effects would be of great value. In this study the effects of four antibiotics, benzylpenicillin, cefuroxime, dicloxacillin and erythromycin, have been evaluated on three types of endothelial cells in culture. The cell types employed were primary culture from human umbilical vein, primary culture from bovine aorta, and the cell line EA-hy 926, a hybride endothelial cell. These cells were exposed to antibiotics for 24 hr and subsequently toxic effects on cells were evaluated by three different assays. Benzylpenicillin was atoxic in all types of cells and in all assays, in contrast to the other antibiotics. The other three antibiotics exerted dose dependent toxic effects in all investigated cells when DNA-synthesis and total cell protein were used as toxicity assays but the results varied between the cell types. There were no significant differences between the effects of cefuroxime, dicloxacillin and erythromycin on bovine endothelial cells. In the other cell types, however, there were significant differences between some drugs but the outcome depended on the cell type. It is concluded that it is possible to show differences between the effect of antibiotics on endothelial cells, but the result depends on the cell type employed.

  18. Endothelial cell phagocytosis of senescent neutrophils decreases procoagulant activity.

    PubMed

    Gao, Chunyan; Xie, Rui; Li, Wen; Zhou, Jin; Liu, Shuchuan; Cao, Fenglin; Liu, Yue; Ma, Ruishuang; Si, Yu; Liu, Yan; Bi, Yayan; Gilbert, Gary E; Shi, Jialan

    2013-06-01

    Abundant senescent neutrophils traverse the vascular compartment and may contribute to pathologic conditions. For example, they become procoagulant when undergoing apoptosis and may contribute to thrombosis or inflammation. Our previous studies demonstrated a dominant clearance pathway in which the neutrophils can be phagocytosed by liver macrophages. The aim of this study was to explore an alternate pathway of neutrophil clearance by endothelial cells. Phagocytosis of the neutrophils by endothelial cells was performed using various experimental approaches includingflow cytometry, confocal microscopy and electron microscopy assays in vitro and in vivo. Procoagulant activity of cultured neutrophils was evaluated by coagulation time, factor Xase and prothrombinase assays. Lactadherin functioned as a novel probe for the detection of phosphatidylserine on apoptotic cells, an opsonin (bridge) between apoptotic cell and phagocyte for promoting phagocytosis, and an efficient anticoagulant for inhibition of factor Xase and thrombin formation. When cultured, purified human neutrophils spontaneously entered apoptosis and developed procoagulant activity that was directly related to the degree of phosphatidylserine exposure. Co-culture of aged neutrophils and endothelial cells resulted in phagocytosis of the neutrophils and prolonged coagulation time. Lactadherin diminished the procoagulant activity and increased the rate of neutrophil clearance. In vivo, neutrophils were sequestered by endothelial cells after blockade of Kupffer cells, a process that was dependent upon both phosphatidylserine exposure and P-selectin expression. Thus, the ability of endothelial cells to clear senescent neutrophils may limit the procoagulant and/or inflammatory impact of these cells.

  19. 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

  20. Stereological Analysis of Neuron, Glial and Endothelial Cell Numbers in the Human Amygdaloid Complex

    PubMed Central

    García-Amado, María; Prensa, Lucía

    2012-01-01

    Cell number alterations in the amygdaloid complex (AC) might coincide with neurological and psychiatric pathologies with anxiety imbalances as well as with changes in brain functionality during aging. This stereological study focused on estimating, in samples from 7 control individuals aged 20 to 75 years old, the number and density of neurons, glia and endothelial cells in the entire AC and in its 5 nuclear groups (including the basolateral (BL), corticomedial and central groups), 5 nuclei and 13 nuclear subdivisions. The volume and total cell number in these territories were determined on Nissl-stained sections with the Cavalieri principle and the optical fractionator. The AC mean volume was 956 mm3 and mean cell numbers (x106) were: 15.3 neurons, 60 glial cells and 16.8 endothelial cells. The numbers of endothelial cells and neurons were similar in each AC region and were one fourth the number of glial cells. Analysis of the influence of the individuals’ age at death on volume, cell number and density in each of these 24 AC regions suggested that aging does not affect regional size or the amount of glial cells, but that neuron and endothelial cell numbers respectively tended to decrease and increase in territories such as AC or BL. These accurate stereological measures of volume and total cell numbers and densities in the AC of control individuals could serve as appropriate reference values to evaluate subtle alterations in this structure in pathological conditions. PMID:22719923

  1. Clinicopathological study of lymphocyte attachment to endothelial cells (endothelialitis) in various liver diseases.

    PubMed

    Nonomura, A; Mizukami, Y; Matsubara, F; Kobayashi, K

    1991-04-01

    An attachment of lymphocytes to the vascular wall, a feature called "endothelialitis" (ETL) or "endotheliitis", was investigated in various liver biopsies, including acute hepatitis (AH), hepatic infectious mononucleosis (IM), drug-induced hepatitis, alcoholic hepatitis and fibrosis, chronic persistent hepatitis (CPH), chronic active hepatitis (CAH), liver cirrhosis (LC), primary biliary cirrhosis (PBC), nonspecific reactive hepatitis (NSRH), and cases with a variety of diseases having almost normal liver histology as control material. Although ETL has been considered to be nearly pathognomic of graft-versus-host disease (GVHD) and acute transplant rejection, ETL was found in both portal and central veins with a variable incidence, not only in all categories of liver diseases, but also in the control group. The incidence of central vein ETL was significantly higher in AH, CAH, PBC, IM, alcoholic fibrosis, and NSRH than that of the control group, and that of portal vein ETL was significantly higher in AH, CPH, CAH, LC, PBC, IM, and alcoholic fibrosis. Even under the light microscope, lymphocytes attached to the endothelial cells had irregular cytoplasmic processes making contact with endothelial cells. Also lymphocytes located beneath the endothelial lining were frequently found. When ETL-positive and -negative cases in the same category were compared, the levels of serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were usually higher in the ETL-positive group, and statistically significant differences were observed in CPH, CAH, LC, PBC and NSRH. In chronic hepatitis, the occurrence of portal vein ETL paralleled the histologic activity of portal inflammation, whereas central vein endothelialitis was associated with active parenchymal inflammation such as sinusoidal lymphocyte infiltration and spotty hepatocyte necrosis, indicating that ETL may be a phenomenon more frequently associated with active hepatic inflammation

  2. 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.

  3. Potential proinflammatory effects of hydroxyapatite nanoparticles on endothelial cells in a monocyte-endothelial cell coculture model.

    PubMed

    Liu, Xin; Sun, Jiao

    2014-01-01

    Currently, synthetic hydroxyapatite nanoparticles (HANPs) are used in nanomedicine fields. The delivery of nanomedicine to the bloodstream exposes the cardiovascular system to a potential threat. However, the possible adverse cardiovascular effects of HANPs remain unclear. Current observations using coculture models of endothelial cells and monocytes with HANPs to mimic the complex physiological functionality of the vascular system demonstrate that monocytes could play an important role in the mechanisms of endothelium dysfunction induced by the exposure to HANPs. Our transmission electron microscopy analysis revealed that both monocytes and endothelial cells could take up HANPs. Moreover, our findings demonstrated that at a subcytotoxic dose, HANPs alone did not cause direct endothelial cell injury, but they did induce an indirect activation of endothelial cells, resulting in increased interleukin-6 production and elevated adhesion molecule expression after coculture with monocytes. The potential proinflammatory effect of HANPs is largely mediated by the release of soluble factors from the activated monocytes, leading to an inflammatory response of the endothelium, which is possibly dependent on p38/c-Jun N-terminal kinase, and nuclear factor-kappa B signaling activation. The use of in vitro monocyte-endothelial cell coculture models for the biocompatibility assessment of HANPs could reveal their potential proinflammatory effects on endothelial cells, suggesting that exposure to HANPs possibly increases the risk of cardiovascular disease.

  4. Tumour-cell-induced endothelial cell necroptosis via death receptor 6 promotes metastasis.

    PubMed

    Strilic, Boris; Yang, Lida; Albarrán-Juárez, Julián; Wachsmuth, Laurens; Han, Kang; Müller, Ulrike C; Pasparakis, Manolis; Offermanns, Stefan

    2016-08-11

    Metastasis is the leading cause of cancer-related death in humans. It is a complex multistep process during which individual tumour cells spread primarily through the circulatory system to colonize distant organs. Once in the circulation, tumour cells remain vulnerable, and their metastatic potential largely depends on a rapid and efficient way to escape from the blood stream by passing the endothelial barrier. Evidence has been provided that tumour cell extravasation resembles leukocyte transendothelial migration. However, it remains unclear how tumour cells interact with endothelial cells during extravasation and how these processes are regulated on a molecular level. Here we show that human and murine tumour cells induce programmed necrosis (necroptosis) of endothelial cells, which promotes tumour cell extravasation and metastasis. Treatment of mice with the receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-inhibitor necrostatin-1 or endothelial-cell-specific deletion of RIPK3 reduced tumour-cell-induced endothelial necroptosis, tumour cell extravasation and metastasis. In contrast, pharmacological caspase inhibition or endothelial-cell-specific loss of caspase-8 promoted these processes. We furthermore show in vitro and in vivo that tumour-cell-induced endothelial necroptosis leading to extravasation and metastasis requires amyloid precursor protein expressed by tumour cells and its receptor, death receptor 6 (DR6), on endothelial cells as the primary mediators of these effects. Our data identify a new mechanism underlying tumour cell extravasation and metastasis, and suggest endothelial DR6-mediated necroptotic signalling pathways as targets for anti-metastatic therapies. PMID:27487218

  5. Orientation of endothelial cell division is regulated by VEGF signaling during blood vessel formation

    PubMed Central

    Zeng, Gefei; Taylor, Sarah M.; McColm, Janet R.; Kappas, Nicholas C.; Kearney, Joseph B.; Williams, Lucy H.; Hartnett, Mary E.; Bautch, Victoria L.

    2007-01-01

    New blood vessel formation requires the coordination of endothelial cell division and the morphogenetic movements of vessel expansion, but it is not known how this integration occurs. Here, we show that endothelial cells regulate division orientation during the earliest stages of blood vessel formation, in response to morphogenetic cues. In embryonic stem (ES) cell–derived vessels that do not experience flow, the plane of endothelial cytokinesis was oriented perpendicular to the vessel long axis. We also demonstrated regulated cleavage orientation in vivo, in flow-exposed forming retinal vessels. Daughter nuclei moved away from the cleavage plane after division, suggesting that regulation of endothelial division orientation effectively extends vessel length in these developing vascular beds. A gain-of-function mutation in VEGF signaling increased randomization of endothelial division orientation, and this effect was rescued by a transgene, indicating that regulation of division orientation is a novel mechanism whereby VEGF signaling affects vessel morphogenesis. Thus, our findings show that endothelial cell division and morphogenesis are integrated in developing vessels by flow-independent mechanisms that involve VEGF signaling, and this cross talk is likely to be critical to proper vessel morphogenesis. PMID:17068148

  6. Methamphetamine is not Toxic but Disrupts the Cell Cycle of Blood-Brain Barrier Endothelial Cells.

    PubMed

    Fisher, D; Gamieldien, K; Mafunda, P S

    2015-07-01

    The cytotoxic effects of methamphetamine (MA) are well established to be caused via induced oxidative stress which in turn compromises the core function of the blood-brain barrier (BBB) by reducing its ability to regulate the homeostatic environment of the brain. While most studies were conducted over a period of 24-48 h, this study investigated the mechanisms by which chronic exposure of MA adversely affect the endothelial cells of BBB over an extended period of 96 h. MA induced significant depression of cell numbers at 96 h. This result was supported by flow cytometric data on the cell cycle which showed that brain endothelial cells (bEnd5) at 96 h were significantly suppressed in the S-phase of the cell cycle. In contrast, at 24-72 h control cell numbers for G1, S and G2-M phases were similar to MA-exposed cells. MA (0-1,000 µM) did not, however, statistically affect the viability and cytotoxicity of the bEnd5 cells, and the profile of ATP production and DNA synthesis (BrdU) across 96 h did not provide a rationale for the suppression of cell division. Our study reports for the first time that chronic exposure to MA results in long-term disruption of the cell cycle phases which eventuates in the attenuation of brain capillary endothelial cell growth after 96 h, compounding and contributing to the already well-known adverse short-term permeability effects of MA exposure on the BBB.

  7. Extrarenal Progenitor Cells Do Not Contribute to Renal Endothelial Repair.

    PubMed

    Sradnick, Jan; Rong, Song; Luedemann, Anika; Parmentier, Simon P; Bartaun, Christoph; Todorov, Vladimir T; Gueler, Faikah; Hugo, Christian P; Hohenstein, Bernd

    2016-06-01

    Endothelial progenitor cells (EPCs) may be relevant contributors to endothelial cell (EC) repair in various organ systems. In this study, we investigated the potential role of EPCs in renal EC repair. We analyzed the major EPC subtypes in murine kidneys, blood, and spleens after induction of selective EC injury using the concanavalin A/anti-concanavalin A model and after ischemia/reperfusion (I/R) injury as well as the potential of extrarenal cells to substitute for injured local EC. Bone marrow transplantation (BMTx), kidney transplantation, or a combination of both were performed before EC injury to allow distinction of extrarenal or BM-derived cells from intrinsic renal cells. During endothelial regeneration, cells expressing markers of endothelial colony-forming cells (ECFCs) were the most abundant EPC subtype in kidneys, but were not detected in blood or spleen. Few cells expressing markers of EC colony-forming units (EC-CFUs) were detected. In BM chimeric mice (C57BL/6 with tandem dimer Tomato-positive [tdT+] BM cells), circulating and splenic EC-CFUs were BM-derived (tdT+), whereas cells positive for ECFC markers in kidneys were not. Indeed, most BM-derived tdT+ cells in injured kidneys were inflammatory cells. Kidneys from C57BL/6 donors transplanted into tdT+ recipients with or without prior BMTx from C57BL/6 mice were negative for BM-derived or extrarenal ECFCs. Overall, extrarenal cells did not substitute for any intrinsic ECs. These results demonstrate that endothelial repair in mouse kidneys with acute endothelial lesions depends exclusively on local mechanisms.

  8. Mesenchymal Stem Cells Ameliorate Atherosclerotic Lesions via Restoring Endothelial Function

    PubMed Central

    Lin, Yu-Ling; Yet, Shaw-Fang; Hsu, Yuan-Tong

    2015-01-01

    Transplantation of mesenchymal stem cells (MSCs) is beneficial in myocardial infarction and hind limb ischemia, but its ability to ameliorate atherosclerosis remains unknown. Here, the effects of MSCs on inhibiting endothelial dysfunction and atherosclerosis were investigated in human/mouse endothelial cells treated with oxidized low-density lipoprotein (oxLDL) and in apolipoprotein E-deficient (apoE−/−) mice fed a high-fat diet. Treatment with oxLDL inactivated the Akt/endothelial nitric-oxide synthase (eNOS) pathway, induced eNOS degradation, and inhibited nitric oxide (NO) production in endothelial cells. Coculture with human MSCs reversed the effects of oxLDL on endothelial cells and restored Akt/eNOS activity, eNOS level, and NO production. Reduction of endothelium-dependent relaxation and subsequent plaque formation were developed in apoE−/− mice fed a high-fat diet. Systemic infusion with mouse MSCs ameliorated endothelial dysfunction and plaque formation in high-fat diet-fed apoE−/− mice. Interestingly, treatment with interleukin-8 (IL8)/macrophage inflammatory protein-2 (MIP-2) alone induced the similar effects of human/mouse MSCs on oxLDL-treated human/mouse endothelial cells. Neutralization antibodies (Abs) against IL8/MIP-2 also blocked the effects of human/mouse MSCs on oxLDL-treated human/mouse endothelial cells. Consistently, MIP-2 injection alone induced the similar effect of MSCs on the endothelial function in high-fat diet-fed apoE−/− mice. The improvement in endothelial dysfunction by mouse MSCs was also blocked when pretreating MSCs with anti-MIP-2 Abs. In conclusion, MSC transplantation improved endothelial function and plaque formation in high-fat diet-fed apoE−/− mice. Activation of the Akt/eNOS pathway in endothelium by IL8/MIP-2 is involved in the protective effect of MSCs. The study helps support the use and clarify the mechanism of MSCs for ameliorating atherosclerosis. PMID:25504897

  9. Characterization of iron uptake from transferrin by murine endothelial cells.

    PubMed

    Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

    2000-01-01

    Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB.

  10. Characterization of iron uptake from transferrin by murine endothelial cells.

    PubMed

    Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

    2000-01-01

    Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB. PMID:10865941

  11. Differential radiation response of cultured endothelial cells and smooth myocytes

    SciTech Connect

    Johnson, L.K.; Longenecker, J.P.; Fajardo, L.F.

    1982-09-01

    In vivo observations have suggested that endothelial cells are the most radiosensitive elements of the vascular wall. To test whether this represents an intrinsic differential sensitivity, the response of bovine aortic endothelial cells and smooth myocytes was investigated in confluent cell cultures exposed to single doses of gamma radiation (250, 500, 1,000 or 2,000 rad). Both cell types showed a dose-dependent decrease in attachment efficiency when dissociated and replated at six hours after radiation. However, the attachment efficiency in both cell types was similar when a 72-hour postirradiation incubation period was used prior to dissociation of the cells. Growth inhibition was significantly greater (7- to 10-fold) in endothelial cells than in myocytes when examined four days after attachment. Confluent endothelial monolayers showed a dose-dependent, progressive cell loss during the 72-hour postirradiation period (70% after 1,000 rad); the myocyte cultures showed no radiation effect on the cell numbers. In spite of the reduction in number, the endothelial cells maintained the continuity of their monolayer by compensation with an increase in mean cell size. Endothelial cells developed multiple structural lesions, including an increase in the number and size of residual and lysosomal bodies, electron-lucent cytoplasmic defects, interruptions in the plasma membrane and irregular aggregation of chromatin, causing electron-lucent nuclei. These changes increased in severity with time and dose and were most pronounced 24 to 72 hours after 1,000 rad. No significant ultrastructural alterations were detected in myocytes four days after 2,000 rad.

  12. Role of Endothelial Cells in Myocardial Ischemia-Reperfusion Injury

    PubMed Central

    Singhal, Arun K.; Symons, J. David; Boudina, Sihem; Jaishy, Bharat; Shiu, Yan-Ting

    2014-01-01

    Minimizing myocardial ischemia-reperfusion injury has broad clinical implications and is a critical mediator of cardiac surgical outcomes. “Ischemic injury” results from a restriction in blood supply leading to a mismatch between oxygen supply and demand of a sufficient intensity and/or duration that leads to cell necrosis, whereas ischemia-reperfusion injury occurs when blood supply is restored after a period of ischemia and is usually associated with apoptosis (i.e. programmed cell death). Compared to vascular endothelial cells, cardiac myocytes are more sensitive to ischemic injury and have received the most attention in preventing myocardial ischemia-reperfusion injury. Many comprehensive reviews exist on various aspects of myocardial ischemia-reperfusion injury. The purpose of this review is to examine the role of vascular endothelial cells in myocardial ischemia-reperfusion injury, and to stimulate further research in this exciting and clinically relevant area. Two specific areas that are addressed include: 1) data suggesting that coronary endothelial cells are critical mediators of myocardial dysfunction after ischemia-reperfusion injury; and 2) the involvement of the mitochondrial permeability transition pore in endothelial cell death as a result of an ischemia-reperfusion insult. Elucidating the cellular signaling pathway(s) that leads to endothelial cell injury and/or death in response to ischemia-reperfusion is a key component to developing clinically applicable strategies that might minimize myocardial ischemia-reperfusion injury. PMID:25558187

  13. Thrombospondin-1 signaling through CD47 inhibits cell cycle progression and induces senescence in endothelial cells

    PubMed Central

    Gao, Qi; Chen, Kexin; Gao, Lu; Zheng, Yang; Yang, Yong-Guang

    2016-01-01

    CD47 signaling in endothelial cells has been shown to suppress angiogenesis, but little is known about the link between CD47 and endothelial senescence. Herein, we demonstrate that the thrombospondin-1 (TSP1)-CD47 signaling pathway is a major mechanism for driving endothelial cell senescence. CD47 deficiency in endothelial cells significantly improved their angiogenic function and attenuated their replicative senescence. Lack of CD47 also suppresses activation of cell cycle inhibitors and upregulates the expression of cell cycle promoters, leading to increased cell cycle progression. Furthermore, TSP1 significantly accelerates replicative senescence and associated cell cycle arrest in a CD47-dependent manner. These findings demonstrate that TSP1-CD47 signaling is an important mechanism driving endothelial cell senescence. Thus, TSP1 and CD47 provide attractive molecular targets for treatment of aging-associated cardiovascular dysfunction and diseases involving endothelial dysregulation. PMID:27607583

  14. Fluid Flow Stimulates Tissue Plasminogen Activator Secretion by Cultured Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Diamond, S. L.; Eskin, S. G.; McIntire, L. V.

    1989-03-01

    Wall shear stress generated by blood flow may regulate the expression of fibrinolytic proteins by endothelial cells. Tissue plasminogen activator (tPA) and plasminogen activator inhibitor, type 1 (PAI-1) secretion by cultured human endothelial cells were not affected by exposure to venous shear stress (4 dynes/cm2). However, at arterial shear stresses of 15 and 25 dynes/cm2, the tPA secretion rate was 2.1 and 3.0 times greater, respectively, than the basal tPA secretion rate. PAI-1 secretion was unaffected by shear stress over the entire physiological range.

  15. Antiangiogenic effects of melatonin in endothelial cell cultures.

    PubMed

    Alvarez-García, Virginia; González, Alicia; Alonso-González, Carolina; Martínez-Campa, Carlos; Cos, Samuel

    2013-05-01

    Endothelial cells represent one of the critical cellular elements in tumor microenvironment playing a crucial role in the growth and progression of cancer through controlling angiogenesis. Vascular endothelial growth factor (VEGF) produced from tumor cells is essential for the expansion of breast cancer and may function in both paracrine and autocrine manners to promote proliferation, growth, survival and migration of endothelial cells. Since melatonin regulates tumor microenvironment by decreasing the secretion of VEGF by malignant epithelial cells and also regulates VEGF expression in human breast cancer cells, the aim of the present study was to investigate the anti-angiogenic activity of melatonin against the pro-angiogenic effects of breast cancer cells. In this work, we demonstrate that melatonin strongly inhibited the proliferation as well as invasion/migration of human umbilical vein endothelial cells (HUVECs). Melatonin disrupted tube formation and counteracted the VEGF-stimulated tubular network formation by HUVEC. In addition, conditioned media collected from human breast cancer cells were angiogenically active and stimulated tubule length formation and this effect was significantly counteracted by the addition of anti-VEGF or melatonin. Melatonin also disintegrated preformed capillary network. All these findings demonstrate that melatonin may play a role in the paracrine interactions that take place between malignant epithelial cells and proximal endothelial cells. Melatonin could be important in reducing endothelial cell proliferation, invasion, migration and tube formation, through a downregulatory action on VEGF. Taken together, our findings suggest that melatonin could potentially be beneficial as an antiangiogenic agent in breast cancer with possible future clinical applications. PMID:23473980

  16. [Pulmonary arterial hypertension, bone marrow, endothelial cell precursors and serotonin].

    PubMed

    Ayme-Dietrich, Estelle; Banas, Sophie M; Monassier, Laurent; Maroteaux, Luc

    2016-01-01

    Serotonin and bone-marrow-derived stem cells participate together in triggering pulmonary hypertension. Our work has shown that the absence of 5-HT2B receptors generates permanent changes in the composition of the blood and bone-marrow in the myeloid lineages, particularly in endothelial cell progenitors. The initial functions of 5-HT2B receptors in pulmonary arterial hypertension (PAH) are restricted to bone-marrow cells. They contribute to the differentiation/proliferation/mobilization of endothelial progenitor cells from the bone-marrow. Those bone-marrow-derived cells have a critical role in the development of pulmonary hypertension and pulmonary vascular remodeling. These data indicate that bone-marrow derived endothelial progenitors play a key role in the pathogenesis of PAH and suggest that interactions involving serotonin and bone morphogenic protein type 2 receptor (BMPR2) could take place at the level of the bone-marrow. PMID:27687599

  17. Mechanisms for SU5416 as a radiosensitizer of endothelial cells.

    PubMed

    Kim, Eun Ho; Kim, Mi-Sook; Jeong, Youn Kyoung; Cho, Ilsung; You, Seung Hoon; Cho, Sung Ho; Lee, Hanna; Jung, Won-Gyun; Kim, Hag Dong; Kim, Joon

    2015-10-01

    Endothelial cells (ECs), that comprise the tumor vasculature, are critical targets for anticancer radiotherapy. The aim of this work was to study the mechanism by which SU5416, a known anti-angiogenesis inhibitor, modifies the radiation responses of human vascular ECs. Two human endothelial cell lines (HUVEC and 2H11) were treated with SU5416 alone, radiation alone, or a combination of both. In vitro tests were performed using colony forming assays, FACS analysis, western blotting, immunohistochemistry, migration assay, invasion assays and endothelial tube formation assays. The combination of radiation and SU5416 significantly inhibited cell survival, the repair of radiation-induced DNA damage, and induced apoptosis. It also caused cell cycle arrest, inhibited cell migration and invasion, and suppressed angiogenesis. In this study, our results first provide a scientific rationale to combine SU5416 with radiotherapy to target ECs and suggest its clinical application in combination cancer treatment with radiotherapy.

  18. Estrogen Stimulates Homing of Endothelial Progenitor Cells to Endometriotic Lesions.

    PubMed

    Rudzitis-Auth, Jeannette; Nenicu, Anca; Nickels, Ruth M; Menger, Michael D; Laschke, Matthias W

    2016-08-01

    The incorporation of endothelial progenitor cells (EPCs) into microvessels contributes to the vascularization of endometriotic lesions. Herein, we analyzed whether this vasculogenic process is regulated by estrogen. Estrogen- and vehicle-treated human EPCs were analyzed for migration and tube formation. Endometriotic lesions were induced in irradiated FVB/N mice, which were reconstituted with bone marrow from FVB/N-TgN (Tie2/green fluorescent protein) 287 Sato mice. The animals were treated with 100 μg/kg β-estradiol 17-valerate or vehicle (control) over 7 and 28 days. Lesion growth, cyst formation, homing of green fluorescent protein(+)/Tie2(+) EPCs, vascularization, cell proliferation, and apoptosis were analyzed by high-resolution ultrasonography, caliper measurements, histology, and immunohistochemistry. Numbers of blood circulating EPCs were assessed by flow cytometry. In vitro, estrogen-treated EPCs exhibited a higher migratory and tube-forming capacity when compared with controls. In vivo, numbers of circulating EPCs were not affected by estrogen. However, estrogen significantly increased the number of EPCs incorporated into the lesions' microvasculature, resulting in an improved early vascularization. Estrogen further stimulated the growth of lesions, which exhibited massively dilated glands with a flattened layer of stroma. This was mainly because of an increased glandular secretory activity, whereas cell proliferation and apoptosis were not markedly affected. These findings indicate that vasculogenesis in endometriotic lesions is dependent on estrogen, which adds a novel hormonally regulated mechanism to the complex pathophysiology of endometriosis. PMID:27315780

  19. Oxidized low-density lipoprotein alters endothelial progenitor cell populations.

    PubMed

    Cui, Yuqi; Narasimhulu, Chandrakala A; Liu, Lingjuan; Li, Xin; Xiao, Yuan; Zhang, Jia; Xie, Xiaoyun; Hao, Hong; Liu, Jason Z; He, Guanglong; Cowan, Peter J; Cui, Lianqun; Zhu, Hua; Parthasarathy, Sampath; Liu, Zhenguo

    2015-06-01

    Oxidized low-density lipoprotein (ox-LDL) is critical to atherosclerosis in hyperlipidemia. Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are important to preventing atherosclerosis, and significantly decreased in hyperlipidemia. This study was to demonstrate ox-LDL and hyperlipidemia could exhibit similar effect on EPC population and the role of reactive oxygen species (ROS). ROS production in BM and blood was significantly increased in male C57BL/6 mice with intravenous ox-LDL treatment, and in hyperlipidemic LDL receptor knockout mice with 4-month high-fat diet. ROS formation was effectively blocked with overexpression of antioxidant enzymes or N-acetylcysteine treatment. In hyperlipidemic and ox-LDL-treated mice, c-Kit(+)/CD31(+) cell number in BM and blood, and Sca-1(+)/Flk-1(+) cell number in blood, not in BM, were significantly decreased, which were not affected by inhibiting ROS production, while blood CD34(+)/Flk-1(+) cell number was significantly increased that was prevented with reduced ROS formation. However, blood CD34(+)/CD133(+) cell number increased in ox-LDL-treated mice, while decreased in hyperlipidemic mice. These data suggested that ox-LDL produced significant changes in BM and blood EPC populations similar (but not identical) to chronic hyperlipidemia with predominantly ROS-independent mechanism(s).

  20. An exquisite cross-control mechanism among endothelial cell fate regulators directs the plasticity and heterogeneity of lymphatic endothelial cells

    PubMed Central

    Kang, Jinjoo; Yoo, Jaehyuk; Lee, Sunju; Tang, Wanli; Aguilar, Berenice; Ramu, Swapnika; Choi, Inho; Otu, Hasan H.; Shin, Jay W.; Dotto, G. Paolo; Koh, Chester J.; Detmar, Michael

    2010-01-01

    Arteriovenous-lymphatic endothelial cell fates are specified by the master regulators, namely, Notch, COUP-TFII, and Prox1. Whereas Notch is expressed in the arteries and COUP-TFII in the veins, the lymphatics express all 3 cell fate regulators. Previous studies show that lymphatic endothelial cell (LEC) fate is highly plastic and reversible, raising a new concept that all 3 endothelial cell fates may coreside in LECs and a subtle alteration can result in a reprogramming of LEC fate. We provide a molecular basis verifying this concept by identifying a cross-control mechanism among these cell fate regulators. We found that Notch signal down-regulates Prox1 and COUP-TFII through Hey1 and Hey2 and that activated Notch receptor suppresses the lymphatic phenotypes and induces the arterial cell fate. On the contrary, Prox1 and COUP-TFII attenuate vascular endothelial growth factor signaling, known to induce Notch, by repressing vascular endothelial growth factor receptor-2 and neuropilin-1. We show that previously reported podoplanin-based LEC heterogeneity is associated with differential expression of Notch1 in human cutaneous lymphatics. We propose that the expression of the 3 cell fate regulators is controlled by an exquisite feedback mechanism working in LECs and that LEC fate is a consequence of the Prox1-directed lymphatic equilibrium among the cell fate regulators. PMID:20351309

  1. Preparation of chitosan films using different neutralizing solutions to improve endothelial cell compatibility.

    PubMed

    He, Qing; Ao, Qiang; Gong, Yandao; Zhang, Xiufang

    2011-12-01

    The development of chitosan-based constructs for application in large-size defects or highly vascularized tissues is still a challenging issue. The poor endothelial cell compatibility of chitosan hinders the colonization of vascular endothelial cells in the chitosan-based constructs, and retards the establishment of a functional microvascular network following implantation. The aim of the present study is to prepare chitosan films with different neutralization methods to improve their endothelial cell compatibility. Chitosan salt films were neutralized with either sodium hydroxide (NaOH) aqueous solution, NaOH ethanol solution, or ethanol solution without NaOH. The physicochemical properties and endothelial cell compatibility of the chitosan films were investigated. Results indicated that neutralization with different solutions affected the surface chemistry, swelling ratio, crystalline conformation, nanotopography, and mechanical properties of the chitosan films. The NaOH ethanol solution-neutralized chitosan film (Chi-NaOH/EtOH film) displayed a nanofiber-dominant surface, while the NaOH aqueous solution-neutralized film (Chi-NaOH/H(2)O film) and the ethanol solution-neutralized film (Chi-EtOH film) displayed nanoparticle-dominant surfaces. Moreover, the Chi-NaOH/EtOH films exhibited a higher stiffness as compared to the Chi-NaOH/H(2)O and Chi-EtOH films. Endothelial cell compatibility of the chitosan films was evaluated with a human microvascular endothelial cell line, HMEC-1. Compared with the Chi-NaOH/H(2)O and Chi-EtOH films, HMECs cultured on the Chi-NaOH/EtOH films fully spread and exhibited significantly higher levels of adhesion and proliferation, with retention of the endothelial phenotype and function. Our findings suggest that the surface nanotopography and mechanical properties contribute to determining the endothelial cell compatibility of chitosan films. The nature of the neutralizing solutions can affect the physicochemical properties and

  2. Generating induced pluripotent stem cell derived endothelial cells and induced endothelial cells for cardiovascular disease modelling and therapeutic angiogenesis.

    PubMed

    Clayton, Z E; Sadeghipour, S; Patel, S

    2015-10-15

    Standard therapy for atherosclerotic coronary and peripheral arterial disease is insufficient in a significant number of patients because extensive disease often precludes effective revascularization. Stem cell therapy holds promise as a supplementary treatment for these patients, as pre-clinical and clinical research has shown transplanted cells can promote angiogenesis via direct and paracrine mechanisms. Induced pluripotent stem cells (iPSCs) are a novel cell type obtained by reprogramming somatic cells using exogenous transcription factor cocktails, which have been introduced to somatic cells via viral or plasmid constructs, modified mRNA or small molecules. IPSCs are now being used in disease modelling and drug testing and are undergoing their first clinical trial, but despite recent advances, the inefficiency of the reprogramming process remains a major limitation, as does the lack of consensus regarding the optimum transcription factor combination and delivery method and the uncertainty surrounding the genetic and epigenetic stability of iPSCs. IPSCs have been successfully differentiated into vascular endothelial cells (iPSC-ECs) and, more recently, induced endothelial cells (iECs) have also been generated by direct differentiation, which bypasses the pluripotent intermediate. IPSC-ECs and iECs demonstrate endothelial functionality in vitro and have been shown to promote neovessel growth and enhance blood flow recovery in animal models of myocardial infarction and peripheral arterial disease. Challenges remain in optimising the efficiency, safety and fidelity of the reprogramming and endothelial differentiation processes and establishing protocols for large-scale production of clinical-grade, patient-derived cells.

  3. Filamin redistribution in an endothelial cell reoxygenation injury model.

    PubMed

    Hastie, L E; Patton, W F; Hechtman, H B; Shepro, D

    1997-01-01

    Ischemia-reperfusion injury increases vascular permeability in part by generating reactive oxygen species that disassemble the endothelial cell actin dense peripheral band. This is followed by an increase in the number and diameter of intercellular gaps. Millimolar concentrations of reactive oxygen metabolites lead to nonspecific endothelial cell injury, but micromolar concentrations activate inflammatory second messenger cascades which produce distributional changes in endothelial cell cytoskeletal proteins. H2O2 (100 microM) causes translocation of filamin, from the membrane to the cytosol within 1 min. Subsequently, gap formation occurs within 10-25 min, which is attributed to rearrangement of the dense peripheral band of F-actin. Plasma membrane blebbing occurs after 90 min and decreases in mitochondrial activity occur after 1-2 h. Deferoxamine (iron chelator) and TEMPO (nonspecific free radical scavenger) inhibit these changes. H2O2 (100-1000 microM) does not increase endothelial cell intracellular Ca2+ through 30 min and pretreating cells with a Ca2+-calmodulin kinase inhibitor or an intracellular Ca2+ chelator does not prevent filamin translocation. Filamin redistribution and actin rearrangement are early events in H2O2-mediated endothelial cell injury that appear to occur through Ca2+-independent pathways.

  4. Endothelial binding of beta toxin to small intestinal mucosal endothelial cells in early stages of experimentally induced Clostridium perfringens type C enteritis in pigs.

    PubMed

    Schumacher, V L; Martel, A; Pasmans, F; Van Immerseel, F; Posthaus, H

    2013-07-01

    Beta toxin (CPB) is known to be an essential virulence factor in the development of lesions of Clostridium perfringens type C enteritis in different animal species. Its target cells and exact mechanism of toxicity have not yet been clearly defined. Here, we evaluate the suitability of a neonatal piglet jejunal loop model to investigate early lesions of C. perfringens type C enteritis. Immunohistochemically, CPB was detected at microvascular endothelial cells in intestinal villi during early and advanced stages of lesions induced by C. perfringens type C. This was first associated with capillary dilatation and subsequently with widespread hemorrhage in affected intestinal segments. CPB was, however, not demonstrated on intestinal epithelial cells. This indicates a tropism of CPB toward endothelial cells and suggests that CPB-induced endothelial damage plays an important role in the early stages of C. perfringens type C enteritis in pigs.

  5. Sub-micron lateral topography affects endothelial migration by modulation of focal adhesion dynamics.

    PubMed

    Antonini, S; Meucci, S; Jacchetti, E; Klingauf, M; Beltram, F; Poulikakos, D; Cecchini, M; Ferrari, A

    2015-06-24

    Through the interaction with topographical features, endothelial cells tune their ability to populate target substrates, both in vivo and in vitro. Basal textures interfere with the establishment and maturation of focal adhesions (FAs) thus inducing specific cell-polarization patterns and regulating a plethora of cell activities that govern the overall endothelial function. In this study, we analyze the effect of topographical features on FAs in primary human endothelial cells. Reported data demonstrate a functional link between FA dynamics and cell polarization and spreading on structured substrates presenting variable lateral feature size. Our results reveal that gratings with 2 µm lateral periodicity maximize contact guidance. The effect is linked to the dynamical state of FAs. We argue that these results are readily applicable to the rational design of active surfaces at the interface with the blood stream.

  6. Vascular endothelial growth factor signalling in endothelial cell survival: A role for NF{kappa}B

    SciTech Connect

    Grosjean, Jennifer . E-mail: Jennifer.grosjean@imperial.ac.uk; Kiriakidis, Serafim; Reilly, Kerri; Feldmann, Marc; Paleolog, Ewa

    2006-02-17

    Angiogenesis is the development of blood capillaries from pre-existing vessels. Vascular endothelial growth factor (VEGF) is a key regulator of vessel growth and regression, and acts as an endothelial survival factor by protecting endothelial cells from apoptosis. Many genes involved in cell proliferation and apoptosis are regulated by the nuclear factor kappa B (NF{kappa}B) transcription factor family. This study aimed to address the hypothesis that VEGF-mediated survival effects on endothelium involve NF{kappa}B. Using an NF{kappa}B-luciferase reporter adenovirus, we observed activation of NF{kappa}B following VEGF treatment of human umbilical vein endothelial cells. This was confirmed using electrophoretic mobility shift assay and found to involve nuclear translocation of NF{kappa}B sub-unit p65. However, NF{kappa}B activation occurred without degradation of inhibitory I{kappa}B proteins (I{kappa}B{alpha}, I{kappa}B{beta}, and I{kappa}B{epsilon}). Instead, tyrosine phosphorylation of I{kappa}B{alpha} was observed following VEGF treatment, suggesting NF{kappa}B activation was mediated by degradation-independent dissociation of I{kappa}B{alpha} from NF{kappa}B. Adenovirus-mediated over-expression of either native I{kappa}B{alpha}, or of I{kappa}B{alpha} in which tyrosine residue 42 was mutated to phenylalanine, inhibited induction of NF{kappa}B-dependent luciferase activity in response to VEGF. Furthermore, VEGF-induced upregulation of mRNA for the anti-apoptotic protein Bcl-2 and cell survival following serum withdrawal was reduced following I{kappa}B{alpha} over-expression. This study highlights that different molecular mechanisms of NF{kappa}B activation may be involved downstream of stimuli which activate the endothelial lining of blood vessels.

  7. Microrheology and ROCK signaling of human endothelial cells embedded in a 3D matrix.

    PubMed

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

    2006-11-01

    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.

  8. Existence of Neural Crest-Derived Progenitor Cells in Normal and Fuchs Endothelial Dystrophy Corneal Endothelium.

    PubMed

    Katikireddy, Kishore Reddy; Schmedt, Thore; Price, Marianne O; Price, Francis W; Jurkunas, Ula V

    2016-10-01

    Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma, aging, and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein, we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crest-derived progenitor (NCDP) cells by showing absence of senescence with passaging, propensity to form spheres, and increased colony forming efficacy compared with the primary cells. The collective expression of stem cell-related genes SOX2, OCT4, LGR5, TP63 (p63), as well as neural crest marker genes PSIP1 (p75(NTR)), PAX3, SOX9, AP2B1 (AP-2β), and NES, generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2, β-III tubulin, and glial fibrillary acidic protein positive neurons and into p75(NTR)-positive human corneal endothelial cells that exhibited transendothelial resistance of functional endothelium. In conclusion, we found that mitotically incompetent ocular tissue cells contain adult NCDPs that exhibit a profile of transcription factors regulating multipotency and neural crest progenitor characteristics. Identification of normal NCDPs in FECD-affected endothelium holds promise for potential autologous cell therapies. PMID:27639969

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

    PubMed

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

    2012-09-01

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

  10. Molecular control of endothelial cell behaviour during blood vessel morphogenesis

    PubMed Central

    Herbert, Shane P.; Stainier, Didier Y.R.

    2012-01-01

    The vertebrate vasculature forms an extensive branched network of blood vessels that supplies tissues with nutrients and oxygen. During vascular development, coordinated control of endothelial cell behaviour at the levels of cell migration, proliferation, polarity, differentiation and cell–cell communication is critical for functional blood vessel morphogenesis. Recent data uncover elaborate transcriptional, post-transcriptional and post-translational mechanisms that fine-tune key signalling pathways (such as the vascular endothelial growth factor and Notch pathways) to control endothelial cell behaviour during blood vessel sprouting (angiogenesis). These emerging frameworks controlling angiogenesis provide unique insights into fundamental biological processes common to other systems, such as tissue branching morphogenesis, mechanotransduction and tubulogenesis. PMID:21860391

  11. Dynamic Endothelial Cell Rearrangements Drive Developmental Vessel Regression

    PubMed Central

    Franco, Claudio A.; Jones, Martin L.; Bernabeu, Miguel O.; Geudens, Ilse; Mathivet, Thomas; Rosa, Andre; Lopes, Felicia M.; Lima, Aida P.; Ragab, Anan; Collins, Russell T.; Phng, Li-Kun; Coveney, Peter V.; Gerhardt, Holger

    2015-01-01

    Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments. PMID:25884288

  12. Brassinosteroids inhibit in vitro angiogenesis in human endothelial cells.

    PubMed

    Rárová, Lucie; Zahler, Stefan; Liebl, Johanna; Kryštof, Vladimír; Sedlák, David; Bartůněk, Petr; Kohout, Ladislav; Strnad, Miroslav

    2012-11-01

    Antiangiogenic activity of the brassinosteroid plant hormones (BRs) and their derivative cholestanon was investigated in human umbilical vein endothelial cells (HUVEC) and in human microvascular endothelial cells (HMEC-1). 24-Epibrassinolide and 28-homocastasterone from group of 21 tested natural BRs inhibited migration of HUVEC cells. Seven tested BRs decreased the number of tubes significantly. Synthetic analogue cholestanon inhibited angiogenesis in vitro more effectively than natural BRs. Because of the similarity of BRs to human steroids, we have also studied interactions of BRs with human steroid receptors. Synthetic BRs cholestanon showed agonistic effects on estrogen-receptor-α, estrogen-receptor-β and androgen receptor. Of the natural BRs, 24-epibrassinolide was found to be a weak antagonist of estrogen-receptor-α (ERα). Our results provide the first evidence that large group of BRs can inhibit in vitro angiogenesis of primary endothelial cells. BRs constitute a novel group of human steroid receptor activators or inhibitors with capacity to inhibit angiogenesis.

  13. Acrylamide induces accelerated endothelial aging in a human cell model.

    PubMed

    Sellier, Cyril; Boulanger, Eric; Maladry, François; Tessier, Frédéric J; Lorenzi, Rodrigo; Nevière, Rémi; Desreumaux, Pierre; Beuscart, Jean-Baptiste; Puisieux, François; Grossin, Nicolas

    2015-09-01

    Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 μM) until growth arrest. To analyze senescence, β-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). β-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 μM) or GA (≥1 μM) (p < 0.05). AAM (≥10 μM) or GA (100 μM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging.

  14. Effects of shear stress on endothelial progenitor cells.

    PubMed

    Obi, Syotaro; Yamamoto, Kimiko; Ando, Joji

    2014-10-01

    Endothelial progenitor cells (EPCs) are adult stem cells that play a central role in neovascularization. EPCs are mobilized from bone marrow into peripheral blood, attach to existing endothelial cells, and then transmigrate across the endothelium into tissues, where they proliferate, differentiate, and form new blood vessels. In the process, EPCs are exposed to shear stress, a biomechanical force generated by flowing blood and tissue fluid flow. When cultured EPCs are exposed to controlled levels of shear stress in a flow-loading device, their bioactivities in terms of proliferation, anti-apoptosis, migration, production of bioactive substances, anti-thrombosis, and tube formation increase markedly. Expression of endothelial marker genes and proteins by EPCs also increases in response to shear stress, and they differentiate into mature endothelial cells. Great advances have been made in elucidating the mechanisms by which mature endothelial cells sense and respond to shear stress, but not in EPCs. Further study of EPC responses to shear stress will be necessary to better understand the physiological and pathophysiological roles of EPCs and to apply EPCs to new therapies in the field of regenerative medicine. PMID:25992410

  15. Fibroblast nemosis induces angiogenic responses of endothelial cells

    SciTech Connect

    Enzerink, Anna; Rantanen, Ville; Vaheri, Antti

    2010-03-10

    Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-{kappa}B. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.

  16. 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.

  17. Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction

    PubMed Central

    Boraldi, Federica; Bartolomeo, Angelica; De Biasi, Sara; Orlando, Stefania; Costa, Sonia; Cossarizza, Andrea; Quaglino, Daniela

    2016-01-01

    Introduction Although rare, circulating endothelial and progenitor cells could be considered as markers of endothelial damage and repair potential, possibly predicting the severity of cardiovascular manifestations. A number of studies highlighted the role of these cells in age-related diseases, including those characterized by ectopic calcification. Nevertheless, their use in clinical practice is still controversial, mainly due to difficulties in finding reproducible and accurate methods for their determination. Methods Circulating mature cells (CMC, CD45-, CD34+, CD133-) and circulating progenitor cells (CPC, CD45dim, CD34bright, CD133+) were investigated by polychromatic high-speed flow cytometry to detect the expression of endothelial (CD309+) or osteogenic (BAP+) differentiation markers in healthy subjects and in patients affected by peripheral vascular manifestations associated with ectopic calcification. Results This study shows that: 1) polychromatic flow cytometry represents a valuable tool to accurately identify rare cells; 2) the balance of CD309+ on CMC/CD309+ on CPC is altered in patients affected by peripheral vascular manifestations, suggesting the occurrence of vascular damage and low repair potential; 3) the increase of circulating cells exhibiting a shift towards an osteoblast-like phenotype (BAP+) is observed in the presence of ectopic calcification. Conclusion Differences between healthy subjects and patients with ectopic calcification indicate that this approach may be useful to better evaluate endothelial dysfunction in a clinical context. PMID:27560136

  18. A dual role for endothelial cells in cytomegalovirus infection? A study of cytomegalovirus infection in a series of rat endothelial cell lines.

    PubMed

    Vossen, R C; Derhaag, J G; Slobbe-van Drunen, M E; Duijvestijn, A M; van Dam-Mieras, M C; Bruggeman, C A

    1996-12-01

    Several clinical findings point to the involvement of microvascular endothelial cells in cytomegalovirus-related pathology. In this study the interactions of cytomegalovirus (CMV) with microvascular endothelial cells was investigated in an in vitro rat model. A series of rat endothelial cell lines, considered representative for the heterogeneity of heart microvascular endothelium in vivo, were infected with rat CMV (RCMV). The course of infection and production of infectious virus were examined using immunofluorescence staining and plaque titration assays, and was compared with infection of fully permissive rat fibroblasts. These endothelial cell lines displayed differences in susceptibility to CMV infection. Two endothelial cell lines (RHEC 50 and 191) were practically non-permissive, while four endothelial cell lines (RHEC 3, 10, 11 and 116) were partly permissive for CMV infection. In contrast to CMV infection in fibroblasts, only limited infection of the permissive endothelial cell lines was observed without spreading of CMV infection through the monolayer, although infectious virus was produced. Detachment of infected endothelial cells and recovery of the monolayer with time was observed. The detached endothelial cells were able to transmit CMV infection to fibroblast monolayers, but not to endothelial monolayers. Our in vitro results demonstrate differences in permissiveness for RCMV between the series of rat endothelial cell lines, which is suggestive for endothelial heterogeneity to CMV infection in vivo. Our findings indicate that endothelial cells are relatively resistant to CMV infection and that, upon infection, the endothelial monolayer may dispose of the virus via detachment of the infected cells. This points to a dual role for the endothelium in CMV infection in vivo: a barrier for CMV infection (by the endothelial monolayer) on the one hand and spreading of CMV infection (by detached infected cells) on the other hand.

  19. Endothelial Cells Stimulate Self-Renewal and Expand Neurogenesis of Neural Stem Cells

    NASA Astrophysics Data System (ADS)

    Shen, Qin; Goderie, Susan K.; Jin, Li; Karanth, Nithin; Sun, Yu; Abramova, Natalia; Vincent, Peter; Pumiglia, Kevin; Temple, Sally

    2004-05-01

    Neural stem cells are reported to lie in a vascular niche, but there is no direct evidence for a functional relationship between the stem cells and blood vessel component cells. We show that endothelial cells but not vascular smooth muscle cells release soluble factors that stimulate the self-renewal of neural stem cells, inhibit their differentiation, and enhance their neuron production. Both embryonic and adult neural stem cells respond, allowing extensive production of both projection neuron and interneuron types in vitro. Endothelial coculture stimulates neuroepithelial cell contact, activating Notch and Hes1 to promote self-renewal. These findings identify endothelial cells as a critical component of the neural stem cell niche.

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

    PubMed Central

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

    2014-01-01

    ABSTRACT The Snail family of zinc-finger transcription factors are evolutionarily conserved proteins that control processes requiring cell movement. Specifically, they regulate epithelial-to-mesenchymal transitions (EMT) where an epithelial cell severs intercellular junctions, degrades basement membrane and becomes a migratory, mesenchymal-like cell. Interestingly, Slug expression has been observed in angiogenic endothelial cells (EC) in vivo, suggesting that angiogenic sprouting may share common attributes with EMT. Here, we demonstrate that sprouting EC in vitro express both Slug and Snail, and that siRNA-mediated knockdown of either inhibits sprouting and migration in multiple in vitro angiogenesis assays. We find that expression of MT1-MMP, but not of VE-Cadherin, is regulated by Slug and that loss of sprouting as a consequence of reduced Slug expression can be reversed by lentiviral-mediated re-expression of MT1-MMP. Activity of MMP2 and MMP9 are also affected by Slug expression, likely through MT1-MMP. Importantly, we find enhanced expression of Slug in EC in human colorectal cancer samples compared with normal colon tissue, suggesting a role for Slug in pathological angiogenesis. In summary, these data implicate Slug as an important regulator of sprouting angiogenesis, particularly in pathological settings. PMID:24554431

  1. Principles of targeting endothelial cell metabolism to treat angiogenesis and endothelial cell dysfunction in disease

    PubMed Central

    Goveia, Jermaine; Stapor, Peter; Carmeliet, Peter

    2014-01-01

    The endothelium is the orchestral conductor of blood vessel function. Pathological blood vessel formation (a process termed pathological angiogenesis) or the inability of endothelial cells (ECs) to perform their physiological function (a condition known as EC dysfunction) are defining features of various diseases. Therapeutic intervention to inhibit aberrant angiogenesis or ameliorate EC dysfunction could be beneficial in diseases such as cancer and cardiovascular disease, respectively, but current strategies have limited efficacy. Based on recent findings that pathological angiogenesis and EC dysfunction are accompanied by EC-specific metabolic alterations, targeting EC metabolism is emerging as a novel therapeutic strategy. Here, we review recent progress in our understanding of how EC metabolism is altered in disease and discuss potential metabolic targets and strategies to reverse EC dysfunction and inhibit pathological angiogenesis. PMID:25063693

  2. Loss of endothelial barrier integrity in mice with conditional ablation of podocalyxin (Podxl) in endothelial cells.

    PubMed

    Horrillo, Angélica; Porras, Gracia; Ayuso, Matilde S; González-Manchón, Consuelo

    2016-08-01

    Podocalyxin (Podxl) has an essential role in the development and function of the kidney glomerular filtration barrier. It is also expressed by vascular endothelia but perinatal lethality of podxl(-/-) mice has precluded understanding of its function in adult vascular endothelial cells (ECs). In this work, we show that conditional knockout mice with deletion of Podxl restricted to the vascular endothelium grow normally but most die spontaneously around three months of age. Histological analysis showed a nonspecific inflammatory infiltrate within the vessel wall frequently associated with degenerative changes, and involving vessels of different caliber in one or more organs. Podxl-deficient lung EC cultures exhibit increased permeability to dextran and macrophage transmigration. After thrombin stimulation, ECs lacking Podxl showed delayed recovery of VE-cadherin cell contacts, persistence of F-actin stress fibers, and sustained phosphorylation of the ERM complex and activation of RhoA, suggesting a failure in endothelial barrier stabilization. The results suggest that Podxl has an essential role in the regulation of endothelial permeability by influencing the mechanisms involved in the restoration of endothelial barrier integrity after injury. PMID:27289182

  3. Targeting brain microvascular endothelial cells: a therapeutic approach to neuroprotection against stroke

    PubMed Central

    Yu, Qi-jin; Tao, Hong; Wang, Xin; Li, Ming-chang

    2015-01-01

    Brain microvascular endothelial cells form the interface between nervous tissue and circulating blood, and regulate central nervous system homeostasis. Brain microvascular endothelial cells differ from peripheral endothelial cells with regards expression of specific ion transporters and receptors, and contain fewer fenestrations and pinocytotic vesicles. Brain microvascular endothelial cells also synthesize several factors that influence blood vessel function. This review describes the morphological characteristics and functions of brain microvascular endothelial cells, and summarizes current knowledge regarding changes in brain microvascular endothelial cells during stroke progression and therapies. Future studies should focus on identifying mechanisms underlying such changes and developing possible neuroprotective therapeutic interventions. PMID:26807131

  4. Directed endothelial cell morphogenesis in micropatterned gelatin methacrylate hydrogels.

    PubMed

    Nikkhah, Mehdi; Eshak, Nouran; Zorlutuna, Pinar; Annabi, Nasim; Castello, Marco; Kim, Keekyoung; Dolatshahi-Pirouz, Alireza; Edalat, Faramarz; Bae, Hojae; Yang, Yunzhi; Khademhosseini, Ali

    2012-12-01

    Engineering of organized vasculature is a crucial step in the development of functional and clinically relevant tissue constructs. A number of previous techniques have been proposed to spatially regulate the distribution of angiogenic biomolecules and vascular cells within biomaterial matrices to promote vascularization. Most of these approaches have been limited to two-dimensional (2D) micropatterned features or have resulted in formation of random vasculature within three-dimensional (3D) microenvironments. In this study, we investigate 3D endothelial cord formation within micropatterned gelatin methacrylate (GelMA) hydrogels with varying geometrical features (50-150 μm height). We demonstrated the significant dependence of endothelial cells proliferation, alignment and cord formation on geometrical dimensions of the patterned features. The cells were able to align and organize within the micropatterned constructs and assemble to form cord structures with organized actin fibers and circular/elliptical cross-sections. The inner layer of the cord structure was filled with gel showing that the micropatterned hydrogel constructs guided the assembly of endothelial cells into cord structures. Notably, the endothelial cords were retained within the hydrogel microconstructs for all geometries after two weeks of culture; however, only the 100 μm-high constructs provided the optimal microenvironment for the formation of circular and stable cord structures. Our findings suggest that endothelial cord formation is a preceding step to tubulogenesis and the proposed system can be used to develop organized vasculature for engineered tissue constructs.

  5. Directed Endothelial Cell Morphogenesis in Micropatterned Gelatin Methacrylate Hydrogels

    PubMed Central

    Nikkhah, Mehdi; Eshak, Nouran; Zorlutuna, Pinar; Annabi, Nasim; Castello, Marco; Kim, Keekyoung; Dolatshahi-Pirouz, Alireza; Edalat, Faramarz; Bae, Hojae; Yang, Yunzhi; Khademhosseini, Ali

    2013-01-01

    Engineering of organized vasculature is a crucial step in the development of functional and clinically relevant tissue constructs. A number of previous techniques have been proposed to spatially regulate the distribution of angiogenic biomolecules and vascular cells within biomaterial matrices to promote vascularization. Most of these approaches have been limited to two-dimensional (2D) micropatterned features or have resulted in formation of random vasculature within three-dimensional (3D) microenvironments. In this study, we investigate 3D endothelial cord formation within micropatterned gelatin methacrylate (GelMA) hydrogels with varying geometrical features (50–150 µm height). We demonstrated the significance dependence of endothelial cells proliferation, alignment and cord formation on geometrical dimensions of the patterned features. The cells were able to align and organize within the micropatterned constructs and assemble to form cord structures with organized actin fibers and circular/elliptical cross-sections. The inner layer of the cord structure was filled with gel showing that the micropatterned hydrogel constructs guided the assembly of endothelial cells into cord structures. Notably, the endothelial cords were retained within the hydrogel microconstructs for all geometries after two weeks of culture; however, only the 100 µm-high constructs provided the optimal microenvironment for the formation of circular and stable cord structures. Our findings suggest that endothelial cord formation is a preceding step to tubulogenesis and the proposed system can be used to develop organized vasculature for engineered tissue constructs. PMID:23018132

  6. A Maillard reaction product enhances eNOS activity in human endothelial cells.

    PubMed

    Schmitt, Christoph A; Heiss, Elke H; Schachner, Daniel; Aristei, Yasmin; Severin, Theodor; Dirsch, Verena M

    2010-07-01

    Nitric oxide (NO) produced by the endothelial nitric oxide synthase (eNOS) is an important signaling molecule in the cardiovascular system. Although dietary factors can modulate eNOS activity, putative effects of processed food are barely investigated. We aimed to examine whether the model Maillard reaction product 3-hydroxy-2-methyl-1-propyl-4(1H)-pyridone (HMPP), formed from maltol or starch and propylamine, affects the eNOS system. Incubation of EA.hy926 endothelial cells with 30-300 microM HMPP for 18 h enhanced endothelial NO release measured with the fluorescent probe diaminofluorescein-2 and eNOS activity determined by the [14C]L-arginine-[14C]L-citrulline conversion assay. HMPP increased NO production also in two different types of primary human endothelial cells. Protein levels of eNOS and inducible NO synthase remained unaltered by HMPP. HMPP inhibited eNOS activity within the first 2-4 h, whereas it potently increased eNOS activity after 12-24 h. Levels of eNOS phosphorylation, expression of heat-shock protein 90, caveolin-1 and various antioxidant enzymes were not affected. Intracellular reactive oxygen species remained unchanged by HMPP. This is the first study to demonstrate positive effects of a Maillard reaction product on eNOS activity and endothelial NO production, which is considered favourable for cardiovascular protection. PMID:20112298

  7. Role of the Retinal Vascular Endothelial Cell in Ocular Disease

    PubMed Central

    Bharadwaj, Arpita S.; Appukuttan, Binoy; Wilmarth, Phillip A.; Pan, Yuzhen; Stempel, Andrew J.; Chipps, Timothy J.; Benedetti, Eric E.; Zamora, David O.; Choi, Dongseok; David, Larry L.; Smith, Justine R.

    2012-01-01

    Retinal endothelial cells line the arborizing microvasculature that supplies and drains the neural retina. The anatomical and physiological characteristics of these endothelial cells are consistent with nutritional requirements and protection of a tissue critical to vision. On the one hand, the endothelium must ensure the supply of oxygen and other nutrients to the metabolically active retina, and allow access to circulating cells that maintain the vasculature or survey the retina for the presence of potential pathogens. On the other hand, the endothelium contributes to the blood-retinal barrier that protects the retina by excluding circulating molecular toxins, microorganisms, and pro-inflammatory leukocytes. Features required to fulfill these functions may also predispose to disease processes, such as retinal vascular leakage and neovascularization, and trafficking of microbes and inflammatory cells. Thus, the retinal endothelial cell is a key participant in retinal ischemic vasculopathies that include diabetic retinopathy and retinopathy of prematurity, and retinal inflammation or infection, as occurs in posterior uveitis. Using gene expression and proteomic profiling, it has been possible to explore the molecular phenotype of the human retinal endothelial cell and contribute to understanding of the pathogenesis of these diseases. In addition to providing support for the involvement of well-characterized endothelial molecules, profiling has the power to identify new players in retinal pathologies. Findings may have implications for the design of new biological therapies. Additional progress in this field is anticipated as other technologies, including epigenetic profiling methods, whole transcriptome shotgun sequencing, and metabolomics, are used to study the human retinal endothelial cell. PMID:22982179

  8. Internalization of Aspergillus fumigatus conidia by epithelial and endothelial cells.

    PubMed Central

    Paris, S; Boisvieux-Ulrich, E; Crestani, B; Houcine, O; Taramelli, D; Lombardi, L; Latgé, J P

    1997-01-01

    The internalization of conidia of the opportunistic fungus Aspergillus fumigatus by primary cell cultures of nonprofessional phagocytes was investigated. This study is the first to show that A. fumigatus conidia were able to be engulfed by tracheal epithelial, alveolar type II, and endothelial cells. PMID:9119494

  9. Tissue Engineered Perivascular Endothelial Cell Implants Regulate Vascular Injury

    NASA Astrophysics Data System (ADS)

    Nathan, Aruna; Nugent, Matthew A.; Edelman, Elazer R.

    1995-08-01

    Molecular biomaterial engineering permits in vivo transplantation of cells and tissues, offering the promise of restoration of physiologic control rather than pharmacologic dosing with isolated compounds. We engrafted endothelial cells on Gelfoam biopolymeric matrices with retention of viability, normal growth kinetics, immunoreactivity, and biochemical activity. The production of heparan sulfate proteoglycan and inhibition of basic fibroblast growth factor binding and activity by engrafted cells were indistinguishable from endothelial cells grown in culture. Perivascular implantation of Gelfoam-endothelial cell scaffolds around balloon-denuded rat carotid arteries reduced intimal hyperplasia 88.1%, far better than the isolated administration of heparin, the most effective endothelial mimic compound. In concert with a reduction in intimal area, cell proliferation was reduced by >90%. To our knowledge, there have been no previous reports of extravascular cell implants controlling vasculoproliferative disease. Tissue engineered cells offer the potential for potent methods of vascular growth regulation and insight into the complex autocrine-paracrine control mechanisms within the blood vessel wall.

  10. Endothelial progenitor cells and burn injury - exploring the relationship.

    PubMed

    Banyard, Derek A; Adnani, Blake O; Melkumyan, Satenik; Araniego, Cheryl Ann; Widgerow, Alan D

    2016-01-01

    Burn wounds result in varying degrees of soft tissue damage that are typically graded clinically. Recently a key participant in neovascularization, the endothelial progenitor cell, has been the subject of intense cardiovascular research to explore whether it can serve as a biomarker for vascular injury. In this review, we examine the identity of the endothelial progenitor cell as well as the evidence that support its role as a key responder after burn insult. While there is conflicting evidence with regards to the delta of endothelial progenitor cell mobilization and burn severity, it is clear that they play an important role in wound healing. Systematic and controlled studies are needed to clarify this relationship, and whether this population can serve as a biomarker for burn severity. PMID:27574674

  11. Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells

    PubMed Central

    1993-01-01

    Polymorphonuclear leukocytes (PMN) traverse an endothelial cell (EC) barrier by crawling between neighboring EC. Whether EC regulate the integrity of their intercellular adhesive and junctional contacts in response to chemotaxing PMN is unresolved. EC respond to the binding of soluble mediators such as histamine by increasing their cytosolic free calcium concentration ([Ca++]i) (Rotrosen, D., and J.I. Gallin. 1986. J. Cell Biol. 103:2379-2387) and undergoing shape changes (Majno, G., S. M. Shea, and M. Leventhal. 1969. J. Cell Biol. 42:617-672). Substances such as leukotriene C4 (LTC4) and thrombin, which increased the permeability of EC monolayers to ions, as measured by the electrical resistance of the monolayers, transiently increased EC [Ca++]i. To determine whether chemotaxing PMN cause similar changes in EC [Ca++]i, human umbilical vein endothelial cells (HUVEC) maintained as monolayers were loaded with fura-2. [Ca++]i was measured in single EC during PMN adhesion to and migration across these monolayers. PMN-EC adhesion and transendothelial PMN migration in response to formyl- methionyl-leucyl-phenylalanine (fMLP) as well as to interleukin 1 (IL- 1) treated EC induced a transient increase in EC [Ca++]i which temporally corresponded with the time course of PMN-EC interactions. When EC [Ca++]i was clamped at resting levels with a cell permeant calcium buffer, PMN migration across EC monolayers and PMN induced changes in EC monolayer permeability were inhibited. However, clamping of EC [Ca++]i did not inhibit PMN-EC adhesion. These studies provide evidence that EC respond to stimulated PMN by increasing their [Ca++]i and that this increase in [Ca++]i causes an increase in EC monolayer permeability. Such [Ca++]i increases are required for PMN transit across an EC barrier. We suggest EC [Ca++]i regulates transendothelial migration of PMN by participating in a signal cascade which stimulates EC to open their intercellular junctions to allow transendothelial

  12. Endothelial Cells Enhance Tumor Cell Invasion through a Crosstalk Mediated by CXC Chemokine Signaling1

    PubMed Central

    Warner, Kristy A; Miyazawa, Marta; Cordeiro, Mabel M R; Love, William J; Pinsky, Matthew S; Neiva, Kathleen G; Spalding, Aaron C; Nör, Jacques E

    2008-01-01

    Field cancerization involves the lateral spread of premalignant or malignant disease and contributes to the recurrence of head and neck tumors. The overall hypothesis underlying this work is that endothelial cells actively participate in tumor cell invasion by secreting chemokines and creating a chemotactic gradient for tumor cells. Here we demonstrate that conditioned medium from head and neck tumor cells enhance Bcl-2 expression in neovascular endothelial cells. Oral squamous cell carcinoma-3 (OSCC3) and Kaposi's sarcoma (SLK) show enhanced invasiveness when cocultured with pools of human dermal microvascular endothelial cells stably expressing Bcl-2 (HDMEC-Bcl-2), compared to cocultures with empty vector controls (HDMEC-LXSN). Xenografted OSCC3 tumors vascularized with HDMEC-Bcl-2 presented higher local invasion than OSCC3 tumors vascularized with control HDMEC-LXSN. CXCL1 and CXCL8 were upregulated in primary endothelial cells exposed to vascular endothelial growth factor (VEGF), as well as in HDMEC-Bcl-2. Notably, blockade of CXCR2 signaling, but not CXCR1, inhibited OSCC3 and SLK invasion toward endothelial cells. These data demonstrate that CXC chemokines secreted by endothelial cells induce tumor cell invasion and suggest that the process of lateral spread of tumor cells observed in field cancerization is guided by chemotactic signals that originated from endothelial cells. PMID:18283335

  13. Regulation of Endothelial Cell Proliferation and Vascular Assembly through Distinct mTORC2 Signaling Pathways

    PubMed Central

    Wang, Shan; Amato, Katherine R.; Song, Wenqiang; Youngblood, Victoria; Lee, Keunwook; Boothby, Mark; Brantley-Sieders, Dana M.

    2015-01-01

    Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates a diverse array of cellular processes, including cell growth, survival, metabolism, and cytoskeleton dynamics. mTOR functions in two distinct complexes, mTORC1 and mTORC2, whose activities and substrate specificities are regulated by complex specific cofactors, including Raptor and Rictor, respectively. Little is known regarding the relative contribution of mTORC1 versus mTORC2 in vascular endothelial cells. Using mouse models of Raptor or Rictor gene targeting, we discovered that Rictor ablation inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation and assembly in vitro and angiogenesis in vivo, whereas the loss of Raptor had only a modest effect on endothelial cells (ECs). Mechanistically, the loss of Rictor reduced the phosphorylation of AKT, protein kinase Cα (PKCα), and NDRG1 without affecting the mTORC1 pathway. In contrast, the loss of Raptor increased the phosphorylation of AKT despite inhibiting the phosphorylation of S6K1, a direct target of mTORC1. Reconstitution of Rictor-null cells with myristoylated AKT (Myr-AKT) rescued vascular assembly in Rictor-deficient endothelial cells, whereas PKCα rescued proliferation defects. Furthermore, tumor neovascularization in vivo was significantly decreased upon EC-specific Rictor deletion in mice. These data indicate that mTORC2 is a critical signaling node required for VEGF-mediated angiogenesis through the regulation of AKT and PKCα in vascular endothelial cells. PMID:25582201

  14. Mast cell heparin stimulates migration of capillary endothelial cells in vitro

    PubMed Central

    1980-01-01

    Migration of capillary endothelial cells is an important component of angiogenesis in vivo. Increased numbers of mast cells have been associated with several types of angiogenesis. We have used a quantitative assay in vitro to demonstrate that mast cells release a factor that significantly increases bovine capillary endothelial cell migration. The factor is present in medium conditioned by mast cells as well as lysates of mast cells. The stimulatory effect of mast cells on migration is specific for capillary endothelial cells. Furthermore, mast cells have no mitogenic activity for capillary endothelial cells. Of all the secretory products of mast cells tested, only heparin stimulated capillary endothelial cell migration in vitro. Heparin preparations from a variety of sources stimulated capillary endothelial cell migration to the same degree but did not stimulate migration of several other cell types. The migration activity of heparin and mast cell conditioned medium was blocked by specific antagonists of heparin (protamine and heparinase), but not by chondroitinase ABC. The migration activity of mast cell conditioned medium was resistant to heat (100 degrees C) and incubation with proteolytic enzymes. These results suggest that the role of mast cells in angiogenesis may be to enhance migration of the endothelial cells of growing capillaries. PMID:7420025

  15. Von Willebrand factor regulates complement on endothelial cells.

    PubMed

    Noone, Damien G; Riedl, Magdalena; Pluthero, Fred G; Bowman, Mackenzie L; Liszewski, M Kathryn; Lu, Lily; Quan, Yi; Balgobin, Steve; Schneppenheim, Reinhard; Schneppenheim, Sonja; Budde, Ulrich; James, Paula; Atkinson, John P; Palaniyar, Nades; Kahr, Walter H A; Licht, Christoph

    2016-07-01

    Atypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura have traditionally been considered separate entities. Defects in the regulation of the complement alternative pathway occur in atypical hemolytic uremic syndrome, and defects in the cleavage of von Willebrand factor (VWF)-multimers arise in thrombotic thrombocytopenic purpura. However, recent studies suggest that both entities are related as defects in the disease-causing pathways overlap or show functional interactions. Here we investigate the possible functional link of VWF-multimers and the complement system on endothelial cells. Blood outgrowth endothelial cells (BOECs) were obtained from 3 healthy individuals and 2 patients with Type 3 von Willebrand disease lacking VWF. Cells were exposed to a standardized complement challenge via the combination of classical and alternative pathway activation and 50% normal human serum resulting in complement fixation to the endothelial surface. Under these conditions we found the expected release of VWF-multimers causing platelet adhesion onto BOECs from healthy individuals. Importantly, in BOECs derived from patients with von Willebrand disease complement C3c deposition and cytotoxicity were more pronounced than on BOECs derived from normal individuals. This is of particular importance as primary glomerular endothelial cells display a heterogeneous expression pattern of VWF with overall reduced VWF abundance. Thus, our results support a mechanistic link between VWF-multimers and the complement system. However, our findings also identify VWF as a new complement regulator on vascular endothelial cells and suggest that VWF has a protective effect on endothelial cells and complement-mediated injury. PMID:27236750

  16. Augmentation of platelet and endothelial cell eNOS activity decreases sepsis-related neutrophil-endothelial cell interactions.

    PubMed

    Khan, Raymond; Kirschenbaum, Linda A; LaRow, Catherine; Berna, Gioiamaria; Griffin, Kelly; Astiz, Mark E

    2010-03-01

    NO is an important mediator of microvascular patency and blood flow. The purpose of this study was to examine the role of enhanced eNOS activity in attenuating sepsis-induced neutrophil-endothelial cell interactions. Microslides coated with human umbilical vein endothelial cells were stimulated with plasma from patients with septic shock. Neutrophil and platelets from control subjects were also stimulated with plasma from patients in septic shock and perfused over stimulated endothelial cells. l-Arginine (LA) with and without NG-monomethyl l-arginine (LNMMA), a nonselective NOS inhibitor, and N-(3-(aminomethyl) benzyl acetamide) ethanimidamide dihydrochloride (1400W), a highly selective iNOS inhibitor, were added to the septic plasma. The number of neutrophils adherent to endothelial cells, neutrophil rolling velocity, and the number of neutrophil aggregates were determined. Cell activation and the formation of platelet-neutrophil aggregates were assessed by flow cytometry. Separate experiments were done with isolated platelets using platelet aggregometry. l-Arginine significantly decreased sepsis-related neutrophil adhesion and aggregation and increased rolling velocity. The addition of LNMMA to LA and cell suspensions reversed the effects of LA on these parameters, whereas the addition of 1400W had no effect on LA-related changes. Platelet-neutrophil aggregation, platelet aggregation, platelet activation, and neutrophil activation induced by septic plasma were also significantly decreased by LA. Again, the addition of LNMMA reversed the effects of LA on these parameters, whereas 1400W had no effect on LA-related changes. These data suggest that enhancement of platelet and endothelial cell eNOS activity decreases sepsis-induced neutrophil-endothelial cell interactions and may play a role in maintaining microvascular patency in septic shock.

  17. Downregulation of Fes inhibits VEGF-A-induced chemotaxis and capillary-like morphogenesis by cultured endothelial cells

    PubMed Central

    Kanda, Shigeru; Kanetake, Hiroshi; Miyata, Yasuyoshi

    2007-01-01

    Abstract The aim of this study was to determine whether the downregulation of endogenous Fes by siRNA in cultured endothelial cells affects vascular endothelial growth factor-A (VEGF-A)-induced chemotaxis and capillary-like morphogenesis, which are considered as angiogenic cellular responses in vitro. VEGF-A-treatment induced autophosphorylation of Fes in cultured endothelial cells.LY294002, a phosphoinositide 3-kinase inhibitor, significantly inhibited VEGF-A-induced chemotaxis and capillary-like morphogenesis.Downregulation of Fes attenuated these VEGF-A-induced cellular responses but LY294002 did not produce further inhibition of these responses. Downregulation of Fes neither affected VEGF-A-induced autophosphorylation of VEGF receptor 2 nor mitogen-activated protein kinase activation, but markedly decreased Akt activation.Taken together, our novel results indicate the involvement of Fes in VEGF-A-induced cellular responses by cultured endothelial cells. PMID:17521372

  18. Treponema pallidum Invades Intercellular Junctions of Endothelial Cell Monolayers

    NASA Astrophysics Data System (ADS)

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

    1988-05-01

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

  19. New insights in endothelial and smooth muscle cell communication.

    PubMed

    Conejo, Víctor Arana; De Haro, Roberto; Sosa-Melgarejo, Jorge; Méndez, José D

    2007-01-01

    Based on immunohistochemical techniques against connexins and the intercellular flux of staining molecules, it has previously been shown that electrotonic communication occurs among endothelial and vascular smooth muscle cells, this due to the presence of myoendothelial gap junctions. The aim of this study was to evaluate the density of myoendothelial contacts in the left coronary and internal mammary arteries as well as in the left saphenous vein by means of electron microscopy, the distance between both cells participating in an myoendothelial contact with a semi-automatic image analysis system and the presence of homocellular and heterocellular gap junctions between endothelial and smooth muscle cells by using the immunohistochemical technique and confocal microscopy in thoracic aorta were also analyzed. The results are that all blood vessels studied present myoendothelial contacts, while density studies show that they are more abundant in the saphenous vein. The myoendothelial contact distance is constant and in no case the cytoplasmic processes reach the plasma membrane of the partner cell toward which they are advanced. Homocellular gap junctions were found between smooth muscle cells and between endothelial cells. Heterocellular gap junctions were absent, evidencing the possibility that signaling molecules between endothelial and smooth muscle cells may be transferred through plasma membranes as was once thought and not necessarily by electrotonic communication. PMID:17383847

  20. Reciprocal interactions between mitral valve endothelial and interstitial cells reduce endothelial-to-mesenchymal transition and myofibroblastic activation

    PubMed Central

    Shapero, Kayle; Wylie-Sears, Jill; Levine, Robert A.; Mayer, John E.; Bischoff, Joyce

    2015-01-01

    Thickening of mitral leaflets, endothelial-to-mesenchymal transition (EndMT), and activated myofibroblast-like interstitial cells have been observed in ischemic mitral valve regurgitation. We set out to determine if interactions between mitral valve endothelial cells (VEC) and interstitial cells (VIC) might affect these alterations. We used in vitro co-culture in Transwell™ inserts to test the hypothesis that VIC secrete factors that inhibit EndMT and conversely, that VEC secrete factors that mitigate the activation of VIC to a myofibroblast-like, activated phenotype. Primary cultures and clonal populations of ovine mitral VIC and VEC were used. Western blot, quantitative reverse transcriptase PCR (qPCR) and functional assays were used to assess changes in cell phenotype and behavior. VIC or conditioned media from VIC inhibited transforming growth factorβ (TGFβ)-induced EndMT in VEC, as indicated by reduced expression of EndMT markers α-smooth muscle actin (α-SMA), Slug, Snai1 and MMP-2 and maintained ability of VEC to mediate leukocyte adhesion, an important endothelial function. VEC or conditioned media from VEC reversed the spontaneous cell culture-induced change in VIC to an activated phenotype, as indicated by reduced expression of α-SMA and type I collagen, increased expression chondromodulin-1 (Chm1), and reduced contractile activity. These results demonstrate that mitral VEC and VIC secrete soluble factors that can reduce VIC activation and inhibit TGFβ-driven EndMT, respectively. These findings suggest that the endothelium of the mitral valve is critical for the maintenance of a quiescent VIC phenotype and that, in turn, VIC prevent EndMT. We speculate that disturbance of the ongoing reciprocal interactions between VEC and VICs in vivo may contribute to the thickened and fibrotic leaflets observed in ischemic mitral regurgitation, and in other types of valve disease. PMID:25633835

  1. Neutrophil Elastase-Generated Fragment of Vascular Endothelial Growth Factor-A Stimulates Macrophage and Endothelial Progenitor Cell Migration

    PubMed Central

    Kurtagic, Elma; Rich, Celeste B.; Buczek-Thomas, Jo Ann; Nugent, Matthew A.

    2015-01-01

    Elastase released from neutrophils as part of the innate immune system has been implicated in chronic diseases such as emphysema and cardiovascular disease. We have previously shown that neutrophil elastase targets vascular endothelial growth factor-A (VEGF) for partial degradation to generate a fragment of VEGF (VEGFf) that has distinct activities. Namely, VEGFf binds to VEGF receptor 1 but not to VEGF receptor 2 and shows altered signaling compared to intact VEGF. In the present study we investigated the chemotactic function of VEGF and VEGFf released from cells by neutrophil elastase. We found that endothelial cells migrated in response to intact VEGF but not VEGFf whereas RAW 264.7 macrophages/monocytes and embryonic endothelial progenitor cells were stimulated to migrate by either VEGF or VEGFf. To investigate the role of elastase-mediated release of VEGF from cells/extracellular matrices, a co-culture system was established. High or low VEGF producing cells were co-cultured with macrophages, endothelial or endothelial progenitor cells and treated with neutrophil elastase. Elastase treatment stimulated macrophage and endothelial progenitor cell migration with the response being greater with the high VEGF expressing cells. However, elastase treatment led to decreased endothelial cell migration due to VEGF cleavage to VEGF fragment. These findings suggest that the tissue response to NE-mediated injury might involve the generation of diffusible VEGF fragments that stimulate inflammatory cell recruitment. PMID:26672607

  2. Primary culture of endothelial cells from atherosclerotic human aorta. Part 1. Identification, morphological and ultrastructural characteristics of two endothelial cell subpopulations.

    PubMed

    Antonov, A S; Nikolaeva, M A; Klueva, T S; Romanov YuA; Babaev, V R; Bystrevskaya, V B; Perov, N A; Repin, V S; Smirnov, V N

    1986-01-01

    Endothelial cells (EC) were harvested by 0.1% collagenase treatment for adult human thoracic aortas obtained 1-3 h after sudden death. At least 35-70% of EC were removed from the intimal surface of aorta, 90-95% of them being viable. Plating efficiency was 70-80%. Monolayer formation was achieved at a seeding density of 5-8 X 10(2) cells/mm2. The cells were identified as endothelium by the presence of Factor VIII antigen, Weigel-Palade bodies and typically endothelial morphology at confluence. Unlike endothelial cultures derived from human umbilical veins and infant aortas, primary cultures obtained from human adult aortas contain multinuclear EC with Factor VIII antigen and Weibel-Palade bodies. The number of multinuclear EC in cultures isolated from aortas affected by atherosclerosis was 2-fold higher (P less than 0.05) than in cultures obtained from grossly normal aortas taken from donors of the same age. EC with numerous lipid inclusions revealed by oil-red-O staining were present in all the EC primary cultures derived from aortas affected by atherosclerosis. No oil-red-O-positive cells were detected among the EC cultured from infant aorta, aorta of young donors, and umbilical vein. An electron microscopic examination of EC from atherosclerotic aorta in culture and in situ failed to reveal any ultrastructural peculiarities distinguishing multinuclear EC from the mononuclear EC. PMID:3004520

  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. Isolation of endothelial cells and pericytes from swine corpus luteum.

    PubMed

    Basini, G; Falasconi, I; Bussolati, S; Grolli, S; Ramoni, R; Grasselli, F

    2014-07-01

    From an angiogenesis perspective, the ovary offers a unique opportunity to study the physiological development of blood vessels. The first purpose of this work was to set up a protocol for the isolation of pig corpus luteum endothelial cells, which were characterized by both morphologic parameters and the expression of typical molecular markers; we also verified their ability to form capillary-like structures in a 3-dimensional matrix, their response to hypoxia and their migration in the presence of vascular endothelial growth factor (VEGF). The effectiveness of our isolation protocol was confirmed by the characteristic "cobblestone shape" of isolated cells at confluence as well as their expression of all the examined endothelial markers. Our data also showed a significant cell production of VEGF and nitric oxide. Isolated endothelial cells were also responsive to hypoxia by increasing the expression and production of VEGF and decreasing that of nitric oxide. In the angiogenesis bioassay, cells displayed the ability of forming capillary-like structures and also exhibited a significant migration in the scratch test. Our data suggest that the isolation of luteal endothelial cells represents a promising tool in experiments designed to clarify the biology of the angiogenic process. Furthermore, we have demonstrated that the isolated population comprises a subset of cells with a multidifferentiative capacity toward the chondrocytic and adipocytic phenotypes. These data suggest the presence of a perivascular or adventitial cell niche in the vascular wall of the corpus luteum populated with cells showing mesenchymal stem cell-like features, as already demonstrated for the adipose tissue and endometrium.

  5. 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.

  6. Endothelial activation by platelets from sickle cell anemia patients.

    PubMed

    Proença-Ferreira, Renata; Brugnerotto, Ana Flávia; Garrido, Vanessa Tonin; Dominical, Venina Marcela; Vital, Daiana Morelli; Ribeiro, Marilene de Fátima Reis; dos Santos, Melissa Ercolin; Traina, Fabíola; Olalla-Saad, Sara T; Costa, Fernando Ferreira; Conran, Nicola

    2014-01-01

    Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association

  7. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.

    PubMed Central

    Connolly, D T; Heuvelman, D M; Nelson, R; Olander, J V; Eppley, B L; Delfino, J J; Siegel, N R; Leimgruber, R M; Feder, J

    1989-01-01

    Vascular permeability factor (VPF) is an Mr 40-kD protein that has been purified from the conditioned medium of guinea pig line 10 tumor cells grown in vitro, and increases fluid permeability from blood vessels when injected intradermally. Addition of VPF to cultures of vascular endothelial cells in vitro unexpectedly stimulated cellular proliferation. VPF promoted the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The identity of the growth factor activity with VPF was established in four ways: (a) the molecular weight of the activity in preparative SDS-PAGE was the same as VPF (Mr approximately 40 kD); (b) multiple isoforms (pI greater than or equal to 8) for both VPF and the growth-promoting activity were observed; (c) a single, unique NH2-terminal amino acid sequence was obtained; (d) both growth factor and permeability-enhancing activities were immunoadsorbed using antipeptide IgG that recognized the amino terminus of VPF. Furthermore, 125I-VPF was shown to bind specifically and with high affinity to endothelial cells in vitro and could be chemically cross-linked to a high-molecular weight cell surface receptor, thus demonstrating a mechanism whereby VPF can interact directly with endothelial cells. Unlike other endothelial cell growth factors, VPF did not stimulate [3H]thymidine incorporation or promote growth of other cell types including mouse 3T3 fibroblasts or bovine smooth muscle cells. VPF, therefore, appears to be unique in its ability to specifically promote increased vascular permeability, endothelial cell growth, and angio-genesis. Images PMID:2478587

  8. Melatonin modulates aromatase activity and expression in endothelial cells.

    PubMed

    Alvarez-García, Virginia; González, Alicia; Martínez-Campa, Carlos; Alonso-González, Carolina; Cos, Samuel

    2013-05-01

    Melatonin is known to suppress the development of endocrine-responsive breast cancers by interacting with the estrogen signaling pathways. Paracrine interactions between malignant epithelial cells and proximal stromal cells are responsible for local estrogen biosynthesis. In human breast cancer cells and peritumoral adipose tissue, melatonin downregulates aromatase, which transforms androgens into estrogens. The presence of aromatase on endothelial cells indicates that endothelial cells may contribute to tumor growth by producing estrogens. Since human umbilical vein endothelial cells (HUVECs) express both aromatase and melatonin receptors, the aim of the present study was to evaluate the ability of melatonin to regulate the activity and expression of aromatase on endothelial cells, thus, modulating local estrogen biosynthesis. In the present study, we demonstrated that melatonin inhibits the growth of HUVECs and reduces the local biosynthesis of estrogens through the downregulation of aromatase. These results are supported by three lines of evidence. Firstly, 1 mM of melatonin counteracted the testosterone-induced cell proliferation of HUVECs, which is dependent on the local biosynthesis of estrogens from testosterone by the aromatase activity of the cells. Secondly, we found that 1 mM of melatonin reduced the aromatase activity of HUVECs. Finally, by real‑time RT-PCR, we demonstrated that melatonin significantly downregulated the expression of aromatase as well as its endothelial-specific aromatase promoter region I.7. We conclude that melatonin inhibits aromatase activity and expression in HUVECs by regulating gene expression of specific aromatase promoter regions, thereby reducing the local production of estrogens. PMID:23450505

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

    NASA Astrophysics Data System (ADS)

    Arbeitman, Claudia R.; del Grosso, Mariela F.; Behar, Moni; García Bermúdez, Gerardo

    2013-11-01

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

  10. Zinc and dexamethasone induce metallothionein accumulation by endothelial cells

    SciTech Connect

    Briske-Anderson, M.; Bobilya, D.J.; Reeves, P.G. )

    1991-03-11

    Several tissues increase their metallothionein (MT) concentration when exposed to elevated amounts of plasma Zn. Endothelial cells form the blood vessels that supply all tissues and constitute a barrier between cells of tissues and the blood. This study examined the ability of endothelial cells to synthesize MT and accumulate Zn in response to high amounts of Zn and dexamethasone. Bovine pulmonary endothelial cells were grown to confluence in Minimum Essential Medium with Earle's salts and 10% fetal calf serum. The monolayer was maintained for 2 d prior to use in medium containing EDTA-dialyzed serum. This low Zn medium was replaced with one containing 1, 6, 25, 50, 100, 150, or 200 {mu}M Zn and incubated for 24 hr before harvesting the cells. MT was quantified by the cadmium binding assay. Cellular Zn concentrations were analyzed by atomic absorption after a nitric acid digestion. The MT concentration was elevated in response to Zn concentrations of 100 {mu}M or more. Cellular Zn concentration was elevated when media Zn was 25 {mu}M or more. MT and cellular Zn concentrations were positively correlated. In another study, inclusion of 0.1 {mu}M dexamethasone in the media increased concentration at all Zn concentrations studied. However, the inclusion of 0.3 {mu}M cis-platinum had no effect. In conclusion, endothelial cells in culture respond to elevated amounts of Zn and dexamethasone in the media by accumulating Zn and MT.

  11. Synthesis of Antihemophilic Factor Antigen by Cultured Human Endothelial Cells

    PubMed Central

    Jaffe, Eric A.; Hoyer, Leon W.; Nachman, Ralph L.

    1973-01-01

    Antihemophilic factor (AHF, Factor VIII) antigen has been demonstrated in cultured human endothelial cells by immunofluorescence studies using monospecific rabbit antibody to human AHF. Control studies with cultured human smooth muscle cells and human fibroblasts were negative. By radioimmunoassay it was demonstrated that cultured human endothelial cells contain AHF antigen which is released into the culture medium. Cultured smooth muscle cells and fibroblasts did not have this property. Cultured endothelial cells incorporated radioactive amino acids into high molecular weight, AHF antigen-rich protein fractions prepared from the culture media, 7% of the radioactive amino acid counts incorporated into this material were precipitated by globulin prepared from rabbit anti-AHF whereas normal rabbit globulin precipitated only 1.5% of the counts. Although cultured endothelial cells actively synthesize AHF antigen, AHF procoagulant activity was not detected in the culture medium. Studies seeking a basis for the lack of procoagulant activity have not clarified this deficiency, but they have established that exogenous AHF procoagulant activity is not inactivated by the tissue culture system. Images PMID:4583980

  12. Cell-based approach for treatment of corneal endothelial dysfunction.

    PubMed

    Okumura, Naoki; Kinoshita, Shigeru; Koizumi, Noriko

    2014-11-01

    Decompensation of the corneal endothelium causes severe visual impairments that lead to blindness. Although corneal transplantation is a well-known effective therapy for corneal endothelial dysfunction, many patients are not afforded that therapeutic opportunity owing to the worldwide shortage of donor corneas. Thus, a tissue engineering-based therapy for treating corneal endothelial dysfunction is highly anticipated. Obstacles associated with the development of tissue engineering therapy include in vitro culture of corneal endothelial cells (CECs) and the techniques used to transplant those cells. Limited proliferation ability, cellular senescence, and fibroblastic transformation during culture are all problems associated with the cultivation of CECs. In addition, transplantation of cultured CECs is technically difficult because the corneal endothelium is composed of a fragile monolayer sheet of cells located at the posterior cornea. In this review article, we present our recent findings using a novel cell culture protocol and show that modulation of CEC adhesion properties through a Rho-kinase inhibitor enables transplantation of CECs in the form of a cell suspension without the use of a carrier. Finally, we provide an update on the clinical application status of a cell-based therapy for treating corneal endothelial dysfunction.

  13. Tumor endothelial cells with distinct patterns of TGFβ-driven endothelial-to-mesenchymal transition

    PubMed Central

    Xiao, Lin; Kim, Dae Joong; Davis, Clayton L.; McCann, James V.; Dunleavey, James M.; Vanderlinden, Alissa; Xu, Nuo; Pattenden, Samantha G.; Frye, Stephen V.; Xu, Xia; Onaitis, Mark; Monaghan-Benson, Elizabeth; Burridge, Keith; Dudley, Andrew C.

    2015-01-01

    Endothelial-to-mesenchymal transition (EndMT) occurs during development and underlies the pathophysiology of multiple diseases. In tumors, unscheduled EndMT generates cancer-associated myofibroblasts that fuel inflammation and fibrosis, and may contribute to vascular dysfunction that promotes tumor progression. We report that freshly isolated subpopulations of tumor-specific endothelial cells (TEC) from a spontaneous mammary tumor model undergo distinct forms of EndMT in response to TGFβ stimulation. Whereas some TEC strikingly up-regulate alpha smooth muscle actin (SMA), a principal marker of EndMT and activated myofibroblasts, counterpart normal mammary gland endothelial cells (NEC) showed little change in SMA expression after TGFβ treatment. Compared with NEC, SMA+ TEC were 40 % less motile in wound healing assays and formed more stable vascular-like networks in vitro when challenged with TGFβ. Lineage tracing using ZsGreenCdh5-Cre reporter mice confirmed that only a fraction of vessels in breast tumors contain SMA+ TEC, suggesting that not all endothelial cells (EC) respond identically to TGFβ in vivo. Indeed, examination of 84 TGFβ-regulated target genes revealed entirely different genetic signatures in TGFβ-stimulated NEC and TEC cultures. Finally, we found that basic FGF (bFGF) exerts potent inhibitory effects on many TGFβ-regulated genes but operates in tandem with TGFβ to up-regulate others. EC challenged with TGFβ secrete bFGF which blocks SMA expression in secondary cultures suggesting a cell-autonomous or lateral-inhibitory mechanism for impeding mesenchymal differentiation. Together, our results suggest that TGFβ-driven EndMT produces a spectrum of EC phenotypes with different functions that could underlie the plasticity and heterogeneity of the tumor vasculature. PMID:25634211

  14. TGF-{beta}2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

    SciTech Connect

    Lu Jiawei; Lu Zhenyu; Reinach, Peter

    2006-11-01

    The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-{beta}2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-{beta}2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-{beta}2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-{beta}2 and FGF-2 oppositely affect BCE cell proliferation and TGF-{beta}2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-{beta}2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-{beta}2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-{beta}2-induced suppression of the PI3-kinase/AKT signaling pathway.

  15. 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.

  16. 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

  17. Mechanical property quantification of endothelial cells using scanning acoustic microscopy

    NASA Astrophysics Data System (ADS)

    Shelke, A.; Brand, S.; Kundu, T.; Bereiter-Hahn, J.; Blase, C.

    2012-04-01

    The mechanical properties of cells reflect dynamic changes of cellular organization which occur during physiologic activities like cell movement, cell volume regulation or cell division. Thus the study of cell mechanical properties can yield important information for understanding these physiologic activities. Endothelial cells form the thin inner lining of blood vessels in the cardiovascular system and are thus exposed to shear stress as well as tensile stress caused by the pulsatile blood flow. Endothelial dysfunction might occur due to reduced resistance to mechanical stress and is an initial step in the development of cardiovascular disease like, e.g., atherosclerosis. Therefore we investigated the mechanical properties of primary human endothelial cells (HUVEC) of different age using scanning acoustic microscopy at 1.2 GHz. The HUVECs are classified as young (tD < 90 h) and old (tD > 90 h) cells depending upon the generation time for the population doubling of the culture (tD). Longitudinal sound velocity and geometrical properties of cells (thickness) were determined using the material signature curve V(z) method for variable culture condition along spatial coordinates. The plane wave technique with normal incidence is assumed to solve two-dimensional wave equation. The size of the cells is modeled using multilayered (solid-fluid) system. The propagation of transversal wave and surface acoustic wave are neglected in soft matter analysis. The biomechanical properties of HUVEC cells are quantified in an age dependent manner.

  18. 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

  19. Metformin improves endothelial function in aortic tissue and microvascular endothelial cells subjected to diabetic hyperglycaemic conditions.

    PubMed

    Ghosh, Suparna; Lakshmanan, Arun P; Hwang, Mu Ji; Kubba, Haidar; Mushannen, Ahmed; Triggle, Chris R; Ding, Hong

    2015-12-01

    The cellular mechanisms whereby metformin, the first line drug for type 2 diabetes (T2DM), mediates its antidiabetic effects remain elusive, particularly as to whether metformin has a direct protective action on the vasculature. This study was designed to determine if a brief 3-h exposure to metformin protects endothelial function against the effects of hyperglycaemia. We investigated the protective effects of metformin on endothelial-dependent vasodilatation (EDV) in thoracic aortae from T2DM db/db mice and on high glucose (HG, 40 mM) induced changes in endothelial nitric oxide synthase (eNOS) signaling in mouse microvascular endothelial cells (MMECs) in culture. Exposure of aortae from db+/? non-diabetic control mice to high glucose (HG, 40 mM) containing Krebs for 3-h significantly (P<0.05) reduced acetylcholine (ACh)-induced EDV compared to ACh-induced EDV in aortae maintained in normal glucose (NG, 11 mM) Krebs. The reduction of EDV was partially reversed following a 3-h exposure to 50 μM metformin; metformin also improved ACh-induced EDV in aortae from diabetic db/db mice. Immunoblot analysis of MMECs cultured in HG versus NG revealed a significant reduction of the ratio of phosphorylated (p-eNOS)/eNOS and p-Akt/Akt, but not the expression of total eNOS or Akt. The 3-h exposure of MMECs to metformin significantly (P<0.05) reversed the HG-induced reduction in phosphorylation of both eNOS and Akt; however, no changes were detected for phosphorylation of AMPK or the expression of SIRT1. Our data indicate that a 3-h exposure to metformin can reverse/reduce the impact of HG on endothelial function, via mechanisms linked to increased phosphorylation of eNOS and Akt.

  20. 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

  1. Binding of ATGs to Endothelial Cells In Vivo.

    PubMed

    Beiras-Fernandez, Andres; Hernandez-Sierra, Astrid; Schulz, Uwe; Richter, Manfred; Thein, Eckart; Moritz, Anton; Werner, Isabella

    2016-05-17

    BACKGROUND Polyclonal anti-thymocyte globulins (ATGs) are immunosuppressive drugs widely used in induction of immunosuppression and treatment of acute rejection after solid organ transplantation. We have previously demonstrated that ATGs bind to endothelial cells in vitro, and are able to modulate ECs. The aim of this study was to investigate the binding of ATGs to endothelial cells under in vivo conditions. MATERIAL AND METHODS Muscle biopsies from extremities of cynomolgus monkeys were obtained after ischemia/reperfusion at 4°C. ATGs (Thymoglobulin, Sanofi-Aventis, France; 1 mg/kg) were added to the blood 30 min prior to the reperfusion. Biopsies (n=10) of patients undergoing heart transplantation and preoperatively treated with ATGs (Thymoglobulin, Sanofi-Aventis, France; 1.5 mg/kg) as induction therapy were also analyzed 6 hours and 7 days after induction. Binding of ATGs to ECs was analyzed with an anti-rabbit IgG antibody by means of immunohistochemistry. RESULTS Binding of ATGs to endothelial cells could be demonstrated in vivo in our animal experiments 4 hours after reperfusion, as well as in the clinical biopsies 6 hours after induction of immunosuppression in heart transplant patients, showing a preferred localization in post-capillary veins. No expression of ATGs on the endothelial surface could be observed after 7 days, suggesting that ATGs may be washed out from the endothelial surface in a time-dependent manner. CONCLUSIONS Our results show that ATGs are able to bind to endothelial cells in an experimental model and in clinical practice, supporting preconditioning strategies with ATGs in solid organ transplantation.

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

    PubMed

    Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

    2015-01-01

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

  3. Activated Factor X Induces Endothelial Cell Senescence Through IGFBP-5

    PubMed Central

    Sanada, Fumihiro; Taniyama, Yoshiaki; Muratsu, Jun; Otsu, Rei; Iwabayashi, Masaaki; Carracedo, Miguel; Rakugi, Hiromi; Morishita, Ryuichi

    2016-01-01

    Uncontrolled coagulation contributes to the pathophysiology of several chronic inflammatory diseases. In these conditions, senescent cells are often observed and is involved in the generation of inflammation. The coincidence of hyper-coagulation, cell senescence, and inflammation suggests the existence of a common underlying mechanism. Recent evidence indicates that activated coagulation factor X (FXa) plays a role in the processes beyond blood coagulation. This non-hematologic function entails the mediation of inflammation and tissue remodeling. We therefore tested the hypothesis that FXa induces cell senescence resulting in tissue inflammation and impaired tissue regeneration. Human umbilical vein endothelial cells were stimulated with FXa for 14 days. The proliferation of cells treated with FXa was significantly smaller, and the fraction of senescence-associated β-galactosidase-positive cells was increased as compared to the control group. RT-qPCR array revealed that FXa increased the expression of IGFBP-5, EGR-1, p53, and p16INK4a. Inhibition of FXa by a direct FXa inhibitor, rivaroxaban, or IGFBP-5 by siRNA decreased FXa-induced cell senescence, restoring cell proliferation. Moreover, in an ischemic hind limb mouse model, FXa inhibited neovascularization by endothelial progenitor cell. However, rivaroxaban significantly restored FXa-induced impaired angiogenesis. In summary, FXa induced endothelial cell senescence through IGFBP-5, resulting in impaired angiogenesis. PMID:27752126

  4. Influence of membrane cholesterol and substrate elasticity on endothelial cell spreading behavior

    PubMed Central

    Hong, Zhongkui; Ersoy, Ilker; Sun, Mingzhai; Bunyak, Filiz; Hampel, Paul; Hong, Zhenling; Sun, Zhe; Li, Zhaohui; Levitan, Irena; Meininger, Gerald A.; Palaniappan, Kannappan

    2012-01-01

    Interactions between implanted materials and the surrounding host cells critically affect the fate of bioengineered materials. In this study, the biomechanical response of bovine aortic endothelial cells (BAECs) with different membrane cholesterol levels to polyacrylamide (PA) gels was investigated by measuring cell adhesion and spreading behaviors at varying PA elasticity. The elasticity of gel substrates was manipulated by cross-linker content. Type I collagen (COL1) was coated on PA gel to provide a biologically functional environment for cell spreading. Precise quantitative characterization of changes in cell area and perimeter of cells across two treatments and three bioengineered substrates were determined using a customized software developed for computational image analysis. We found that the initial response of endothelial cells to changes in substrate elasticity was determined by membrane cholesterol levels, and that the extent of endothelial cell spreading increases with membrane cholesterol content. All of the BAECs with different cholesterol levels showed little growth on substrates with elasticity below 20 kPa, but increased spreading at higher substrate elasticity. Cholesterol-depleted cells were consistently smaller than control and cholesterol-enriched cells regardless of substrate elasticity. These observations indicate that membrane-cholesterol plays an important role in cell spreading on soft materials constructed with appropriate elasticity. PMID:23239612

  5. Influence of membrane cholesterol and substrate elasticity on endothelial cell spreading behavior.

    PubMed

    Hong, Zhongkui; Ersoy, Ilker; Sun, Mingzhai; Bunyak, Filiz; Hampel, Paul; Hong, Zhenling; Sun, Zhe; Li, Zhaohui; Levitan, Irena; Meininger, Gerald A; Palaniappan, Kannappan

    2013-07-01

    Interactions between implanted materials and the surrounding host cells critically affect the fate of bioengineered materials. In this study, the biomechanical response of bovine aortic endothelial cells (BAECs) with different membrane cholesterol levels to polyacrylamide (PA) gels was investigated by measuring cell adhesion and spreading behaviors at varying PA elasticity. The elasticity of gel substrates was manipulated by cross-linker content. Type I collagen (COL1) was coated on PA gel to provide a biologically functional environment for cell spreading. Precise quantitative characterization of changes in cell area and perimeter of cells across two treatments and three bioengineered substrates were determined using a customized software developed for computational image analysis. We found that the initial response of endothelial cells to changes in substrate elasticity was determined by membrane cholesterol levels, and that the extent of endothelial cell spreading increases with membrane cholesterol content. All of the BAECs with different cholesterol levels showed little growth on substrates with elasticity below 20 kPa, but increased spreading at higher substrate elasticity. Cholesterol-depleted cells were consistently smaller than control and cholesterol-enriched cells regardless of substrate elasticity. These observations indicate that membrane cholesterol plays an important role in cell spreading on soft biomimetic materials constructed with appropriate elasticity.

  6. Ionizing radiation decreases capillary-like structure formation by endothelial cells in vitro.

    PubMed

    Ahmad, Mansur; Khurana, Neerja R; Jaberi, Joby E

    2007-01-01

    For successful tissue engineering in surgical radiotherapy patients, irradiated endothelial cells (EC) must form new blood vessels to nourish and build connections with the engineered segment. Therefore, it is critical to understand neovasculogenesis by irradiated EC. The objective of this study was to determine the effects of ionizing radiation on endothelial cell proliferation and capillary-like structures (CLS) formation. Human Umbilical Vein Endothelial Cells (HUVEC) were irradiated with single or fractionated doses of radiation. Proliferation was determined by counting cells. CLS morphology was analyzed from photomicrographs. A single dose of 8 Gy radiation was highly lethal to HUVEC compared to lower dosage. A single dose had more of an inhibitory effect on cell proliferation compared to the same dose delivered in a fractionated manner. CLS formation began after cells reached confluency. To form a CLS, a single cell expanded, and a number of cells rearranged around its periphery in an oval fashion (mimicking a vessel wall). The central cell later disintegrated leaving a void, mimicking the lumen. Irradiated EC can form CLS, although they are fewer and smaller compared to those by sham cells. By disrupting the peripheral cells, >or=4 Gy doses significantly reduced the number of CLS. The disruptive affect was seen more with large CLS compared to small CLS. At different doses, the shapes of CLS were not significantly different. PMID:17028041

  7. Conditioned medium from human umbilical vein endothelial cells markedly improves the proliferation and differentiation of circulating endothelial progenitors.

    PubMed

    Castelli, Germana; Parolini, Isabella; Cerio, Anna Maria; D'Angiò, Agnese; Pasquini, Luca; Carollo, Maria; Sargiacomo, Massimo; Testa, Ugo; Pelosi, Elvira

    2016-10-01

    Circulating endothelial progenitor cells (EPCs) have been suggested as a precious source for generating functionally competent endothelial cells (ECs), candidate for various clinical applications. However, the paucity of these progenitor cells and the technical difficulties for their in vitro growth represent a main limitation to their use. In the present study we hypothesized that the paracrine effects of human umbilical vein endothelial cells (HUVECs) may improve endothelial cell generation from cord blood (CB) EPCs. In line with this hypothesis we showed that HUVEC conditioned medium (CM) or co-culture with HUVECs markedly improved the proliferation and differentiation and delayed the senescence of CB EPCs. The endothelial-promoting effect of CM seems to be related to smaller vesicles including exosomes (sEV/exo) contained in this medium and transferred to CB CD34(+) EPCs: in fact, purified preparations of sEV/exo isolated from CM mimicked the effect of CM to sustain endothelial formation. These observations provided the interesting indication that mature ECs exert a stimulatory effect on endothelial cell differentiation from CD34(+) cells. PMID:27667168

  8. Atorvastatin neutralises the thrombin-induced tissue factor expresion in endothelial cells via geranylgeranyl pyrophosphate.

    PubMed

    Martínez-Sales, Vicenta; Vila, Virtudes; Ferrando, Marcos; Reganon, Edelmiro

    2011-01-01

    Statins may have beneficial effects in atherogenesis given their antithrombotic properties involving non-lipid mechanisms that modify endothelial function of tissue factor induction by thrombin. In this study, we investigate the effect of atorvastatin on tissue factor (TF) activity in thrombin-stimulated endothelial cells and its regulation through mevalonate or its derivatives. First subculture of human umbilical endothelial cells was used for this study. Cells were treated with thrombin and atorvastatin for different time intervals and dosage. Tissue factor activity was measured as Factor Xa generation induced by Tissue Factor-Factor VIIa complex on confluent cells. Our results show that atorvastatin prevents the thrombin-induced up-regulation of tissue factor activity in a concentration-dependent manner. Mevalonate and geranylgeranyl pyrophosphate reversed this inhibitory effect of atorvastatin on tissue factor activity, while the presence of farnesyl pyrophosphate did not prevent the atorvastatin effect on thrombin-induced tissue factor activity. Rho-kinase inhibitor did not affect the thrombin stimulation of tissue factor activity. High amount of hydrophobic isoprenoid groups decreases the thrombin-induced TF activity and may promote endothelial cell anti-thrombotic action. Rho kinase pathways do not have a major role in the thrombin-mediated TF activity. The inhibitory effect of atorvastatin on thrombin-induced TF activity was partially reversed by MVA and GGPP but not FPP.

  9. Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells.

    PubMed

    Nakashima, Yukiko; Takahashi, Satoru

    2014-08-22

    Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells.

  10. Capture of endothelial cells under flow using immobilized vascular endothelial growth factor

    PubMed Central

    Smith, Randall J.; Koobatian, Maxwell T.; Shahini, Aref; Swartz, Daniel D.; Andreadis, Stelios T.

    2015-01-01

    We demonstrate the ability of immobilized vascular endothelial growth factor (VEGF) to capture endothelial cells (EC) with high specificity under fluid flow. To this end, we engineered a surface consisting of heparin bound to poly-L-lysine to permit immobilization of VEGF through the C-terminal heparin-binding domain. The immobilized growth factor retained its biological activity as shown by proliferation of EC and prolonged activation of KDR signaling. Using a microfluidic device we assessed the ability to capture EC under a range of shear stresses from low (0.5 dyne/cm2) to physiological (15 dyne/cm2). Capture was significant for all shear stresses tested. Immobilized VEGF was highly selective for EC as evidenced by significant capture of human umbilical vein and ovine pulmonary artery EC but no capture of human dermal fibroblasts, human hair follicle derived mesenchymal stem cells, or mouse fibroblasts. Further, VEGF could capture EC from mixtures with non-EC under low and high shear conditions as well as from complex fluids like whole human blood under high shear. Our findings may have far reaching implications, as they suggest that VEGF could be used to promote endothelialization of vascular grafts or neovascularization of implanted tissues by rare but continuously circulating EC. PMID:25771020

  11. Endothelial cell expression of haemoglobin α regulates nitric oxide signalling.

    PubMed

    Straub, Adam C; Lohman, Alexander W; Billaud, Marie; Johnstone, Scott R; Dwyer, Scott T; Lee, Monica Y; Bortz, Pamela Schoppee; Best, Angela K; Columbus, Linda; Gaston, Benjamin; Isakson, Brant E

    2012-11-15

    Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe(3+) state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe(2+) state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells. PMID:23123858

  12. METABOLIC CAPACITY REGULATES IRON HOMEOSTATIS IN ENDOTHELIAL CELLS

    EPA Science Inventory

    The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuat...

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

    PubMed

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

    2015-12-01

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

  14. Cilostazol strengthens barrier integrity in brain endothelial cells.

    PubMed

    Horai, Shoji; Nakagawa, Shinsuke; Tanaka, Kunihiko; Morofuji, Yoichi; Couraud, Pierre-Oliver; Deli, Maria A; Ozawa, Masaki; Niwa, Masami

    2013-03-01

    We studied the effect of cilostazol, a selective inhibitor of phosphodiesterase 3, on barrier functions of blood-brain barrier (BBB)-related endothelial cells, primary rat brain capillary endothelial cells (RBEC), and the immortalized human brain endothelial cell line hCMEC/D3. The pharmacological potency of cilostazol was also evaluated on ischemia-related BBB dysfunction using a triple co-culture BBB model (BBB Kit™) subjected to 6-h oxygen glucose deprivation (OGD) and 3-h reoxygenation. There was expression of phosphodiesterase 3B mRNA in RBEC, and a significant increase in intracellular cyclic AMP (cAMP) content was detected in RBEC treated with both 1 and 10 μM cilostazol. Cilostazol increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), and decreased the endothelial permeability of sodium fluorescein through the RBEC monolayer. The effects on these barrier functions were significantly reduced in the presence of protein kinase A (PKA) inhibitor H-89. Microscopic observation revealed smooth and even localization of occludin immunostaining at TJs and F-actin fibers at the cell borders in cilostazol-treated RBEC. In hCMEC/D3 cells treated with 1 and 10 μM cilostazol for 24 and 96 h, P-glycoprotein transporter activity was increased, as assessed by rhodamine 123 accumulation. Cilostazol improved the TEER in our triple co-culture BBB model with 6-h OGD and 3-h reoxygenation. As cilostazol stabilized barrier integrity in BBB-related endothelial cells, probably via cAMP/PKA signaling, the possibility that cilostazol acts as a BBB-protective drug against cerebral ischemic insults to neurons has to be considered. PMID:23224787

  15. Changes of myoid and endothelial cells in the peritubular wall during contraction of the seminiferous tubule.

    PubMed

    Losinno, Antonella D; Sorrivas, Viviana; Ezquer, Marcelo; Ezquer, Fernando; López, Luis A; Morales, Alfonsina

    2016-08-01

    The wall of the seminiferous tubule in rodents consists of an inner layer of myoid cells covered by an outer layer of endothelial cells. Myoid cells are a type of smooth muscle cell containing α-actin filaments arranged in two independent layers that contract when stimulated by endothelin-1. The irregular surface relief of the tubular wall is often considered a hallmark of contraction induced by a variety of stimuli. We examine morphological changes of the rat seminiferous tubule wall during contraction by a combination of light, confocal, transmission and scanning electron microscopy. During ET-1-induced contraction, myoid cells changed from a flat to a conical shape, but their actin filaments remained in independent layers. As a consequence of myoid cell contraction, the basement membrane became wavy, orientation of collagen fibers in the extracellular matrix was altered and the endothelial cell layer became folded. To observe the basement of the myoid cell cone, the endothelial cell monolayer was removed by collagenase digestion prior to SEM study. In contracted tubules, it is possible to distinguish cell relief: myoid cells have large folds on the external surface oriented parallel to the tubular axis, whereas endothelial cells have numerous cytoplasmic projections facing the interstitium. The myoid cell cytoskeleton is unusual in that the actin filaments are arranged in two orthogonal layers, which adopt differing shapes during contraction with myoid cells becoming cone-shaped. This arrangement impacts on other components of the seminiferous tubule wall and affects the propulsion of the tubular contents to the rete testis. PMID:26987820

  16. Angiotensin I converting enzyme activity in rabbit corneal endothelial cells.

    PubMed

    Neels, H M; Vanden Berghe, D A; Neetens, A J; Delgadillo, R A; Scharpe, S L

    1983-01-01

    Angiotensin I converting enzyme (ACE) was studied in Vero cells, rabbit corneal fibroblasts, and rabbit corneal endothelial cells. The enzyme activity was determined by means of an assay employing hippuryl-glycyl-glycine as a substrate. The hippuric acid end product was separated from the substrate by reversed phase liquid chromatography and measured spectrophotometrically at 228 nm. The enzyme was further characterized by a captopril inhibition study. Significant ACE activity was found in rabbit corneal endothelial cells but not in other types of cells tested. This is the first report of the presence of this enzyme in a specific ocular cell type and suggests that angiotensin II may play a role in normal ocular physiology.

  17. Dynamics of receptor-mediated nanoparticle internalization into endothelial cells.

    PubMed

    Gonzalez-Rodriguez, David; Barakat, Abdul I

    2015-01-01

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

  18. Brown spider (Loxosceles intermedia) venom triggers endothelial cells death by anoikis.

    PubMed

    Nowatzki, Jenifer; Sene, Reginaldo Vieira; Paludo, Katia Sabrina; Rizzo, Luiz Eduardo; Souza-Fonseca-Guimarães, Fernando; Veiga, Silvio Sanches; Nader, Helena Bonciani; Franco, Célia Regina C; Trindade, Edvaldo S

    2012-09-01

    Brown spider (Loxosceles sp.) venom affects the endothelium of vessels and triggers disruptive activity in the subendothelial matrix. The vascular disorders observed after venom exposure include leukocyte and platelet activation, disseminated intravascular coagulation, an increase in vessel permeability and hemorrhage into the dermis. In this study, we report additional evidence regarding the mechanism of endothelial cell cytotoxicity induced by Loxosceles intermedia venom. Exposure to venom led to endothelial cell detachment in a time-dependent manner. Loss of cell anchorage and cell-cell adhesion following venom exposure was accompanied by changes in the distribution of the α₅β₁ integrin and VE-cadherin. An ultrastructural analysis of cells treated with venom revealed morphological alterations characteristic of apoptosis. Moreover, after venom exposure, the ratio between Bax and Bcl-2 proteins was disturbed in favor of Bax. In addition, late apoptosis was only observed in cells detached by the action of venom. Accordingly, there was no increase in apoptosis when cells were exposed to L. intermedia venom in suspension, suggesting that the loss of cell anchorage provides the signal to initiate apoptosis. Thus, L. intermedia venom likely triggers endothelial cell death indirectly through an apoptotic mechanism known as anoikis.

  19. Toll-Like Receptor 4 Mediates Endothelial Cell Activation Through NF-κB but Is Not Associated with Endothelial Dysfunction in Patients with Rheumatoid Arthritis

    PubMed Central

    Menghini, Rossella; Campia, Umberto; Tesauro, Manfredi; Marino, Arianna; Rovella, Valentina; Rodia, Giuseppe; Schinzari, Francesca; Tolusso, Barbara; di Daniele, Nicola; Federici, Massimo; Zoli, Angelo; Ferraccioli, Gianfranco; Cardillo, Carmine

    2014-01-01

    Objective To investigate the effects of TLR4 antagonism on human endothelial cells activation and cytokine expression, and whether the Asp299Gly TLR4 polymorphism is associated with better endothelial function in patients with rheumatoid arthritis (RA). Methods Human aortic endothelial cells (HAECs) were treated with lipopolysaccharide (LPS), OxPAPC, and free fatty acids (FFA) at baseline and after incubation with the TLR4 antagonist eritoran (E5564). Cytokine expression was assessed by quantitative real-time PCR. In vivo endothelial function was assessed as brachial artery flow-mediated dilation (FMD) in RA patients with the wild type gene (aa) and with the Asp299Gly TLR4 polymorphic variant (ag). Results In HAEC, TLR4 antagonism with eritoran inhibited LPS-induced mRNA expression of IL-6, IL-8, TNFα, CCL-2, VCAM and ICAM (P<0.05 for all) and inhibited Ox-PAPC-induced mRNA expression of IL-8 (P<0.05) and IL-6, albeit not to a statistically significant level (p = 0.07). In contrast, eritoran did not affect FFA-induced mRNA expression of IL-6 (P>0.05). In 30 patients with RA (15 with the ag allele) undergoing measurement of FMD, no differences in FMD and plasma levels of IL-6, IL-8, VCAM, and ICAM were found between the aa and the ag phenotype (P>0.05 for all). Conclusions TLR4 signaling in endothelial cells may be triggered by LPS and oxidized phospholipids, leading to endothelial activation and inflammation, which are inhibited by eritoran. Our in vivo investigation, however, does not support an association between the Asp299Gly TLR4 polymorphism and improved endothelium-dependent vasodilator function in patients with RA. Further study is needed to better understand the potential role of TLR4 on endothelial dysfunction in this and other patient populations. PMID:24918924

  20. Pancreatic Tumor Cell Secreted CCN1/Cyr61 Promotes Endothelial cell migration and Aberrant Neovascularization

    PubMed Central

    Maity, Gargi; Mehta, Smita; Haque, Inamul; Dhar, Kakali; Sarkar, Sandipto; Banerjee, Sushanta K.; Banerjee, Snigdha

    2014-01-01

    The complex signaling networks between cancer cells and adjacent endothelial cells make it challenging to unravel how cancer cells send extracellular messages to promote aberrant vascularization or tumor angiogenesis. Here, in vitro and in vivo models show that pancreatic cancer cell generated unique microenvironments can underlie endothelial cell migration and tumor angiogenesis. Mechanistically, we find that pancreatic cancer cell secreted CCN1/Cyr61 matricellular protein rewires the microenvironment to promote endothelial cell migration and tumor angiogenesis. This event can be overcome by Sonic Hedgehog (SHh) antibody treatment. Collectively, these studies identify a novel CCN1 signaling program in pancreatic cancer cells which activates SHh through autocrine-paracrine circuits to promote endothelial cell migration and tumor angiogenesis and suggests that CCN1 signaling of pancreatic cancer cells is vital for the regulation of tumor angiogenesis. Thus CCN1 signaling could be an ideal target for tumor vascular disruption in pancreatic cancer. PMID:24833309

  1. Role of androgen and vitamin D receptors in endothelial cells from benign and malignant human prostate

    PubMed Central

    Chung, Ivy; Montecinos, Viviana P.; Buttyan, Ralph; Johnson, Candace S.; Smith, Gary J.

    2013-01-01

    Forty years ago, Judah Folkman (Folkman. N Engl J Med 285: 1182–1186, 1971) proposed that tumor growth might be controlled by limiting formation of new blood vessels (angiogenesis) needed to supply a growing tumor with oxygen and nutrients. To this end, numerous “antiangiogenic” agents have been developed and tested for therapeutic efficacy in cancer patients, including prostate cancer (CaP) patients, with limited success. Despite the lack of clinical efficacy of lead anti-angiogenic therapeutics in CaP patients, recent published evidence continues to support the idea that prostate tumor vasculature provides a reasonable target for development of new therapeutics. Particularly relevant to antiangiogenic therapies targeted to the prostate is the observation that specific hormones can affect the survival and vascular function of prostate endothelial cells within normal and malignant prostate tissues. Here, we review the evidence demonstrating that both androgen(s) and vitamin D significantly impact the growth and survival of endothelial cells residing within prostate cancer and that systemic changes in circulating androgen or vitamin D drastically affect blood flow and vascularity of prostate tissue. Furthermore, recent evidence will be discussed about the expression of the receptors for both androgen and vitamin D in prostate endothelial cells that argues for direct effects of these hormone-activated receptors on the biology of endothelial cells. Based on this literature, we propose that prostate tumor vasculature represents an unexplored target for modulation of tumor growth. A better understanding of androgen and vitamin D effects on prostate endothelial cells will support development of more effective angiogenesis-targeting therapeutics for CaP patients. PMID:23548616

  2. Brown spider venom toxins interact with cell surface and are endocytosed by rabbit endothelial cells.

    PubMed

    Nowatzki, Jenifer; de Sene, Reginaldo Vieira; Paludo, Katia Sabrina; Veiga, Silvio Sanches; Oliver, Constance; Jamur, Maria Célia; Nader, Helena Bonciani; Trindade, Edvaldo S; Franco, Célia Regina C

    2010-09-15

    Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations. Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom. After treating endothelial cells with venom toxins, we observed that the venom interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates. When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells. The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L. intermedia venom on endothelial cells is not mediated by venom internalization.

  3. Propofol ameliorates endothelial inflammation induced by hypoxia/reoxygenation in human umbilical vein endothelial cells: Role of phosphatase A2.

    PubMed

    Zhu, Minmin; Ding, Juan; Jiang, Hui; Kong, Lingchao; Sun, Zhirong; Chen, Jiawei; Miao, Changhong

    2015-10-01

    Hypoxia/reoxygenation (H/R) induces endothelial inflammation with augmentation of endothelial adhesion molecules over-expression. Propofol was reported to attenuate endothelial adhesion molecule expression in some situations. Here, we examined the molecular mechanism for how propofol restored H/R-mediated up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs). Compared with the control group, H/R up-regulated expression of Pin-1 and PP2A, increased p66(Shc)-Ser(36) phosphorylation, induced p66(Shc) mitochondrial translocation, O2(-) accumulation and NF-κB activation, and decreased eNOS-Ser(1177) phosphorylation and nitric oxide (NO) production, thus up-regulating expression of endothelial adhesion molecules and increasing mononuclear-endothelial interaction. More importantly, except that propofol had no effect on H/R-induced p66(Shc)-Ser(36) phosphorylation, most of H/R-mediated changes were alleviated by propofol, resulting in the reduction of endothelial adhesion molecules expression and mononuclear-endothelial adhesion. Moreover, we demonstrated the protective effect of propofol on H/R-induced endothelial inflammation was similar to that of calyculin A, an inhibitor of PP2A. In contrast, FTY720, an activator of PP2A, antagonized the effect of propofol. Our data indicated that propofol down-regulated PP2A expression, leading to reduced dephosphorylation of p66(Shc)-Ser(36) and eNOS-Ser(1177), which is associated with ROS accumulation and NO reduction, resulting in inhibition of endothelial adhesion molecule expression and mononuclear-endothelial interaction.

  4. Sphingosine 1-phosphate induced synthesis of glycocalyx on endothelial cells.

    PubMed

    Zeng, Ye; Liu, Xiao-Heng; Tarbell, John; Fu, Bingmei

    2015-11-15

    Sphingosine 1-phosphate (S1P) protects glycocalyx against shedding, playing important roles in endothelial functions. We previously found that glycocalyx on endothelial cells (ECs) was shed after plasma protein depletion. In the present study, we investigated the role of S1P on the recovery of glycocalyx, and tested whether it is mediated by phosphoinositide 3-kinase (PI3K) pathway. After depletion of plasma protein, ECs were treated with S1P for another 6h. And then, the major components of glycocalyx including syndecan-1 with attached heparan sulfate (HS) and chondroitin sulfate (CS) on endothelial cells were detected using confocal fluorescence microscopy. Role of PI3K in the S1P-induced synthesis of glycocalyx was confirmed by using the PI3K inhibitor (LY294002). Syndecan-1 with attached HS and CS were degraded with duration of plasma protein depletion. S1P induced recovery of syndecan-1 with attached HS and CS. The PI3K inhibitor LY294002 abolished the effect of S1P on recovery of glycocalyx. Thus, S1P induced synthesis of glycocalyx on endothelial cells and it is mediated by PI3K pathway.

  5. Sildenafil Reduces Insulin-Resistance in Human Endothelial Cells

    PubMed Central

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

    2011-01-01

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

  6. Cilengitide inhibits proliferation and differentiation of human endothelial progenitor cells in vitro

    SciTech Connect

    Loges, Sonja; Butzal, Martin; Otten, Jasmin; Schweizer, Michaela; Fischer, Uta; Bokemeyer, Carsten; Hossfeld, Dieter K.; Schuch, Gunter; Fiedler, Walter . E-mail: fiedler@uke.uni-hamburg.de

    2007-06-15

    Bone marrow derived hematopoietic stem cells can function as endothelial progenitor cells. They are recruited to malignant tumors and differentiate into endothelial cells. This mechanism of neovascularization termed vasculogenesis is distinct from proliferation of pre-existing vessels. To better understand vasculogenesis we developed a cell culture model with expansion and subsequent endothelial differentiation of human CD133{sup +} progenitor cells in vitro. {alpha}{sub v}{beta}{sub 3}-integrins are expressed by endothelial cells and play a role in the attachment of endothelial cells to the extracellular matrix. We investigated the effect of Cilengitide, a peptide-like, high affinity inhibitor of {alpha}{sub v}{beta}{sub 3}- and {alpha}{sub v}{beta}{sub 5}-integrins in our in vitro system. We could show expression of {alpha}{sub v}{beta}{sub 3}-integrin on 60 {+-} 9% of non-adherent endothelial progenitors and on 91 {+-} 7% of differentiated endothelial cells. {alpha}{sub v}{beta}{sub 3}-integrin was absent on CD133{sup +} hematopoietic stem cells. Cilengitide inhibited proliferation of CD133{sup +} cells in a dose-dependent manner. The development of adherent endothelial cells from expanded CD133{sup +} cells was reduced even stronger by Cilengitide underlining its effect on integrin mediated cell adhesion. Expression of endothelial antigens CD144 and von Willebrand factor on differentiating endothelial precursors was decreased by Cilengitide. In summary, Cilengitide inhibits proliferation and differentiation of human endothelial precursor cells underlining its anti-angiogenic effects.

  7. Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices.

    PubMed

    Baldo, C; Lopes, D S; Faquim-Mauro, E L; Jacysyn, J F; Niland, S; Eble, J A; Clissa, P B; Moura-da-Silva, A M

    2015-12-15

    Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2β1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms.

  8. Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages.

    PubMed

    Veszelka, Szilvia; Pásztói, Mária; Farkas, Attila E; Krizbai, István; Ngo, Thi Khue Dung; Niwa, Masami; Abrahám, Csongor S; Deli, Mária A

    2007-01-01

    Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.

  9. Arteries are formed by vein-derived endothelial tip cells.

    PubMed

    Xu, Cong; Hasan, Sana S; Schmidt, Inga; Rocha, Susana F; Pitulescu, Mara E; Bussmann, Jeroen; Meyen, Dana; Raz, Erez; Adams, Ralf H; Siekmann, Arndt F

    2014-12-15

    Tissue vascularization entails the formation of a blood vessel plexus, which remodels into arteries and veins. Here we show, by using time-lapse imaging of zebrafish fin regeneration and genetic lineage tracing of endothelial cells in the mouse retina, that vein-derived endothelial tip cells contribute to emerging arteries. Our movies uncover that arterial-fated tip cells change migration direction and migrate backwards within the expanding vascular plexus. This behaviour critically depends on chemokine receptor cxcr4a function. We show that the relevant Cxcr4a ligand Cxcl12a selectively accumulates in newly forming bone tissue even when ubiquitously overexpressed, pointing towards a tissue-intrinsic mode of chemokine gradient formation. Furthermore, we find that cxcr4a mutant cells can contribute to developing arteries when in association with wild-type cells, suggesting collective migration of endothelial cells. Together, our findings reveal specific cell migratory behaviours in the developing blood vessel plexus and uncover a conserved mode of artery formation.

  10. Arteries are formed by vein-derived endothelial tip cells

    PubMed Central

    Xu, Cong; Hasan, Sana S.; Schmidt, Inga; Rocha, Susana F.; Pitulescu, Mara E.; Bussmann, Jeroen; Meyen, Dana; Raz, Erez; Adams, Ralf H.; Siekmann, Arndt F.

    2014-01-01

    Tissue vascularization entails the formation of a blood vessel plexus, which remodels into arteries and veins. Here we show, by using time-lapse imaging of zebrafish fin regeneration and genetic lineage tracing of endothelial cells in the mouse retina, that vein-derived endothelial tip cells contribute to emerging arteries. Our movies uncover that arterial-fated tip cells change migration direction and migrate backwards within the expanding vascular plexus. This behaviour critically depends on chemokine receptor cxcr4a function. We show that the relevant Cxcr4a ligand Cxcl12a selectively accumulates in newly forming bone tissue even when ubiquitously overexpressed, pointing towards a tissue-intrinsic mode of chemokine gradient formation. Furthermore, we find that cxcr4a mutant cells can contribute to developing arteries when in association with wild-type cells, suggesting collective migration of endothelial cells. Together, our findings reveal specific cell migratory behaviours in the developing blood vessel plexus and uncover a conserved mode of artery formation. PMID:25502622

  11. Vanadium pentoxide induces activation and death of endothelial cells.

    PubMed

    Montiel-Dávalos, Angélica; Gonzalez-Villava, Adriana; Rodriguez-Lara, Vianey; Montaño, Luis Felipe; Fortoul, Teresa I; López-Marure, Rebeca

    2012-01-01

    Vanadium is a transition metal released into the atmosphere, as air-suspended particles, as a result of the combustion of fossil fuels and some metallurgic industry activities. Air-suspended particle pollution causes inflammation-related processes such as thrombosis and other cardiovascular events. Our aim was to evaluate the effect of vanadium pentoxide (V2O5) on endothelial cells since they are key participants in the pathogenesis of several cardiovascular and inflammatory diseases. Cell adhesion, the expression of adhesion molecules and oxidative stress, as well as proliferation, morphology and cell death of human umbilical vein endothelial cells (HUVECs) exposed to V2O5, were evaluated. Vanadium pentoxide at a 3.12 µg cm(-2) concentration induced an enhanced adhesion of the U937 macrophage cell line to HUVECs, owing to an increased expression of late adhesion molecules. HUVECs exposed to V2O5 showed an increase in ROS and nitric oxide production, and a diminished proliferation. These changes in vanadium-treated HUVECs were accompanied by severe morphological changes and apoptotic cell death. Vanadium pentoxide induced serious endothelial cell damage, probably related to the increased cardiovascular morbidity and mortality observed in individuals living in highly air-polluted areas. PMID:21721017

  12. Isolation and Characterization of Human Lung Lymphatic Endothelial Cells.

    PubMed

    Lorusso, Bruno; Falco, Angela; Madeddu, Denise; Frati, Caterina; Cavalli, Stefano; Graiani, Gallia; Gervasi, Andrea; Rinaldi, Laura; Lagrasta, Costanza; Maselli, Davide; Gnetti, Letizia; Silini, Enrico M; Quaini, Eugenio; Ampollini, Luca; Carbognani, Paolo; Quaini, Federico

    2015-01-01

    Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy. PMID:26137493

  13. Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

    PubMed Central

    Lorusso, Bruno; Falco, Angela; Madeddu, Denise; Frati, Caterina; Cavalli, Stefano; Graiani, Gallia; Gervasi, Andrea; Rinaldi, Laura; Lagrasta, Costanza; Maselli, Davide; Gnetti, Letizia; Silini, Enrico M.; Quaini, Eugenio; Ampollini, Luca; Carbognani, Paolo; Quaini, Federico

    2015-01-01

    Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy. PMID:26137493

  14. Binding of recombinant annexin V to endothelial cells: effect of annexin V binding on endothelial-cell-mediated thrombin formation.

    PubMed Central

    van Heerde, W L; Poort, S; van 't Veer, C; Reutelingsperger, C P; de Groot, P G

    1994-01-01

    Annexin V binds with high affinity to procoagulant phospholipid vesicles and thereby inhibits the procoagulant reactions catalysed by these surfaces in vitro. In vivo, vascular endothelial cells are known to catalyse the formation of thrombin by the expression of binding sites at which procoagulant complexes can assemble. Here, we have studied the binding capacity of recombinant annexin V (rANV) to quiescent, phorbol 12-myristate 13-acetate (PMA)- and tumour necrosis factor alpha (TNF-alpha)-stimulated cultured human umbilical-vein endothelial cells (HUVEC). The dissociation constant (Kd) was 15.5 +/- 3.3 nM and the number of binding sites was 8.8 (+/- 3.9) x 10(6)/cell. These binding parameters did not change significantly during a 30 h incubation period with PMA or TNF-alpha. rANV inhibited HUVEC-mediated factor Xa formation via the extrinsic as well as the intrinsic route. Activation of factor X by the tissue factor-factor VII-factor X complex and tenase complex was inhibited with IC50 values of 43 +/- 30 nM and 33 +/- 24 nM respectively. Endothelial-cell-mediated generation of thrombin by the prothrombinase complex was inhibited by rANV with an IC50 of 16 +/- 12 nM. Preincubation of rANV with the endothelial cells did not significantly influence the IC50 values. These results show that rANV binds to the same extent to quiescent, PMA- and TNF-stimulated HUVEC, and, as a result of this binding, rANV efficiently inhibits endothelial-cell-mediated thrombin formation. PMID:8068019

  15. Primary Phenomenon in the Network Formation of Endothelial Cells: Effect of Charge.

    PubMed

    Arai, Shunto

    2015-01-01

    Blood vessels are essential organs that are involved in the supply of nutrients and oxygen and play an important role in regulating the body's internal environment, including pH, body temperature, and water homeostasis. Many studies have examined the formation of networks of endothelial cells. The results of these studies have revealed that vascular endothelial growth factor (VEGF) affects the interactions of these cells and modulates the network structure. Though almost all previous simulation studies have assumed that the chemoattractant VEGF is present before network formation, vascular endothelial cells secrete VEGF only after the cells bind to the substrate. This suggests VEGF is not essential for vasculogenesis especially at the early stage. Using a simple experiment, we find chain-like structures which last quite longer than it is expected, unless the energetically stable cluster should be compact. Using a purely physical model and simulation, we find that the hydrodynamic interaction retard the compaction of clusters and that the chains are stabilized through the effects of charge. The charge at the surface of the cells affect the interparticle potential, and the resulting repulsive forces prevent the chains from folding. The ions surrounding the cells may also be involved in this process. PMID:26690133

  16. Primary Phenomenon in the Network Formation of Endothelial Cells: Effect of Charge.

    PubMed

    Arai, Shunto

    2015-12-07

    Blood vessels are essential organs that are involved in the supply of nutrients and oxygen and play an important role in regulating the body's internal environment, including pH, body temperature, and water homeostasis. Many studies have examined the formation of networks of endothelial cells. The results of these studies have revealed that vascular endothelial growth factor (VEGF) affects the interactions of these cells and modulates the network structure. Though almost all previous simulation studies have assumed that the chemoattractant VEGF is present before network formation, vascular endothelial cells secrete VEGF only after the cells bind to the substrate. This suggests VEGF is not essential for vasculogenesis especially at the early stage. Using a simple experiment, we find chain-like structures which last quite longer than it is expected, unless the energetically stable cluster should be compact. Using a purely physical model and simulation, we find that the hydrodynamic interaction retard the compaction of clusters and that the chains are stabilized through the effects of charge. The charge at the surface of the cells affect the interparticle potential, and the resulting repulsive forces prevent the chains from folding. The ions surrounding the cells may also be involved in this process.

  17. Primary Phenomenon in the Network Formation of Endothelial Cells: Effect of Charge

    PubMed Central

    Arai, Shunto

    2015-01-01

    Blood vessels are essential organs that are involved in the supply of nutrients and oxygen and play an important role in regulating the body’s internal environment, including pH, body temperature, and water homeostasis. Many studies have examined the formation of networks of endothelial cells. The results of these studies have revealed that vascular endothelial growth factor (VEGF) affects the interactions of these cells and modulates the network structure. Though almost all previous simulation studies have assumed that the chemoattractant VEGF is present before network formation, vascular endothelial cells secrete VEGF only after the cells bind to the substrate. This suggests VEGF is not essential for vasculogenesis especially at the early stage. Using a simple experiment, we find chain-like structures which last quite longer than it is expected, unless the energetically stable cluster should be compact. Using a purely physical model and simulation, we find that the hydrodynamic interaction retard the compaction of clusters and that the chains are stabilized through the effects of charge. The charge at the surface of the cells affect the interparticle potential, and the resulting repulsive forces prevent the chains from folding. The ions surrounding the cells may also be involved in this process. PMID:26690133

  18. Radioligand binding to muscarinic receptors of bovine aortic endothelial cells.

    PubMed

    Brunner, F; Kukovetz, W R

    1991-02-01

    1. Muscarinic receptors on endothelial cells of bovine thoracic aorta were characterized by binding assays in which (-)-[3H]-N-methyl quinuclidinyl benzilate ([3H]-NMeQNB) was used as radioligand. 2. Binding of [3H]-NMeQNB to crude membranes of freshly isolated endothelial cells was atropine-displaceable and of high affinity (KD = 0.48 nM) to a single class of sites (maximum binding capacity: 14 +/- 3 fmol mg-1 protein). Stereospecificity of the binding sites was demonstrated in experiments in which [3H]-NMeQNB binding was inhibited by dexetimide in the nanomolar range (KI = 0.63 nM) and by levetimide, its stereoisomer in the micromolar range (KI = 3.2 microM) (selectivity factor: approximately 5000). 3. Drug competition curves indicated a single class of binding sites for antagonists and the following apparent affinities (KI, nM): methyl atropine: 1.1: 4-diphenylacetoxy N-methyl piperidine methyl bromide (4-DAMP): 3.4; pirenzepine: 16; 11-[2-diethylamino-methyl)-1-piperidinyl- acetyl]-5,11-dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one (AF-DX 116); 2.500. Competition of acetylcholine with [3H]-NMeQNB was best described by two affinity sites (or states) (KH = 0.82 microM, KL = 1.6 microM). In the presence of guanylimido diphosphate [Gpp(NH)p] (100 microM), acetylcholine affinity (IC50) was slightly, but significantly reduced (factor approximately 4). 4. Binding of [3H]-NMeQNB to freshly harvested intact cells was also atropine-displaceable, stereospecific (selectivity factor: approximately 3500) and of high affinity (KD = 0.35 nM). The maximum binding capacity (9 +/- 2 fmol mg-1 total cell protein) was comparable to that of membranes and corresponded to approximately 900 binding sites per endothelial cell. Binding to enzymatically harvested and cultured endothelial cells, or membranes derived therefrom, showed no atropine-displaceable binding. 5. The results suggest that (1) bovine aortic endothelial cells contain muscarinic binding sites with all necessary

  19. 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.

  20. 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.

  1. Endothelial cells and cathepsins: Biochemical and biomechanical regulation.

    PubMed

    Platt, Manu O; Shockey, W Andrew

    2016-03-01

    Cathepsins are mechanosensitive proteases that are regulated not only by biochemical factors, but are also responsive to biomechanical forces in the cardiovascular system that regulate their expression and activity to participate in cardiovascular tissue remodeling. Their elastinolytic and collagenolytic activity have been implicated in atherosclerosis, abdominal aortic aneurysms, and in heart valve disease, all of which are lined by endothelial cells that are the mechanosensitive monolayer of cells that sense and respond to fluid shear stress as the blood flows across the surfaces of the arteries and valve leaflets. Inflammatory cytokine signaling is integrated with biomechanical signaling pathways by the endothelial cells to transcribe, translate, and activate either the cysteine cathepsins to remodel the tissue or to express their inhibitors to maintain healthy cardiovascular tissue structure. Other cardiovascular diseases should now be included in the study of the cysteine cathepsin activation because of the additional biochemical cues they provide that merges with the already existing hemodynamics driving cardiovascular disease. Sickle cell disease causes a chronic inflammation including elevated TNFα and increased numbers of circulating monocytes that alter the biochemical stimulation while the more viscous red blood cells due to the sickling of hemoglobin alters the hemodynamics and is associated with accelerated elastin remodeling causing pediatric strokes. HIV-mediated cardiovascular disease also occurs earlier in than the broader population and the influence of HIV-proteins and antiretrovirals on endothelial cells must be considered to understand these accelerated mechanisms in order to identify new therapeutic targets for prevention.

  2. Lymphatic endothelial cells support tumor growth in breast cancer

    PubMed Central

    Lee, Esak; Pandey, Niranjan B.; Popel, Aleksander S.

    2014-01-01

    Tumor lymphatic vessels (LV) serve as a conduit of tumor cell dissemination, due to their leaky nature and secretion of tumor-recruiting factors. Though lymphatic endothelial cells (LEC) lining the LV express distinct factors (also called lymphangiocrine factors), these factors and their roles in the tumor microenvironment are not well understood. Here we employ LEC, microvascular endothelial cells (MEC), and human umbilical vein endothelial cells (HUVEC) cultured in triple-negative MDA-MB-231 tumor-conditioned media (TCM) to determine the factors that may be secreted by various EC in the MDA-MB-231 breast tumor. These factors will serve as endothelium derived signaling molecules in the tumor microenvironment. We co-injected these EC with MDA-MB-231 breast cancer cells into animals and showed that LEC support tumor growth, HUVEC have no significant effect on tumor growth, whereas MEC suppress it. Focusing on LEC-mediated tumor growth, we discovered that TCM-treated LEC (‘tumor-educated LEC') secrete high amounts of EGF and PDGF-BB, compared to normal LEC. LEC-secreted EGF promotes tumor cell proliferation. LEC-secreted PDGF-BB induces pericyte infiltration and angiogenesis. These lymphangiocrine factors may support tumor growth in the tumor microenvironment. This study shows that LV serve a novel role in the tumor microenvironment apart from their classical role as conduits of metastasis. PMID:25068296

  3. Shell matters: Magnetic targeting of SPIONs and in vitro effects on endothelial and monocytic cell function.

    PubMed

    Matuszak, Jasmin; Dörfler, Philipp; Zaloga, Jan; Unterweger, Harald; Lyer, Stefan; Dietel, Barbara; Alexiou, Christoph; Cicha, Iwona

    2015-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) are versatile and easily functionalized agents with high potential for diagnostic and therapeutic intravascular applications. In this study, we analyzed the responses of endothelial (ECs) and monocytic cells to three different types of SPIONs, in order to assess the influence of physico-chemical properties on the biological reactions to SPIONs. The following formulations were used: (1) Lauric acid-coated and BSA-stabilized SPION-1,(2) Lauric acid/BSA-coated SPION-2 and (3) dextran-coated SPION-3. SPION-1 were strongly internalized by ECs and reduced their viability in static conditions. Additionally, they had a dose-dependent inhibitory effect on monocytic cell chemotaxis to MCP-1, but did not affect monocytic cell recruitment by ECs. SPION-2 uptake was less pronounced, both in ECs and monocytic cells, and these particles were better tolerated by the vascular cells. Not being internalized by endothelial or monocytic cells, SPION-3 did not induce relevant effects on cell viability, motility or endothelial-monocytic cell interactions.Taken together, localized accumulation of circulating SPION under physiologic-like flow conditions and their cellular uptake depends on the physicochemical characteristics. Our findings suggest that SPION-2 are suitable for magnetic targeting of atherosclerotic plaques. Due to their excellent biocompatibility and low internalization, SPION-3 may represent a suitable imaging agent for intravascular applications. PMID:26410877

  4. Angiotensin-converting enzyme kinetics in an endothelial cell column

    SciTech Connect

    Howell, R.E.; Haselton, F.R.; Mueller, S.N. )

    1990-04-01

    The kinetics of saturable endothelial metabolic functions have been assessed in vivo by transient (indicator-dilution) measurements and in culture by steady-state measurements, but comparisons between the two are difficult. Therefore, we used indicator-dilution methods to assess the kinetics of angiotensin-converting enzyme (ACE) activity in cultured endothelium. Bovine fetal aortic endothelial cells were grown to confluence on microcarrier beads. Cell-covered beads were poured into polypropylene columns and perfused with serum-free culture medium. Six injections, containing (3H)benzol-Phe-Ala-Pro (( 3H)BPAP, an ACE substrate) and varying amounts of unlabeled BPAP, were applied to each column and effluent was collected in serial samples. The apparent kinetics of BPAP metabolism were determined by four models used previously to determine pulmonary endothelial ACE kinetics in vivo, the most useful model incorporating transit time heterogeneity. The Km averaged 5 microM, which is close to values determined previously in vivo and in vitro. The Amax (Vmax.reaction volume) and Amax/Km averaged 6 nmol/min and 1.5 ml/min, respectively, which are lower than estimates in vivo. In conclusion, we have developed a new method for investigating saturable metabolic activity in cultured endothelium, which after further exploration should also enable better comparisons of endothelial metabolic functions in vivo and in culture.

  5. Low density lipoprotein uptake by an endothelial-smooth muscle cell bilayer

    SciTech Connect

    Alexander, J.J.; Miguel, R.; Graham, D. )

    1991-03-01

    To study the interaction of endothelial and smooth muscle cells, and the means by which such interaction may affect lipid permeability of the arterial wall, cell bilayers were established by use of a transwell culture system. After confluent growth of both cell types had been achieved, iodine 125 bound to low-density lipoprotein (10 ng protein/ml) was added to the media of the upper well. After a 3-hour incubation period, the iodine 125-bound low-density lipoprotein content of the upper and lower media demonstrated an impedance to lipoprotein movement across the endothelial cell monolayer as compared to the bare porous polycarbonate filter of the transwell (p less than 10(-6)). The presence of smooth muscle cells in the bottom well significantly enhanced the permeability of the endothelial cell layer (p less than 10(-60)). This effect remained unchanged over a 9-day time course. Membrane binding and cellular uptake of low-density lipoprotein by endothelial cells was not altered by smooth muscle cells, indicating that this change in permeability could not be easily attributed to changes in receptor-mediated transport or transcytosis. Membrane binding (p less than 0.02) and cellular uptake (p less than 10(-6)) of low-density lipoprotein by smooth muscle cells in the bilayer, when adjusted for counts available in the smooth muscle cell media, were both reduced in the early incubation period as compared to isolated smooth muscle cells. The disproportionate reduction in uptake as compared to binding would suggest that this was not entirely a receptor-dependent process.

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

    SciTech Connect

    Ahn, Sunyoung; Kim, Hyun Jeong; Chi, Sung-Gil; Park, Heonyong

    2012-03-09

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

  7. Arterial identity of endothelial cells is controlled by local cues.

    PubMed

    Othman-Hassan, K; Patel, K; Papoutsi, M; Rodriguez-Niedenführ, M; Christ, B; Wilting, J

    2001-09-15

    The ephrins and their Eph receptors comprise the largest family of receptor tyrosine kinases. Studies on mice have revealed an important function of ephrin-B2 and Eph-B4 for the development of the arterial and venous vasculature, respectively, but the mechanisms regulating their expression have not been studied yet. We have cloned a chick ephrin-B2 cDNA probe. Expression was observed in endothelial cells of extra- and intraembryonic arteries and arterioles in all embryos studied from day 2 (stage 10 HH, before perfusion of the vessels) to day 16. Additionally, expression was found in the somites and neural tube in early stages, and later also in the smooth muscle cells of the aorta, parts of the Müllerian duct, dosal neural tube, and joints of the limbs. We isolated endothelial cells from the internal carotid artery and the vena cava of 14-day-old quail embryos and grafted them separately into day-3 chick embryos. Reincubation was performed until day 6 and the quail endothelial cells were identified with the QH1 antibody. The grafted arterial and venous endothelial cells expressed ephrin-B2 when they integrated into the lining of arteries. Cells that were not integrated into vessels, or into vessels other than arteries, were ephrin-B2-negative. The studies show that the expression of the arterial marker ephrin-B2 is controlled by local cues in arterial vessels of older embryos. Physical forces or the media smooth muscle cells may be involved in this process.

  8. Nitrative Stress Participates in Endothelial Progenitor Cell Injury in Hyperhomocysteinemia

    PubMed Central

    Dong, Yu; Sun, Qi; Liu, Teng; Wang, Huanyuan; Jiao, Kun; Xu, Jiahui; Liu, Xin; Liu, Huirong; Wang, Wen

    2016-01-01

    In order to investigate the role of nitrative stress in vascular endothelial injury in hyperhomocysteinemia (HHcy), thirty healthy adult female Wistar rats were randomly divided into three groups: control, hyperhomocysteinemia model, and hyperhomocysteinemia with FeTMPyP (peroxynitrite scavenger) treatment. The endothelium-dependent dilatation of thoracic aorta in vitro was determined by response to acetylcholine (ACh). The histological changes in endothelium were assessed by HE staining and scanning electron microscopy (SEM). The expression of 3-nitrotyrosine (NT) in thoracic aorta was demonstrated by immunohistochemistry and immunofluorescence, and the number of circulating endothelial progenitor cells (EPCs) was quantified by flow cytometry. Hyperhomocysteinemia caused significant endothelial injury and dysfunction including vasodilative and histologic changes, associated with higher expression of NT in thoracic aorta. FeTMPyP treatment reversed these injuries significantly. Further, the effect of nitrative stress on cultured EPCs in vitro was investigated by administering peroxynitrite donor (3-morpholino-sydnonimine, SIN-1) and peroxynitrite scavenger (FeTMPyP). The roles of nitrative stress on cell viability, necrosis and apoptosis were evaluated with 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, respectively. Also, the phospho-eNOS expression and tube formation in Matrigel of cultured EPCs was detected. Our data showed that the survival of EPCs was much lower in SIN-1 group than in vehicle group, both the apoptosis and necrosis of EPCs were much more severe, and the p-eNOS expression and tube formation in Matrigel were obviously declined. Subsequent pretreatment with FeTMPyP reversed these changes. Further, pretreatment with FeTMPyP reversed homocysteine-induced EPC injury. In conclusion, this study indicates that

  9. Nitrative Stress Participates in Endothelial Progenitor Cell Injury in Hyperhomocysteinemia.

    PubMed

    Dong, Yu; Sun, Qi; Liu, Teng; Wang, Huanyuan; Jiao, Kun; Xu, Jiahui; Liu, Xin; Liu, Huirong; Wang, Wen

    2016-01-01

    In order to investigate the role of nitrative stress in vascular endothelial injury in hyperhomocysteinemia (HHcy), thirty healthy adult female Wistar rats were randomly divided into three groups: control, hyperhomocysteinemia model, and hyperhomocysteinemia with FeTMPyP (peroxynitrite scavenger) treatment. The endothelium-dependent dilatation of thoracic aorta in vitro was determined by response to acetylcholine (ACh). The histological changes in endothelium were assessed by HE staining and scanning electron microscopy (SEM). The expression of 3-nitrotyrosine (NT) in thoracic aorta was demonstrated by immunohistochemistry and immunofluorescence, and the number of circulating endothelial progenitor cells (EPCs) was quantified by flow cytometry. Hyperhomocysteinemia caused significant endothelial injury and dysfunction including vasodilative and histologic changes, associated with higher expression of NT in thoracic aorta. FeTMPyP treatment reversed these injuries significantly. Further, the effect of nitrative stress on cultured EPCs in vitro was investigated by administering peroxynitrite donor (3-morpholino-sydnonimine, SIN-1) and peroxynitrite scavenger (FeTMPyP). The roles of nitrative stress on cell viability, necrosis and apoptosis were evaluated with 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, respectively. Also, the phospho-eNOS expression and tube formation in Matrigel of cultured EPCs was detected. Our data showed that the survival of EPCs was much lower in SIN-1 group than in vehicle group, both the apoptosis and necrosis of EPCs were much more severe, and the p-eNOS expression and tube formation in Matrigel were obviously declined. Subsequent pretreatment with FeTMPyP reversed these changes. Further, pretreatment with FeTMPyP reversed homocysteine-induced EPC injury. In conclusion, this study indicates that

  10. Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells

    PubMed Central

    1994-01-01

    When cultivated on substrates that prevent cell adhesion (the polymer polyhydroxyethylmethacrylate, bovine serum albumin, and Teflon), human endothelial cells (EC) rapidly lost viability with a half-life of approximately 10 h. Dying EC showed the morphological and biochemical characteristics of apoptosis. The apoptotic process of suspended EC was delayed by the protein synthesis inhibitor cycloheximide. To obtain information as to the mechanism involved in the apoptosis of suspended EC, we investigated whether adhesion to matrix proteins or integrin occupancy in EC retaining a round shape may affect EC suicide. EC bound to low coating concentration of either fibronectin or vitronectin, retaining a round shape and failing to organize actin microfilaments, underwent to rapid cell death; by contrast, cells on high substrate concentrations became flattened, showed actin microfilament organization, and retained viability. Addition of saturating amounts of soluble vitronectin to suspended round-shaped EC did not reduce the process of apoptosis. Finally, when suspended EC bound Gly-Arg-Gly-Asp- Ser-coated microbeads (approximately 10 microbeads/cell), yet retaining a round shape, the apoptotic process was not affected. Oncogene- transformed EC in suspension were less susceptible to cell death and apoptosis than normal EC. Overall, these data indicate that cell attachment to matrix or integrin binding per se is not sufficient for maintaining cell viability, and that cells need to undergo some minimal degree of shape change to survive. Modulation of interaction with the extracellular matrix can, therefore, be an important target for the control of angiogenesis. PMID:7523422

  11. Time analysis of corneal endothelial cell density after cataract extraction.

    PubMed

    Galin, M A; Lin, L L; Fetherolf, E; Obstbaum, S A; Sugar, A

    1979-07-01

    Serial endothelial photographs were taken preoperatively and postoperatively in 200 eyes; 111 eyes contained a Rayner iris clip lens, 54 eyes contained a Fyodorov Sputnik lens, and 35 eyes had no lens. Central endothelial cell density was changed in all instances, with counts in implanted eyes declining 25 to 30%, and in nonimplanted eyes 10 to 15%. In both instances, the decline essentially ceased at about three months. The cause of the greater decline in implanted eyes appeared to be mechanical and subsequent cell loss after the 90-day period was virtually equal for the two groups. Methods that may be used to alter the difference in cell density occurring with implantation are best analyzed by using the 90-day period data for comparison. PMID:464015

  12. Isolation and characterization of endothelial progenitor cells from human blood.

    PubMed

    Mead, Laura E; Prater, Daniel; Yoder, Mervin C; Ingram, David A

    2008-07-01

    Circulating endothelial progenitor cells (EPCs) in adult human peripheral blood were originally identified in 1997 by Asahara et al., which challenged the paradigm that vasculogenesis is a process restricted to embryonic development. Since their original identification, EPCs have been extensively studied as biomarkers to assess the risk of cardiovascular disease in human subjects and as a potential cell therapeutic for vascular regeneration. Endothelial colony-forming cells (ECFCs), which are a subtype of EPCs, were recently identified from circulating adult and human umbilical cord blood. In contrast to other types of EPCs, which display various monocyte/macrophage phenotypes and functions, ECFCs are characterized by robust proliferative potential, secondary and tertiary colony formation upon replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In this unit, we describe detailed methodologies for isolation and characterization of ECFCs from both human peripheral and umbilical cord blood.

  13. Nitrones reverse hyperglycemia-induced endothelial dysfunction in bovine aortic endothelial cells.

    PubMed

    Headley, Colwyn A; DiSilvestro, David; Bryant, Kelsey E; Hemann, Craig; Chen, Chun-An; Das, Amlan; Ziouzenkova, Ouliana; Durand, Grégory; Villamena, Frederick A

    2016-03-15

    Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN-LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5mM glucose, LG) or high glucose (50mM, HG) for 14 days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin (BH4)​ levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC with nitrones for 24h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased (BH4) levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50 μM) treatment of BAEC grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted.

  14. Nitrones reverse hyperglycemia-induced endothelial dysfunction in bovine aortic endothelial cells.

    PubMed

    Headley, Colwyn A; DiSilvestro, David; Bryant, Kelsey E; Hemann, Craig; Chen, Chun-An; Das, Amlan; Ziouzenkova, Ouliana; Durand, Grégory; Villamena, Frederick A

    2016-03-15

    Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN-LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5mM glucose, LG) or high glucose (50mM, HG) for 14 days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin (BH4)​ levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC with nitrones for 24h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased (BH4) levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50 μM) treatment of BAEC grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted. PMID:26774452

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

    NASA Astrophysics Data System (ADS)

    Kador, Karl Erich

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

  16. Repressed Ca(2+) clearance in parthenolide-treated murine brain bEND.3 endothelial cells.

    PubMed

    Tsai, Tien-Yao; Lou, Shyh-Liang; Cheng, Ka-Shun; Wong, Kar-Lok; Wang, Mei-Ling; Su, Tzu-Hui; Chan, Paul; Leung, Yuk-Man

    2015-12-15

    Parthenolide is a sesquiterpene lactone compound isolated from the leaves and flowerheads of the plant feverfew (Tanacetum parthenium). The anticancer effects of parthenolide have been well studied and this lactone compound is currently under clinical trials. Parthenolide is also a protective agent in cardiac reperfusion injury via its inhibition of nuclear factor-κB (NF-κB). Not much is known if this compound affects signal transduction in non-tumor cells. We investigated whether parthenolide affected Ca(2+) signaling in endothelial cells, key components in regulating the vascular tone. In this work using mouse cortical microvascular bEND.3 endothelial cells, we found that a 15-h treatment with parthenolide resulted in amplified ATP-triggered Ca(2+) signal; the latter had a very slow decay rate suggesting suppression of Ca(2+) clearance. Evidence suggests parthenolide suppressed Ca(2+) clearance by inhibiting the plasmalemmal Ca(2+) pump; such suppression did not result from decreased expression of the plasmalemmal Ca(2+) pump protein. Rather, such suppression was possibly a consequence of endoplasmic reticulum (ER) stress, since salubrinal (an ER stress protector) was able to alleviate parthenolide-induced Ca(2+) clearance suppression. Given the current deployment of parthenolide as an anti-cancer drug in clinical trials and the potential usage of this lactone as a cardioprotectant, it is important to examine in details the perturbing effects of parthenolide on Ca(2+) homeostasis in endothelial cells and neighboring vascular smooth muscle cells, activities of which exert profound effects on hemodynamics. PMID:26607466

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

    PubMed Central

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

    2015-01-01

    Candida parapsilosis is a fungal pathogen that is associated with hematogenously disseminated disease in premature neonates, acutely ill or immunocompromised patients. In cell culture, C. parapsilosis cells are actively and avidly endocytosed by endothelial cells via actin polymerization mediated by N-WASP. Here we present evidence that C. parapsilosis that were internalized by endothelial cells remained alive, and avoided being acidified or otherwise damaged via the host cell. Internalized fungal cells reproduced intracellularly and eventually burst out of the host endothelial cell. When neutrophils were added to endothelium and C. parapsilosis, they patrolled the endothelial surface and efficiently killed most adherent fungal cells prior to endocytosis. But after endocytosis by endothelial cells, internalized fungal cells evaded neutrophil killing. Silencing endothelial N-WASP blocked endocytosis of C. parapsilosis and left fungal cells stranded on the cell surface, where they were susceptible to neutrophil killing. These observations suggest that for C. parapsilosis to escape from the bloodstream, fungi may adhere to and be internalized by endothelial cells before being confronted and phagocytosed by a patrolling leukocyte. Once internalized by endothelial cells, C. parapsilosis may safely replicate to cause further rounds of infection. Immunosurveillance of the intravascular lumen by leukocytes crawling on the endothelial surface and rapid killing of adherent yeast may play a major role in controlling C. parapsilosis dissemination and infected endothelial cells may be a significant reservoir for fungal persistence. PMID:26039751

  18. Microenvironmental Regulation of the Sinusoidal Endothelial Cell Phenotype In Vitro

    PubMed Central

    March, Sandra; Hui, Elliot E.; Underhill, Gregory H.; Khetani, Salman; Bhatia, Sangeeta N.

    2010-01-01

    Liver Sinusoidal Endothelial Cells (LSEC) differ, both structurally and functionally, from endothelial cells (EC) lining blood vessels of other tissues. For example, in contrast to other EC, LSEC posses fenestrations, have low detectable levels of PECAM-1 expression, and in rat tissue, they distinctively express a cell surface marker recognized by the SE-1 antibody. These unique phenotypic characteristics seen in hepatic tissue are lost over time upon culture in vitro; therefore, this study sought to systematically examine the effects of microenvironmental stimuli, namely, extracellular matrix (ECM) and neighboring cells, on the LSEC phenotype in vitro. In probing the role of the underlying extracellular matrix, we identified collagen I and collagen III as well as mixtures of collagen I/collagen IV/fibronectin as having a positive effect on LSEC survival. Furthermore, using a stable hepatocellular model (hepatocyte-fibroblast) we were able to prolong the expression of both SE-1 and phenotypic functions of LSEC such as Factor VIII activity in co-cultured LSECs through the production of short-range paracrine signals. In the course of these experiments, we identified the antigen recognized by SE-1 as CD32b. Collectively, this study has identified several microenvironmental regulators of liver sinusoidal endothelial cells that prolong their phenotypic functions for up to 2 weeks in culture, enabling the development of better in vitro models of liver physiology and disease. PMID:19585615

  19. Cholesterol depletion increases membrane stiffness of aortic endothelial cells.

    PubMed

    Byfield, Fitzroy J; Aranda-Espinoza, Helim; Romanenko, Victor G; Rothblat, George H; Levitan, Irena

    2004-11-01

    This study has investigated the effect of cellular cholesterol on membrane deformability of bovine aortic endothelial cells. Cellular cholesterol content was depleted by exposing the cells to methyl-beta-cyclodextrin or enriched by exposing the cells to methyl-beta-cyclodextrin saturated with cholesterol. Control cells were treated with methyl-beta-cyclodextrin-cholesterol at a molar ratio that had no effect on the level of cellular cholesterol. Mechanical properties of the cells with different cholesterol contents were compared by measuring the degree of membrane deformation in response to a step in negative pressure applied to the membrane by a micropipette. The experiments were performed on substrate-attached cells that maintained normal morphology. The data were analyzed using a standard linear elastic half-space model to calculate Young elastic modulus. Our observations show that, in contrast to the known effect of cholesterol on membrane stiffness of lipid bilayers, cholesterol depletion of bovine aortic endothelial cells resulted in a significant decrease in membrane deformability and a corresponding increase in the value of the elastic coefficient of the membrane, indicating that cholesterol-depleted cells are stiffer than control cells. Repleting the cells with cholesterol reversed the effect. An increase in cellular cholesterol to a level higher than that of normal cells, however, had no effect on the elastic properties of bovine aortic endothelial cells. We also show that although cholesterol depletion had no apparent effect on the intensity of F-actin-specific fluorescence, disrupting F-actin with latrunculin A abrogated the stiffening effect. We suggest that cholesterol depletion increases the stiffness of the membrane by altering the properties of the submembrane F-actin and/or its attachment to the membrane.

  20. Junctional communication is induced in migrating capillary endothelial cells

    PubMed Central

    1989-01-01

    Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis inhibitor mitomycin C, but was prevented by the protein synthesis inhibitor, cycloheximide. The induction of wound-associated communication was also inhibited when migration was prevented by placing the cells immediately after wounding at 22 degrees C or after exposure to cytochalasin D, suggesting that the increase in communication is dependent on cells migrating into the wound area. In contrast, migration was not prevented when coupling was blocked by exposure of the cells to retinoic acid, although this agent did disrupt the characteristic sheet-like pattern of migration typically seen during endothelial repair. These results suggest that junctional communication may play an important role in wound repair, possibly by coordinating capillary endothelial cell migration. PMID:2592412

  1. Increased circulating inflammatory endothelial cells in blacks with essential hypertension.

    PubMed

    Eirin, Alfonso; Zhu, Xiang-Yang; Woollard, John R; Herrmann, Sandra M; Gloviczki, Monika L; Saad, Ahmed; Juncos, Luis A; Calhoun, David A; Rule, Andrew D; Lerman, Amir; Textor, Stephen C; Lerman, Lilach O

    2013-09-01

    Morbidity and mortality attributable to hypertension are higher in black essential hypertensive (EH) compared with white EH patients, possibly related to differential effects on vascular injury and repair. Although circulating endothelial progenitor cells (EPCs) preserve endothelial integrity, inflammatory endothelial cells (IECs) detach from sites of injury and represent markers of vascular damage. We hypothesized that blood levels of IECs and inflammatory markers would be higher in black EH compared with white EH patients. Inferior vena cava and renal vein levels of CD34+/KDR+ (EPC) and VAP-1+ (IEC) cells were measured by fluorescence-activated cell sorting in white EH and black EH patients under fixed sodium intake and blockade of the renin-angiotensin system, and compared with systemic levels in normotensive control subjects (n=19 each). Renal vein and inferior vena cava levels of inflammatory cytokines and EPC homing factors were measured by Luminex. Blood pressure, serum creatinine, lipids, and antihypertensive medications did not differ between white and black EH patients, and EPC levels were decreased in both. Circulating IEC levels were elevated in black EH patients, and inversely correlated with EPC levels (R(2)=0.58; P=0.0001). Systemic levels of inflammatory cytokines and EPC homing factors were higher in black EH compared with white EH patients, and correlated directly with IECs. Renal vein inflammatory cytokines, EPCs, and IECs did not differ from their circulating levels. Most IECs expressed endothelial markers, fewer expressed progenitor cell markers, but none showed lymphocyte or phagocytic cell markers. Thus, increased release of cytokines and IECs in black EH patients may impair EPC reparative capacity and aggravate vascular damage, and accelerate hypertension-related complications.

  2. Fluid shear, intercellular stress, and endothelial cell alignment

    PubMed Central

    Steward, Robert; Tambe, Dhananjay; Hardin, C. Corey; Krishnan, Ramaswamy

    2015-01-01

    Endothelial cell alignment along the direction of laminar fluid flow is widely understood to be a defining morphological feature of vascular homeostasis. While the role of associated signaling and structural events have been well studied, associated intercellular stresses under laminar fluid shear have remained ill-defined and the role of these stresses in the alignment process has remained obscure. To fill this gap, we report here the tractions as well as the complete in-plane intercellular stress fields measured within the human umbilical vein endothelial cell (HUVEC) monolayer subjected to a steady laminar fluid shear of 1 Pa. Tractions, intercellular stresses, as well as their time course, heterogeneity, and anisotropy, were measured using monolayer traction microscopy and monolayer stress microscopy. Prior to application of laminar fluid flow, intercellular stresses were largely tensile but fluctuated dramatically in space and in time (317 ± 122 Pa). Within 12 h of the onset of laminar fluid flow, the intercellular stresses decreased substantially but continued to fluctuate dramatically (142 ± 84 Pa). Moreover, tractions and intercellular stresses aligned strongly and promptly (within 1 h) along the direction of fluid flow, whereas the endothelial cell body aligned less strongly and substantially more slowly (12 h). Taken together, these results reveal that steady laminar fluid flow induces prompt reduction in magnitude and alignment of tractions and intercellular stress tensor components followed by the retarded elongation and alignment of the endothelial cell body. Appreciably smaller intercellular stresses supported by cell-cell junctions logically favor smaller incidence of gap formation and thus improved barrier integrity. PMID:25652451

  3. Dengue Virus Infection of Mast Cells Triggers Endothelial Cell Activation ▿

    PubMed Central

    Brown, Michael G.; Hermann, Laura L.; Issekutz, Andrew C.; Marshall, Jean S.; Rowter, Derek; Al-Afif, Ayham; Anderson, Robert

    2011-01-01

    Vascular perturbation is a hallmark of severe forms of dengue disease. We show here that antibody-enhanced dengue virus infection of primary human cord blood-derived mast cells (CBMCs) and the human mast cell-like line HMC-1 results in the release of factor(s) which activate human endothelial cells, as evidenced by increased expression of the adhesion molecules ICAM-1 and VCAM-1. Endothelial cell activation was prevented by pretreatment of mast cell-derived supernatants with a tumor necrosis factor (TNF)-specific blocking antibody, thus identifying TNF as the endothelial cell-activating factor. Our findings suggest that mast cells may represent an important source of TNF, promoting vascular endothelial perturbation following antibody-enhanced dengue virus infection. PMID:21068256

  4. The Secretome of Endothelial Progenitor Cells Promotes Brain Endothelial Cell Activity through PI3-Kinase and MAP-Kinase

    PubMed Central

    Di Santo, Stefano; Seiler, Stefanie; Fuchs, Anna-Lena; Staudigl, Jennifer; Widmer, Hans Rudolf

    2014-01-01

    Background Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. Methods Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. Results Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. Conclusion The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects. PMID:24755675

  5. Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures

    PubMed Central

    Doulet, Nicolas; Donnadieu, Emmanuel; Laran-Chich, Marie-Pierre; Niedergang, Florence; Nassif, Xavier; Couraud, Pierre Olivier; Bourdoulous, Sandrine

    2006-01-01

    Neisseria meningitidis elicits the formation of membrane protrusions on vascular endothelial cells, enabling its internalization and transcytosis. We provide evidence that this process interferes with the transendothelial migration of leukocytes. Bacteria adhering to endothelial cells actively recruit ezrin, moesin, and ezrin binding adhesion molecules. These molecules no longer accumulate at sites of leukocyte–endothelial contact, preventing the formation of the endothelial docking structures required for proper leukocyte diapedesis. Overexpression of exogenous ezrin or moesin is sufficient to rescue the formation of docking structures on and leukocyte migration through infected endothelial monolayers. Inversely, expression of the dominant-negative NH2-terminal domain of ezrin markedly inhibits the formation of docking structures and leukocyte diapedesis through noninfected monolayers. Ezrin and moesin thus appear as pivotal endothelial proteins required for leukocyte diapedesis that are titrated away by N. meningitidis. These results highlight a novel strategy developed by a bacterial pathogen to hamper the host inflammatory response by interfering with leukocyte–endothelial cell interaction. PMID:16717131

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

    PubMed

    Schweighofer, Bernhard; Rohringer, Sabrina; Pröll, Johannes; Holnthoner, Wolfgang

    2015-06-01

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

  7. Dihydroartemisinin induces endothelial cell anoikis through the activation of the JNK signaling pathway

    PubMed Central

    Zhang, Jiao; Guo, Ling; Zhou, Xia; Dong, Fengyun; Li, Liqun; Cheng, Zuowang; Xu, Yinghua; Liang, Jiyong; Xie, Qi; Liu, Ju

    2016-01-01

    Angiogenesis is required for the growth and metastasis of solid tumors. The anti-malarial agent dihydroartemisinin (DHA) demonstrates potent anti-angiogenic activity, but the underlying molecular mechanisms are not yet fully understood. During the process of angiogenesis, endothelial cells migrating from existing capillaries may undergo programmed cell death after detaching from the extracellular matrix, a process that is defined as anchorage-dependent apoptosis or anoikis. In the present study, DHA-induced cell death was compared in human umbilical vein endothelial cells (HUVECs) cultured in suspension and attached to culture plates. In suspended HUVECs, the cell viability was decreased and apoptosis was increased with the treatment of 50 µM DHA for 5 h, while the same treatment did not affect the attached HUVECs. In addition, 50 µM DHA increased the phosphorylation of c-Jun N-terminal kinase (JNK) in suspended HUVECs, but not in attached HUVECs, for up to 5 h of treatment. The JNK inhibitor, SP600125, reversed DHA-induced cell death in suspended HUVECs, suggesting that the JNK pathway may mediate DHA-induced endothelial cell anoikis. The data from the present study indicates a novel mechanism for understanding the anti-angiogenic effects of DHA, which may be used as a component for chemotherapy. PMID:27602117

  8. Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63

    NASA Astrophysics Data System (ADS)

    Poeter, Michaela; Brandherm, Ines; Rossaint, Jan; Rosso, Gonzalo; Shahin, Victor; Skryabin, Boris V.; Zarbock, Alexander; Gerke, Volker; Rescher, Ursula

    2014-04-01

    To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63.

  9. Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63.

    PubMed

    Poeter, Michaela; Brandherm, Ines; Rossaint, Jan; Rosso, Gonzalo; Shahin, Victor; Skryabin, Boris V; Zarbock, Alexander; Gerke, Volker; Rescher, Ursula

    2014-01-01

    To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63. PMID:24769558

  10. Albumin-derived advanced glycation end-products trigger the disruption of the vascular endothelial cadherin complex in cultured human and murine endothelial cells.

    PubMed Central

    Otero, K; Martínez, F; Beltrán, A; González, D; Herrera, B; Quintero, G; Delgado, R; Rojas, A

    2001-01-01

    Endothelial cell (EC) junctions regulate in large part the integrity and barrier function of the vascular endothelium. Advanced glycation end-products (AGEs), the irreversibly formed reactive derivatives of non-enzymic glucose-protein condensation reactions, are strongly implicated in endothelial dysfunction that distinguishes diabetes- and aging-associated vascular complications. The aim of the present study was to determine whether AGEs affect EC lateral junction proteins, with particular regard to the vascular endothelial cadherin (VE-cadherin) complex. Our results indicate that AGE-modified BSA (AGE-BSA), a prototype of advanced glycated proteins, disrupts the VE-cadherin complex when administered to ECs. AGE-BSA, but not unmodified BSA, was found to induce decreases in the levels of VE-cadherin, beta-catenin and gamma-catenin in the complex and in total cell extracts, as well as a marked reduction in the amount of VE-cadherin present at the cell surface. In contrast, the level of platelet endothelial cell adhesion molecule-1 (PECAM-1), which is located at lateral junctions, was not altered. Supplementation of the cellular antioxidative defences abolished these effects. Finally, the loss of components of the VE-cadherin complex was correlated with increases in vascular permeability and in EC migration. These findings suggest that some of the AGE-induced biological effects on the endothelium could be mediated, at least in part, by the weakening of intercellular contacts caused by decreases in the amount of VE-cadherin present. PMID:11672430

  11. Action of Shiga Toxin Type-2 and Subtilase Cytotoxin on Human Microvascular Endothelial Cells

    PubMed Central

    Amaral, María M.; Sacerdoti, Flavia; Jancic, Carolina; Repetto, Horacio A.; Paton, Adrienne W.; Paton, James C.; Ibarra, Cristina

    2013-01-01

    The hemolytic uremic syndrome (HUS) associated with diarrhea is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB) was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC) has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF) and platelet/endothelial cell adhesion molecule 1 (PECAM-1). HGEC also expressed the globotriaosylceramide (Gb3) receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 −a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis. PMID:23936204

  12. Interleukin 3 stimulates proliferation and triggers endothelial-leukocyte adhesion molecule 1 gene activation of human endothelial cells.

    PubMed

    Brizzi, M F; Garbarino, G; Rossi, P R; Pagliardi, G L; Arduino, C; Avanzi, G C; Pegoraro, L

    1993-06-01

    Proliferation and functional activation of endothelial cells within a tissue site of inflammation are regulated by humoral factors released by cells, such as T lymphocytes and monocytes, infiltrating the perivascular space. In the present study we investigated the effects of interleukin 3 (IL-3), an activated T lymphocyte-derived cytokine, on cultured human umbilical vein endothelial cells (HUVEC). Proliferative activity, evaluated both by estimation of the fraction of cells in the S phase and by direct cell count demonstrated that IL-3, at the dose of 25 ng/ml, enhances more than threefold both DNA synthesis and cell proliferation above baseline control conditions. Binding studies with radioiodinated ligand demonstrated that HUVEC constitutively express a smaller number of IL-3 binding sites (approximately 99 binding sites per cell, with an apparent Kd of 149 pM). Accordingly, molecular analysis showed the presence of transcripts for both alpha and beta subunits of the IL-3 receptor. Functional activation of endothelial cells was evaluated by the expression of the endothelial-leukocyte adhesion molecule 1 (ELAM-1) transcript and by leukocyte adhesion. The ELAM-1 gene transcript was clearly detectable 4 h after IL-3 addition and started to decrease after 12 h. Moreover, IL-3-induced ELAM-1 transcription was followed by enhanced adhesion of neutrophils and CD4+ T cells to HUVEC. The findings that IL-3 can stimulate both proliferation and functional activation of endothelial cells suggest that this cytokine can be involved in sustaining the process of chronic inflammation.

  13. Biomechanics and Intracellular Dynamics of Vascular Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Ou-Yang, H. Daniel

    2004-03-01

    Understanding the internal mechanical properties of living cells is essential to gain insight to basic cellular functions ranging from cellular signal transduction, intracellular traffics to cell motility. Vascular endothelial cells form a single cell layer that lines all blood vessels and serves to regulate exchanges between the blood stream and the surrounding tissues. Endothelial cells are one of the most studied cell types because of their roles in cardiovascular diseases and the linkage between their growth control and strategies of cancer treatments. This talk reports the application of a novel methodology by which scientists can explore cellular functions and study cytoskeleton dynamics of living cells at the subcellular level with minimal invasion. The methodology is based on the realization that optical tweezers can be used to measure the mechanical properties of the cytoskeleton in the vicinity of organelles and cellular structures. Optical tweezers is a technique based on the physics that dielectric materials, such as silica beads, latex particles or protein aggregates are attracted to and thus trapped at the focal point of a tightly focused laser beam in an aqueous medium. It has been shown that viscoelasticity can be determined from the movements of the trapped object in an oscillating optical tweezers. Applying the oscillating tweezers to intracellular cellular structures, we were able to determine the frequency dependent mechanical properties of the interior of cultured bovine endothelial cells. In contrast to the viscoelastic behavior expected of a network of cytoskelatal proteins, we found unusually large fluctuations in both elastic and loss moduli of the cell interior. More surprisingly, both mechanical moduli showed rhythmic behavior with a periodicity in the range of 20 - 30 seconds in healthy living cells. The rhythm could be altered by drug treatments, and the amplitude of the fluctuations diminished when cells were depleted of nutrients

  14. Endothelial activation by platelets from sickle cell anemia patients.

    PubMed

    Proença-Ferreira, Renata; Brugnerotto, Ana Flávia; Garrido, Vanessa Tonin; Dominical, Venina Marcela; Vital, Daiana Morelli; Ribeiro, Marilene de Fátima Reis; dos Santos, Melissa Ercolin; Traina, Fabíola; Olalla-Saad, Sara T; Costa, Fernando Ferreira; Conran, Nicola

    2014-01-01

    Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association

  15. Endothelial Activation by Platelets from Sickle Cell Anemia Patients

    PubMed Central

    Proença-Ferreira, Renata; Brugnerotto, Ana Flávia; Garrido, Vanessa Tonin; Dominical, Venina Marcela; Vital, Daiana Morelli; Ribeiro, Marilene de Fátima Reis; dos Santos, Melissa Ercolin; Traina, Fabíola; Olalla-Saad, Sara T.; Costa, Fernando Ferreira; Conran, Nicola

    2014-01-01

    Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association

  16. S1PR1 Tyr143 phosphorylation downregulates endothelial cell surface S1PR1 expression and responsiveness

    PubMed Central

    Chavez, Alejandra; Schmidt, Tracy Thennes; Yazbeck, Pascal; Rajput, Charu; Desai, Bhushan; Sukriti, Sukriti; Giantsos-Adams, Kristina; Knezevic, Nebojsa; Malik, Asrar B.; Mehta, Dolly

    2015-01-01

    ABSTRACT Activation of sphingosine-1-phosphate receptor 1 (S1PR1) plays a key role in repairing endothelial barrier function. We addressed the role of phosphorylation of the three intracellular tyrosine residues of S1PR1 in endothelial cells in regulating the receptor responsiveness and endothelial barrier function regulated by sphingosine 1-phosphate (S1P)-mediated activation of S1PR1. We demonstrated that phosphorylation of only Y143 site was required for S1PR1 internalization in response to S1P. Maximal S1PR1 internalization was seen in 20 min but S1PR1 returned to the cell surface within 1 h accompanied by Y143-dephosphorylation. Cell surface S1PR1 loss paralleled defective endothelial barrier enhancement induced by S1P. Expression of phospho-defective (Y143F) or phospho-mimicking (Y143D) mutants, respectively, failed to internalize or showed unusually high receptor internalization, consistent with the requirement of Y143 in regulating cell surface S1PR1 expression. Phosphorylation of the five S1PR1 C-terminal serine residues did not affect the role of Y143 phosphorylation in signaling S1PR1 internalization. Thus, rapid reduction of endothelial cell surface expression of S1PR1 subsequent to Y143 phosphorylation is a crucial mechanism of modulating S1PR1 signaling, and hence the endothelial barrier repair function of S1P. PMID:25588843

  17. Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo.

    PubMed

    Ribatti, D; Presta, M; Vacca, A; Ria, R; Giuliani, R; Dell'Era, P; Nico, B; Roncali, L; Dammacco, F

    1999-04-15

    Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor.

  18. Abrupt reflow enhances cytokine-induced proinflammatory activation of endothelial cells during simulated shock and resuscitation.

    PubMed

    Li, Ranran; Zijlstra, Jan G; Kamps, Jan A A M; van Meurs, Matijs; Molema, Grietje

    2014-10-01

    Circulatory shock and resuscitation are associated with systemic hemodynamic changes, which may contribute to the development of MODS (multiple organ dysfunction syndrome). In this study, we used an in vitro flow system to simulate the consecutive changes in blood flow as occurring during hemorrhagic shock and resuscitation in vivo. We examined the kinetic responses of different endothelial genes in human umbilical vein endothelial cells preconditioned to 20 dyne/cm unidirectional laminar shear stress for 48 h to flow cessation and abrupt reflow, respectively, as well as the effect of flow cessation and reflow on tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory activation. Endothelial CD31 and VE-cadherin were not affected by the changes in flow in the absence or presence of TNF-α. The messenger RNA levels of proinflammatory molecules E-selectin, VCAM-1 (vascular cell adhesion molecule 1), and IL-8 (interleukin 8) were significantly induced by flow cessation respectively acute reflow, whereas ICAM-1 (intercellular adhesion molecule 1) was downregulated on flow cessation and induced by subsequent acute reflow. Flow cessation also affected the Ang/Tie2 (Angiopoietin/Tie2 receptor tyrosine kinase) system by downregulating Tie2 and inducing its endothelial ligand Ang2, an effect that was further extended on acute reflow. Furthermore, the induction of proinflammatory adhesion molecules by TNF-α under flow cessation was significantly enhanced on subsequent acute reflow. This study demonstrated that flow alterations per se during shock and resuscitation contribute to endothelial activation and that these alterations interact with proinflammatory factors coexisting in vivo such as TNF-α. The abrupt reflow-related enhancement of cytokine-induced endothelial proinflammatory activation supports the concept that sudden regain of flow during resuscitation has an aggravating effect on endothelial activation, which may play a significant role in vascular

  19. Enhancement of tumor necrosis factor-induced endothelial cell injury by cycloheximide

    SciTech Connect

    Nolop, K.B.; Ryan, U.S. )

    1990-08-01

    Tumor necrosis factor (TNF), a potent polypeptide mediator released by activated monocytes and macrophages, has a number of proinflammatory effects on endothelial cells. TNF is cytotoxic to tumor cells in vivo and in vitro, but TNF-induced toxicity to endothelial cells is less well established. We now report that cycloheximide (CHX), an inhibitor of protein synthesis, renders endothelial cells highly susceptible to TNF-induced lysis. TNF alone did not change the overall rate of protein synthesis by endothelial cells, whereas the addition of CHX completely abolished protein synthesis. Endothelial cells incubated in TNF alone in high concentrations (up to 1,000 U/ml) showed minimal rounding up and release of 51Cr. Likewise, CHX alone (5 micrograms/ml) had no significant effect on endothelial cell morphology and release of 51Cr. However, incubation of endothelial cells in both CHX and TNF caused injury in a dose-dependent manner. Morphological evidence of cell retraction, rounding, and detachment began within 2 h, but specific 51Cr release did not begin to rise until after 4 h. These changes were not observed when endothelial cells were incubated with TNF/CHX at 4 degrees C. The combination of TNF/CHX was lethal to all endothelial cells tested (bovine pulmonary artery, human umbilical vein, and human aorta), with human aortic cells showing the most pronounced changes. We conclude that healthy endothelial cells are resistant to TNF-induced lysis, but inhibition of their ability to make protein renders them highly susceptible.

  20. Solid tumor therapy by selectively targeting stromal endothelial cells.

    PubMed

    Liu, Shihui; Liu, Jie; Ma, Qian; Cao, Liu; Fattah, Rasem J; Yu, Zuxi; Bugge, Thomas H; Finkel, Toren; Leppla, Stephen H

    2016-07-12

    Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors. PMID:27357689

  1. 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.

  2. Binding of tissue plasminogen activator to cultured human endothelial cells.

    PubMed Central

    Hajjar, K A; Hamel, N M; Harpel, P C; Nachman, R L

    1987-01-01

    Tissue plasminogen activator (t-PA) and urokinase (u-PA), the major activators of plasminogen, are synthesized and released from endothelial cells. We previously demonstrated specific and functional binding of plasminogen to cultured human umbilical vein endothelial cells (HUVEC). In the present study we found that t-PA could bind to HUVEC. Binding of t-PA to HUVEC was specific, saturable, plasminogen-independent, and did not require lysine binding sites. The t-PA bound in a rapid and reversible manner, involving binding sites of both high (Kd, 28.7 +/- 10.8 pM; Bmax, 3,700 +/- 300) and low (Kd, 18.1 +/- 3.8 nM; Bmax 815,000 +/- 146,000) affinity. t-PA binding was 70% inhibited by a 100-fold molar excess of u-PA. When t-PA was bound to HUVEC, its apparent catalytic efficiency increased by three- or fourfold as measured by plasminogen activation. HUVEC-bound t-PA was active site-protected from its rapidly acting inhibitor: plasminogen activator inhibitor. These results demonstrate that t-PA specifically binds to HUVEC and that such binding preserves catalytic efficiency with respect to plasminogen activation. Therefore, endothelial cells can modulate hemostatic and thrombotic events at the cell surface by providing specific binding sites for activation of plasminogen. PMID:3119664

  3. Interrogating TGF-β Function and Regulation in Endothelial Cells.

    PubMed

    Maring, J A; van Meeteren, L A; Goumans, M J; Ten Dijke, Peter

    2016-01-01

    Transforming growth factor-β (TGF-β) is a multifunctional cytokine with important roles in embryogenesis and maintaining tissue homeostasis during adult life. There are three isoforms of TGF-β, i.e., TGF-β1, -β2, and -β3, which signal by binding to a complex of transmembrane type I and type II serine/threonine kinase receptors and intracellular Smad transcription factors. In most cell types TGF-β signals via TGF-β type II receptor (TβRII) and TβRI, also termed activin receptor-like kinase 5 (ALK5). In endothelial cells, TGF-β signals via ALK5 and ALK1. These two type I receptors mediate opposite cellular response for TGF-β. The co-receptor endoglin, highly expressed on proliferating endothelial cells, facilitates TGF-β/ALK1 and inhibits TGF-β/ALK5 signaling. Knockout of TGF-β receptors in mice all result in embryonic lethality during midgestation from defects in angiogenesis, illustrating the pivotal role of TGF-β in this process. This chapter introduces methods for examining the function and regulation of TGF-β in angiogenesis in in vitro assays using cultured endothelial cells and ex vivo metatarsal explants.

  4. 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

  5. Tumors skew endothelial cells to disrupt NK cell, T-cell and macrophage functions

    PubMed Central

    Mulligan, Jennifer K.; Lathers, Deanne M. R.

    2012-01-01

    Introduction Patients and mice with solid tumors, such as Lewis lung carcinoma (LLC), have defects in functions of immune effector cells. Endothelial cells, a component of the tumor vasculature, are potential regulators of immune cell functions. Therefore, these studies examined the impact of exposure to LLC tumor on the ability of endothelial cells to modulate immune cell functions. Materials and methods Endothelial cells were pre-treated with LLC tumor-conditioned medium (EndoT-sup) for 24 h. Control endothelial cells that were exposed to medium (EndoMedia) or epithelial cell-conditioned medium (EndoEpi-sup). After the initial 24 h incubation, endothelial cells were washed and fresh media was added. Cells were allowed to incubate for an additional 24 h. Supernatants from EndoMedia, EndoEpi-sup or EndoT-sup were collected and assayed for immune modulatory products and for immune modulatory activity. Results Supernatant from EndoT-sup contained increased levels of PGE2, IL-6 and VEGF as compared to EndoMedia and EndoEpi-sup controls. NK cell activity, as measured by TNF-α and IFN-γ secretion, was increased following exposure to media conditioned by EndoMedia and EndoEpi-sup. Exposure of NK cells to supernatants of EndoT-sup, also increases TNF-α and IFN-γ secretion, but to a lesser extent than by EndoMedia and EndoEpi-sup. Examination of macrophage functions demonstrated that supernatant from EndoT-sup decreased microbead phagocytosis and increased production of the immune suppressive mediators, IL-10 and PGE2. Lastly, T-cell responses to stimulation with anti-CD3 in the presence of supernatants from EndoT-sup were examined. IFN-γ production by CD8+ T-cells was reduced after exposure to EndoT-sup-conditioned medium, as compared to cells treatments with medium or control conditioned medium. Production of IFN-γ by CD4+ T-cells exposed to EndoT-sup was not altered. Conclusions Taken together, these studies demonstrate that tumors skew endothelial cells to

  6. Rat brain endothelial cells are a target of manganese toxicity

    PubMed Central

    Marreilha dos Santos, Ana Paula; Milatovic, Dejan; Au, Catherine; Yin, Zhaobao; Batoreu, Maria Camila C.; Aschner, Michael

    2010-01-01

    Manganese (Mn) is an essential trace metal, however exposure to high Mn levels can result in neurodegenerative changes resembling Parkinson´s disease (PD). Information on Mn´s effects on endothelial cells of the blood-brain barrier (BBB) is lacking. Accordingly, we tested the hypothesis that BBB endothelial cells are a primary target for Mn-induced neurotoxicity. The studies were conducted in an in vitro BBB model of immortalized rat brain endothelial (RBE4) cells. ROS production was determined by F2-Isoprostane (F2-IsoPs) measurement. The relationship between Mn toxicity and redox status was investigated upon intracellular glutathione (GSH) depletion with diethylmaleate (DEM) or L-buthionine sulfoximine (BSO). Mn exposure (200 or 800 µM MnCl2 or MnSO4) for 4 or 24h led to significant decrease in cell viability vs. controls. DEM or BSO pre-treatment led to further enhancement in cytotoxicity vs. exposure to Mn alone, with more pronounced cell death after 24h DEM pre-treatment. F2-IsoPs levels in cells exposed to MnCl2 (200 or 800 µM), were significantly increased after 4h and remained elevated 24h after exposure compared with controls. Consistent with the effects on cell viability and F2-IsoPs, treatment with MnCl2 (200 or 800 µM) was also associated with a significant decrease in membrane potential. This effect was more pronounced in cells exposed to DEM plus MnCl2 vs. cells exposed to Mn alone. We conclude that Mn induces direct injury to mitochondria in RBE4 cells. The ensuing impairment in energy metabolism and redox status may modify the restrictive properties of the BBB compromising its function. PMID:20170646

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. "Decoding" Angiogenesis: New Facets Controlling Endothelial Cell Behavior.

    PubMed

    Sewduth, Raj; Santoro, Massimo M

    2016-01-01

    Angiogenesis, the formation of new blood vessels, is a unique and crucial biological process occurring during both development and adulthood. A better understanding of the mechanisms that regulates such process is mandatory to intervene in pathophysiological conditions. Here we highlight some recent argument on new players that are critical in endothelial cells, by summarizing novel discoveries that regulate notorious vascular pathways such as Vascular Endothelial Growth Factor (VEGF), Notch and Planar Cell Polarity (PCP), and by discussing more recent findings that put metabolism, redox signaling and hemodynamic forces as novel unforeseen facets in angiogenesis. These new aspects, that critically regulate angiogenesis and vascular homeostasis in health and diseased, represent unforeseen new ground to develop anti-angiogenic therapies. PMID:27493632

  9. “Decoding” Angiogenesis: New Facets Controlling Endothelial Cell Behavior

    PubMed Central

    Sewduth, Raj; Santoro, Massimo M.

    2016-01-01

    Angiogenesis, the formation of new blood vessels, is a unique and crucial biological process occurring during both development and adulthood. A better understanding of the mechanisms that regulates such process is mandatory to intervene in pathophysiological conditions. Here we highlight some recent argument on new players that are critical in endothelial cells, by summarizing novel discoveries that regulate notorious vascular pathways such as Vascular Endothelial Growth Factor (VEGF), Notch and Planar Cell Polarity (PCP), and by discussing more recent findings that put metabolism, redox signaling and hemodynamic forces as novel unforeseen facets in angiogenesis. These new aspects, that critically regulate angiogenesis and vascular homeostasis in health and diseased, represent unforeseen new ground to develop anti-angiogenic therapies. PMID:27493632

  10. Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells

    PubMed Central

    van den Biggelaar, Maartje; Hernández-Fernaud, Juan Ramon; van den Eshof, Bart L.; Neilson, Lisa J.; Meijer, Alexander B.; Mertens, Koen

    2014-01-01

    Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor 1 (PAR1)-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Because of its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phosphopeptide enrichment, and high-resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which have not been previously related to thrombin. Those sites were localized on proteins that are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions, and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites that may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function. PMID:24501219

  11. Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells.

    PubMed

    van den Biggelaar, Maartje; Hernández-Fernaud, Juan Ramon; van den Eshof, Bart L; Neilson, Lisa J; Meijer, Alexander B; Mertens, Koen; Zanivan, Sara

    2014-03-20

    Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor 1 (PAR1)-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Because of its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phosphopeptide enrichment, and high-resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which have not been previously related to thrombin. Those sites were localized on proteins that are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions, and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites that may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function. PMID:24501219

  12. Endothelial cell sensing, restructuring, and invasion in collagen hydrogel structures.

    PubMed

    Hosseini, Y; Agah, M; Verbridge, S S

    2015-11-01

    Experimental tools to model cell-tissue interactions will likely lead to new ways to both understand and treat cancer. While the mechanical properties and regulation of invasion have been recently studied for tumor cells, they have received less attention in the context of tumor vascular dynamics. In this article, we have investigated the interaction between the surfaces of structures encountered by endothelial cells invading their surrounding extracellular matrix (ECM) during angiogenesis. For this purpose, we have fabricated round and sharp geometries with various curvature and sharpness indices in collagen hydrogel over a wide range of stiffness to mimic different microenvironments varying from normal to tumor tissues. We have then cultured endothelial cells on these structures to investigate the bi-directional interaction between the cells and ECM. We have observed that cell invasion frequency is higher from the structures with the highest sharpness and curvature index, while interestingly the dependence of invasion on the local micro-geometry is strongest for the highest density matrices. Notably, structures with the highest invasion length are linked with higher deformation of side structures, which may be related to traction force-activated signaling suggesting further investigation. We have noted that round structures are more favorable for cell adhesion and in some cases round structures drive cell invasion faster than sharp ones. These results highlight the ability of endothelial cells to sense small variations in ECM geometry, and respond with a balance of matrix invasion as well as deformation, with potential implications for feedback mechanisms that may enhance vascular abnormality in response to tumor-induced ECM alterations.

  13. Endothelial cell sensing, restructuring, and invasion in collagen hydrogel structures.

    PubMed

    Hosseini, Y; Agah, M; Verbridge, S S

    2015-11-01

    Experimental tools to model cell-tissue interactions will likely lead to new ways to both understand and treat cancer. While the mechanical properties and regulation of invasion have been recently studied for tumor cells, they have received less attention in the context of tumor vascular dynamics. In this article, we have investigated the interaction between the surfaces of structures encountered by endothelial cells invading their surrounding extracellular matrix (ECM) during angiogenesis. For this purpose, we have fabricated round and sharp geometries with various curvature and sharpness indices in collagen hydrogel over a wide range of stiffness to mimic different microenvironments varying from normal to tumor tissues. We have then cultured endothelial cells on these structures to investigate the bi-directional interaction between the cells and ECM. We have observed that cell invasion frequency is higher from the structures with the highest sharpness and curvature index, while interestingly the dependence of invasion on the local micro-geometry is strongest for the highest density matrices. Notably, structures with the highest invasion length are linked with higher deformation of side structures, which may be related to traction force-activated signaling suggesting further investigation. We have noted that round structures are more favorable for cell adhesion and in some cases round structures drive cell invasion faster than sharp ones. These results highlight the ability of endothelial cells to sense small variations in ECM geometry, and respond with a balance of matrix invasion as well as deformation, with potential implications for feedback mechanisms that may enhance vascular abnormality in response to tumor-induced ECM alterations. PMID:26379187

  14. Effect of Polyelectrolyte Film Stiffness on Endothelial Cells During Endothelial-to-Mesenchymal Transition.

    PubMed

    Zhang, He; Chang, Hao; Wang, Li-mei; Ren, Ke-feng; Martins, M Cristina L; Barbosa, Mário A; Ji, Jian

    2015-11-01

    Endothelial-to-mesenchymal transition (EndMT), during which endothelial cells (ECs) transdifferentiate into mesenchymal phenotype, plays a key role in the development of vascular implant complications such as endothelium dysfunction and in-stent restenosis. Substrate stiffness has been confirmed as a key factor to influence EC behaviors; however, so far, the relationship between substrate stiffness and EndMT has been rarely studied. Here, ECs were cultured on the (poly(L-lysine)/hyaluronate acid) (PLL/HA) multilayer films with controlled stiffness for 2 weeks, and their EndMT behaviors were studied. We demonstrated that ECs lost their markers (vWf and CD31) in a stiffness-dependent manner even without supplement of growth factors, and the softer film favored the maintaining of EC phenotype. Further, induced by transforming growth factor β1 (TGF-β1), ECs underwent EndMT, as characterized by losing their typical cobblestone morphology and markers and gaining smooth muscle cell markers (α-smooth muscle actin and calponin). Interestingly, stronger EndMT was observed when ECs were cultured on the stiffer film. Collectively, our findings suggest that substrate stiffness has significant effects on EndMT, and a softer substrate is beneficial to ECs by keeping their phenotype and inhibiting EndMT, which presents a new strategy for surface design of vascular implant materials. PMID:26477358

  15. Titanium dioxide nanoparticles increase inflammatory responses in vascular endothelial cells.

    PubMed

    Han, Sung Gu; Newsome, Bradley; Hennig, Bernhard

    2013-04-01

    Atherosclerosis is a chronic inflammatory disease that remains the leading cause of death in the United States. Numerous risk factors for endothelial cell inflammation and the development of atherosclerosis have been identified, including inhalation of ultrafine particles. Recently, engineered nanoparticles (NPs) such as titanium (TiO2) NPs have attracted much attention due to their wide range of applications. However, there are also great concerns surrounding potential adverse health effects in vascular systems. Although TiO2 NPs are known to induce oxidative stress and inflammation, the associated signaling pathways have not been well studied. The focus of this work, therefore, deals with examination of the cellular signaling pathways responsible for TiO2 NP-induced endothelial oxidative stress and inflammation. In this study, primary vascular endothelial cells were treated with TiO2 NPs for 2-16h at concentrations of 0-50 μg/mL. TiO2 NP exposure increased cellular oxidative stress and DNA binding of NF-κB. Further, phosphorylation of Akt, ERK, JNK and p38 was increased in cells exposed to TiO2 NPs. TiO2 NPs also significantly increased induction of mRNA and protein levels of vascular cell adhesion molecule-1 (VCAM-1) and mRNA levels of monocyte chemoattractant protein-1 (MCP-1). Pretreatment with inhibitors for NF-κB (pyrrolidine dithiocarbamate), oxidative stress (epigallocatechin gallate and apocynin), Akt (LY294002), ERK (PD98059), JNK (SP600125) and p38 (SB203580) significantly attenuated TiO2 NP-induced MCP-1 and VCAM-1 gene expression. These data indicate that TiO2 NPs can induce endothelial inflammatory responses via redox-sensitive cellular signaling pathways.

  16. The fundamental role of endothelial cells in hantavirus pathogenesis

    PubMed Central

    Hepojoki, Jussi; Vaheri, Antti; Strandin, Tomas

    2014-01-01

    Hantavirus, a genus of rodent- and insectivore-borne viruses in the family Bunyaviridae, is a group of emerging zoonotic pathogens. Hantaviruses cause hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome in man, often with severe consequences. Vascular leakage is evident in severe hantavirus infections, and increased permeability contributes to the pathogenesis. This review summarizes the current knowledge on hantavirus interactions with hematopoietic and endothelial cells, and their effects on the increased vascular permeability. PMID:25566236

  17. Endothelial cell proliferation associated with lesions of murine systemic candidiasis.

    PubMed Central

    Ashman, R B; Papadimitriou, J M

    1994-01-01

    Neovascularization is associated with tumor growth and some inflammatory diseases but has not been reported to be induced by infectious agents. In a mouse model of systemic Candida albicans infection, extensive endothelial cell proliferation was seen in the periphery of brain abscesses and in the areas of fungal pyelonephritis in the kidney. This finding is important for an understanding of the pathogenesis of fungal infections and may contribute to an analysis of the mechanisms of angiogenesis. Images PMID:7523305

  18. Fibroblast growth factor rescues brain endothelial cells lacking presenilin 1 from apoptotic cell death following serum starvation

    PubMed Central

    Gama Sosa, Miguel A.; De Gasperi, Rita; Hof, Patrick R.; Elder, Gregory A.

    2016-01-01

    Presenilin 1 (Psen1) is important for vascular brain development and is known to influence cellular stress responses. To understand the role of Psen1 in endothelial stress responses, we investigated the effects of serum withdrawal on wild type (wt) and Psen1−/− embryonic brain endothelial cells. Serum starvation induced apoptosis in Psen1−/− cells but did not affect wt cells. PI3K/AKT signaling was reduced in serum-starved Psen1−/− cells, and this was associated with elevated levels of phospho-p38 consistent with decreased pro-survival AKT signaling in the absence of Psen1. Fibroblast growth factor (FGF1 and FGF2), but not vascular endothelial growth factor (VEGF) rescued Psen1−/− cells from serum starvation induced apoptosis. Inhibition of FGF signaling induced apoptosis in wt cells under serum withdrawal, while blocking γ-secretase activity had no effect. In the absence of serum, FGF2 immunoreactivity was distributed diffusely in cytoplasmic and nuclear vesicles of wt and Psen1−/− cells, as levels of FGF2 in nuclear and cytosolic fractions were not significantly different. Thus, sensitivity of Psen1−/− cells to serum starvation is not due to lack of FGF synthesis but likely to effects of Psen1 on FGF release onto the cell surface and impaired activation of the PI3K/AKT survival pathway. PMID:27443835

  19. Endothelial cells downregulate apolipoprotein D expression in mural cells through paracrine secretion and Notch signaling

    PubMed Central

    Pajaniappan, Mohanasundari; Glober, Nancy K.; Kennard, Simone; Liu, Hua; Zhao, Ning

    2011-01-01

    Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion. PMID:21705670

  20. Endothelial cells downregulate apolipoprotein D expression in mural cells through paracrine secretion and Notch signaling.

    PubMed

    Pajaniappan, Mohanasundari; Glober, Nancy K; Kennard, Simone; Liu, Hua; Zhao, Ning; Lilly, Brenda

    2011-09-01

    Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion.

  1. 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.

  2. The Neurorepellent Slit2 Inhibits Postadhesion Stabilization of Monocytes Tethered to Vascular Endothelial Cells.

    PubMed

    Mukovozov, Ilya; Huang, Yi-Wei; Zhang, Qiuwang; Liu, Guang Ying; Siu, Allan; Sokolskyy, Yaroslav; Patel, Sajedabanu; Hyduk, Sharon J; Kutryk, Michael J B; Cybulsky, Myron I; Robinson, Lisa A

    2015-10-01

    The secreted neurorepellent Slit2, acting through its transmembrane receptor, Roundabout (Robo)-1, inhibits chemotaxis of varied cell types, including leukocytes, endothelial cells, and vascular smooth muscle cells, toward diverse attractants. The role of Slit2 in regulating the steps involved in recruitment of monocytes in vascular inflammation is not well understood. In this study, we showed that Slit2 inhibited adhesion of monocytic cells to activated human endothelial cells, as well as to immobilized ICAM-1 and VCAM-1. Microfluidic live cell imaging showed that Slit2 inhibited the ability of monocytes tethered to endothelial cells to stabilize their actin-associated anchors and to resist detachment in response to increasing shear forces. Transfection of constitutively active plasmids revealed that Slit2 inhibited postadhesion stabilization of monocytes on endothelial cells by preventing activation of Rac1. We further found that Slit2 inhibited chemotaxis of monocytes toward CXCL12 and CCL2. To determine whether Slit2 and Robo-1 modulate pathologic monocyte recruitment associated with vascular inflammation and cardiovascular disease, we tested PBMC from patients with coronary artery disease. PBMC from these patients had reduced surface levels of Robo-1 compared with healthy age- and sex-matched subjects, and Slit2 failed to inhibit chemotaxis of PBMC of affected patients, but not healthy control subjects, toward CCL2. Furthermore, administration of Slit2 to atherosclerosis-prone LDL receptor-deficient mice inhibited monocyte recruitment to nascent atherosclerotic lesions. These results demonstrate that Slit2 inhibits chemotaxis of monocytes, as well as their ability to stabilize adhesions and resist detachment forces. Slit2 may represent a powerful new tool to inhibit pathologic monocyte recruitment in vascular inflammation and atherosclerosis.

  3. Angiocrine functions of organ-specific endothelial cells

    PubMed Central

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

    2016-01-01

    Preface Endothelial cells lining blood vessel capillaries are not just passive conduits for delivering blood. Tissue-specific endothelium establish specialized vascular niches that deploy specific sets of growth factors, known as angiocrine factors, which actively participate in inducing, specifying, patterning, and guiding organ regeneration and maintaining homeostasis and metabolism. Angiocrine factors upregulated in response to injury orchestrates self-renewal and differentiation of tissue-specific repopulating resident stem and progenitor cells into functional organs. Uncovering the precise mechanisms whereby physiological-levels of angiocrine factors are spatially and temporally produced, and distributed by organotypic endothelium to repopulating cells, will lay the foundation for driving organ repair without scarring. PMID:26791722

  4. Endothelial cell metabolism in normal and diseased vasculature

    PubMed Central

    Eelen, Guy; de Zeeuw, Pauline; Simons, Michael; Carmeliet, Peter

    2015-01-01

    Higher organisms rely on a closed cardiovascular circulatory system with blood vessels supplying vital nutrients and oxygen to distant tissues. Not surprisingly, vascular pathologies rank among the most life-threatening diseases. At the crux of most of these vascular pathologies are (dysfunctional) endothelial cells (ECs), the cells lining the blood vessel lumen. ECs display the remarkable capability to switch rapidly from a quiescent state to a highly migratory and proliferative state during vessel sprouting. This angiogenic switch has long been considered to be dictated by angiogenic growth factors (eg vascular endothelial growth factor; VEGF) and other signals (eg Notch) alone, but recent findings show that it is also driven by a metabolic switch in ECs. Furthermore, these changes in metabolism may even override signals inducing vessel sprouting. Here, we review how EC metabolism differs between the normal and dysfunctional/diseased vasculature and how it relates to or impacts the metabolism of other cell types contributing to the pathology. We focus on the biology of ECs in tumor blood vessel and diabetic ECs in atherosclerosis as examples of the role of endothelial metabolism in key pathological processes. Finally, current as well as unexplored ‘EC metabolism’-centric therapeutic avenues are discussed. PMID:25814684

  5. Shear-Induced Nitric Oxide Production by Endothelial Cells.

    PubMed

    Sriram, Krishna; Laughlin, Justin G; Rangamani, Padmini; Tartakovsky, Daniel M

    2016-07-12

    We present a biochemical model of the wall shear stress-induced activation of endothelial nitric oxide synthase (eNOS) in an endothelial cell. The model includes three key mechanotransducers: mechanosensing ion channels, integrins, and G protein-coupled receptors. The reaction cascade consists of two interconnected parts. The first is rapid activation of calcium, which results in formation of calcium-calmodulin complexes, followed by recruitment of eNOS from caveolae. The second is phosphorylation of eNOS by protein kinases PKC and AKT. The model also includes a negative feedback loop due to inhibition of calcium influx into the cell by cyclic guanosine monophosphate (cGMP). In this feedback, increased nitric oxide (NO) levels cause an increase in cGMP levels, so that cGMP inhibition of calcium influx can limit NO production. The model was used to predict the dynamics of NO production by an endothelial cell subjected to a step increase of wall shear stress from zero to a finite physiologically relevant value. Among several experimentally observed features, the model predicts a highly nonlinear, biphasic transient behavior of eNOS activation and NO production: a rapid initial activation due to the very rapid influx of calcium into the cytosol (occurring within 1-5 min) is followed by a sustained period of activation due to protein kinases. PMID:27410748

  6. The soyabean isoflavone genistein modulates endothelial cell behaviour.

    PubMed

    Sandoval, Marisa J; Cutini, Pablo H; Rauschemberger, María Belén; Massheimer, Virginia L

    2010-07-01

    The aim of the present study was to investigate the direct action of the phyto-oestrogen genistein (Gen) on vascular endothelial behaviour, either in the presence or absence of proinflammatory agents. In rat aortic endothelial cell (EC) cultures, 24 h of treatment with Gen significantly increased cell proliferation in a wide range of concentration (0.001-10 nm). This mitogenic action was prevented by the oestrogen receptor (ER) antagonist ICI 182780 or by the presence of the specific NO synthase inhibitor l-nitro-arginine methyl ester. When monocytes adhesion to EC was measured, Gen partially attenuated leucocyte adhesion not only under basal conditions, but also in the presence of bacterial lipopolysaccharides (LPS). The effect of the phyto-oestrogen on the expression of EC adhesion molecules was evaluated. Gen down-regulated the enhancement in mRNA levels of E-selectin, vascular cell adhesion molecule-1 and P-selectin elicited by the proinflammatory agent bacterial LPS. The regulation of EC programmed death induced by the isoflavone was also demonstrated. Incubation with 10 nm Gen prevented DNA fragmentation induced by the apoptosis inductor H2O2. The results presented suggest that Gen would exert a protective effect on vascular endothelium, due to its regulatory action on endothelial proliferation, apoptosis and leucocyte adhesion, events that play a critical role in vascular diseases. The molecular mechanism displayed by the phyto-oestrogen involved the participation of the ER and the activation of the NO pathway.

  7. Decrease of fibrinolytic activity in human endothelial cells by arsenite.

    PubMed

    Jiang, Shinn-Jong; Lin, Tsun-Mei; Wu, Hua-Lin; Han, Huai-Song; Shi, Guey-Yueh

    2002-01-01

    Blackfoot disease (BFD) is an endemic peripheral vascular occlusive disease that occurred in the southwest coast of Taiwan. It is believed that arsenic in the drinking water from artesian wells plays an important role in the development of the disease. We have previously shown that BFD patients had significant lower tissue-type plasminogen activator (t-PA) antigen level and higher plasminogen activator inhibitor, Type 1 (PAI-1) antigen level than normal controls. The purpose of this study was to investigate the effects of arsenite on the fibrinolytic and anticoagulant activities of cultured macrovascular and microvascular endothelial cells. Incubation of human microvascular endothelial cells (HMEC-1), but not human umbilical vein endothelial cells (HUVECs), with arsenite caused a decrease of t-PA mRNA level, a rise of both PAI-1 mRNA level and PAI activity. Arsenite could also inhibit the thrombomodulin (TM) mRNA expression and reduce the TM antigen level in HMEC-1. In conclusion, arsenite had a greater effect on HMEC-1 as compared to HUVECs in lowering the fibrinolytic activity and may be responsible for the reduced capacity of fibrinolysis associated with BFD.

  8. Interaction of recombinant octameric hemoglobin with endothelial cells.

    PubMed

    Gaucher, Caroline; Domingues-Hamdi, Élisa; Prin-Mathieu, Christine; Menu, Patrick; Baudin-Creuza, Véronique

    2015-02-01

    Hemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbβG83C. We investigate the potential side effects of rHbβG83C on endothelial cells. The rHbβG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbβG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb βG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.

  9. Homocysteine injures vascular endothelial cells by inhibiting mitochondrial activity

    PubMed Central

    Yang, Fengyong; Qi, Xiujing; Gao, Zheng; Yang, Xingju; Zheng, Xingfeng; Duan, Chonghao; Zheng, Jian

    2016-01-01

    The aim of the present study was to investigate the role of homocysteine (Hcy) in the pathogenesis of pulmonary embolism (PE) and the associated molecular mechanisms in human umbilical vein endothelial cells (HUVECs). Hcy contents were detected with high-performance liquid chromatography. Apoptosis was detected by flow cytometry using Annexin-V staining. Cytochrome c oxidase (COX) activity was assessed with an enzyme activity assay, and the expression levels of COX 17 were determined by western blot analysis. Intracellular reactive oxygen species levels were measured using a microplate reader with a fluorescence probe. The results demonstrated that, compared with the control group, the serum Hcy levels were significantly elevated in the PE group, suggesting that Hcy may be an indicator for PE. Following treatment with Hcy, the apoptosis rate was markedly elevated in HUVECs. Moreover, Hcy decreased COX activity and downregulated the expression of COX 17 in HUVECs. Furthermore, Hcy increased the ROS levels in these endothelial cells. However, all the above-mentioned physiopathological changes induced by Hcy in HUVECs could be restored by folic acid. In conclusion, the results of the present study demonstrated that Hcy inhibited COX activity, downregulated COX 17 expression, increased intracellular ROS levels and enhanced apoptosis in endothelial cells.

  10. Single-cell analysis of endothelial morphogenesis in vivo

    PubMed Central

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

    2015-01-01

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

  11. Vascular endothelial platelet endothelial cell adhesion molecule 1 (PECAM-1) regulates advanced metastatic progression

    PubMed Central

    DeLisser, Horace; Liu, Yong; Desprez, Pierre-Yves; Thor, Ann; Briasouli, Paraskevei; Handumrongkul, Chakrapong; Wilfong, Jonathon; Yount, Garret; Nosrati, Mehdi; Fong, Sylvia; Shtivelman, Emma; Fehrenbach, Melane; Cao, Gaoyuan; Moore, Dan H.; Nayak, Shruti; Liggitt, Denny; Kashani-Sabet, Mohammed; Debs, Robert

    2010-01-01

    Most patients who die from cancer succumb to treatment-refractory advanced metastatic progression. Although the early stages of tumor metastasis result in the formation of clinically silent micrometastatic foci, its later stages primarily reflect the progressive, organ-destructive growth of already advanced metastases. Early-stage metastasis is regulated by multiple factors within tumor cells as well as by the tumor microenvironment (TME). In contrast, the molecular determinants that control advanced metastatic progression remain essentially uncharacterized, precluding the development of therapies targeted against it. Here we show that the TME, functioning in part through platelet endothelial cell adhesion molecule 1 (PECAM-1), drives advanced metastatic progression and is essential for progression through its preterminal end stage. PECAM-1–KO and chimeric mice revealed that its metastasis-promoting effects are mediated specifically through vascular endothelial cell (VEC) PECAM-1. Anti–PECAM-1 mAb therapy suppresses both end-stage metastatic progression and tumor-induced cachexia in tumor-bearing mice. It reduces proliferation, but not angiogenesis or apoptosis, within advanced tumor metastases. Because its antimetastatic effects are mediated by binding to VEC rather than to tumor cells, anti–PECAM-1 mAb appears to act independently of tumor type. A modified 3D coculture assay showed that anti–PECAM-1 mAb inhibits the proliferation of PECAM-1–negative tumor cells by altering the concentrations of secreted factors. Our studies indicate that a complex interplay between elements of the TME and advanced tumor metastases directs end-stage metastatic progression. They also suggest that some therapeutic interventions may target late-stage metastases specifically. mAb-based targeting of PECAM-1 represents a TME-targeted therapeutic approach that suppresses the end stages of metastatic progression, until now a refractory clinical entity. PMID:20926749

  12. Deleterious effects of endotoxin on cultured endothelial cells: an in vitro model of vascular injury

    SciTech Connect

    Yamada, O.; Moldow, C.F.; Sacks, T.; Craddock, P.R.; Boogaerts, M.A.; Jacob, H.S.

    1981-06-01

    The effects of endotoxin-triggered granulocytes on the viability of endothelial cells in vitro was investigated. Endotoxin or its lipid A component caused granulocytes to adhere to and significantly damage cultured endothelial cells. Fresh serum is not necessary but does amplify both adherence and endothelial injury. Much of the endothelial injury was inhibited by free-radical scavengers or by blocking granulocyte adhesion to endothelial cells and appears to result from free radical production by the stimulated granulocyte. Studies in this model suggest a pathogenic role for the endotoxin-triggered granulocyte in the Shwartzman reaction and perhaps related clinical disorders.

  13. Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    NASA Technical Reports Server (NTRS)

    Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1998-01-01

    Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

  14. Biomarkers of endothelial cell activation in early sepsis

    PubMed Central

    Skibsted, Simon; Jones, Alan E; Puskarich, Michael A.; Arnold, Ryan; Sherwin, Robert; Trzeciak, Stephen; Schuetz, Philipp; Aird, William C.; Shapiro, Nathan I

    2013-01-01

    PURPOSE To investigate the association of endothelial-related markers with organ dysfunction and in-hospital mortality to validate our earlier findings in a multicenter study. We hypothesize that: 1) endothelial biomarkers will be associated with organ dysfunction and mortality in sepsis; and, that sFlt-1, holds promise as novel prognostic markers in sepsis. METHODS A prospective, multicenter, observational study of a convenience sample of Emergency Department (ED) patients with a suspected infection presenting to one of four urban, academic medical center EDs between January 2009 and January 2010. We collected plasma while the patients were in the ED, and subsequently assayed endothelial-related biomarkers, namely sFlt-1, sE-Selectin, sICAM-1, sVCAM-1, and PAI-1. Outcomes were organ dysfunction and in-hospital mortality. RESULTS We enrolled at a total of 166 patients: 63 with sepsis (38%), 61 with severe sepsis (37%) and 42 with septic shock (25%). All endothelial biomarkers were significantly associated with sepsis severity, P < 0.002. We found a significant inter-correlation between all biomarkers, strongest between sFlt1 and PAI-1 (r=0.61, P < 0.001) and PAI-1 and sE-selectin and sICAM-1 (r=0.49, P < 0.001). Among the endothelial biomarkers, sFlt-1 had the strongest association with SOFA score (r=0.58, P < 0.001). sFlt-1 and PAI-1 had the highest area under the operating receiver characteristic curve for mortality of 0.87. CONCLUSIONS This multi-center validation study confirms that markers of endothelial activation are associated with sepsis severity, organ dysfunction and mortality in sepsis. This supports the hypothesis that the endothelium plays a central role in the pathophysiology of sepsis and may serve as a more accurate prediction tool and a target for therapies aimed at ameliorating endothelial cell dysfunction. Additionally, sFLT-1 holds promise as a novel sepsis severity biomarker. PMID:23524845

  15. In vitro differentiation of porcine aortic vascular precursor cells to endothelial and vascular smooth muscle cells.

    PubMed

    Zaniboni, Andrea; Bernardini, Chiara; Bertocchi, Martina; Zannoni, Augusta; Bianchi, Francesca; Avallone, Giancarlo; Mangano, Chiara; Sarli, Giuseppe; Calzà, Laura; Bacci, Maria Laura; Forni, Monica

    2015-09-01

    Recent findings suggest that progenitor and multipotent mesenchymal stromal cells (MSCs) are associated with vascular niches. Cells displaying mesenchymal properties and differentiating to whole components of a functional blood vessel, including endothelial and smooth muscle cells, can be defined as vascular stem cells (VSCs). Recently, we isolated a population of porcine aortic vascular precursor cells (pAVPCs), which have MSC- and pericyte-like properties. The aim of the present work was to investigate whether pAVPCs possess VSC-like properties and assess their differentiation potential toward endothelial and smooth muscle lineages. pAVPCs, maintained in a specific pericyte growth medium, were cultured in high-glucose DMEM + 10% FBS (long-term medium, LTM) or in human endothelial serum-free medium + 5% FBS and 50 ng/ml of hVEGF (endothelial differentiation medium, EDM). After 21 days of culture in LTM, pAVPCs showed an elongated fibroblast-like morphology, and they seem to organize in cord-like structures. qPCR analysis of smooth muscle markers [α-smooth muscle actin (α-SMA), calponin, and smooth muscle myosin (SMM) heavy chain] showed a significant increment of the transcripts, and immunofluorescence analysis confirmed the presence of α-SMA and SMM proteins. After 21 days of culture in EDM, pAVPCs displayed an endothelial cell-like morphology and revealed the upregulation of the expression of endothelial markers (CD31, vascular endothelial-cadherin, von Willebrand factor, and endothelial nitric oxide synthase) showing the CD31-typical pattern. In conclusion, pAVPCs could be defined as a VSC-like population considering that, if they are maintained in a specific pericyte medium, they express MSC markers, and they have, in addition to the classical mesenchymal trilineage differentiation potential, the capacity to differentiate in vitro toward the smooth muscle and the endothelial cell phenotypes.

  16. 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

  17. The role of BMPs in endothelial cell function and dysfunction.

    PubMed

    Dyer, Laura A; Pi, Xinchun; Patterson, Cam

    2014-09-01

    The bone morphogenetic protein (BMP) family of proteins has a multitude of roles throughout the body. In embryonic development, BMPs promote endothelial specification and subsequent venous differentiation. The BMP pathway also plays important roles in the adult vascular endothelium, promoting angiogenesis and mediating shear and oxidative stress. The canonical BMP pathway functions through the Smad transcription factors; however, other intracellular signaling cascades can be activated, and receptor complexes beyond the traditional type I and type II receptors add additional layers of regulation. Dysregulated BMP signaling has been linked to vascular diseases including pulmonary hypertension and atherosclerosis. This review addresses recent advances in the roles of BMP signaling in the endothelium and how BMPs affect endothelial dysfunction and human disease. PMID:24908616

  18. In Vitro Expansion of Corneal Endothelial Cells on Biomimetic Substrates

    PubMed Central

    Palchesko, Rachelle N.; Lathrop, Kira L.; Funderburgh, James L.; Feinberg, Adam W.

    2015-01-01

    Corneal endothelial (CE) cells do not divide in vivo, leading to edema, corneal clouding and vision loss when the density drops below a critical level. The endothelium can be replaced by transplanting allogeneic tissue; however, access to donated tissue is limited worldwide resulting in critical need for new sources of corneal grafts. In vitro expansion of CE cells is a potential solution, but is challenging due to limited proliferation and loss of phenotype in vitro via endothelial to mesenchymal transformation (EMT) and senescence. We hypothesized that a bioengineered substrate recapitulating chemo-mechanical properties of Descemet's membrane would improve the in vitro expansion of CE cells while maintaining phenotype. Results show that bovine CE cells cultured on a polydimethylsiloxane surface with elastic modulus of 50 kPa and collagen IV coating achieved >3000-fold expansion. Cells grew in higher-density monolayers with polygonal morphology and ZO-1 localization at cell-cell junctions in contrast to control cells on polystyrene that lost these phenotypic markers coupled with increased α-smooth muscle actin expression and fibronectin fibril assembly. In total, these results demonstrate that a biomimetic substrate presenting native basement membrane ECM proteins and mechanical environment may be a key element in bioengineering functional CE layers for potential therapeutic applications. PMID:25609008

  19. In vitro expansion of corneal endothelial cells on biomimetic substrates.

    PubMed

    Palchesko, Rachelle N; Lathrop, Kira L; Funderburgh, James L; Feinberg, Adam W

    2015-01-01

    Corneal endothelial (CE) cells do not divide in vivo, leading to edema, corneal clouding and vision loss when the density drops below a critical level. The endothelium can be replaced by transplanting allogeneic tissue; however, access to donated tissue is limited worldwide resulting in critical need for new sources of corneal grafts. In vitro expansion of CE cells is a potential solution, but is challenging due to limited proliferation and loss of phenotype in vitro via endothelial to mesenchymal transformation (EMT) and senescence. We hypothesized that a bioengineered substrate recapitulating chemo-mechanical properties of Descemet's membrane would improve the in vitro expansion of CE cells while maintaining phenotype. Results show that bovine CE cells cultured on a polydimethylsiloxane surface with elastic modulus of 50 kPa and collagen IV coating achieved >3000-fold expansion. Cells grew in higher-density monolayers with polygonal morphology and ZO-1 localization at cell-cell junctions in contrast to control cells on polystyrene that lost these phenotypic markers coupled with increased α-smooth muscle actin expression and fibronectin fibril assembly. In total, these results demonstrate that a biomimetic substrate presenting native basement membrane ECM proteins and mechanical environment may be a key element in bioengineering functional CE layers for potential therapeutic applications. PMID:25609008

  20. WR-1065 and radioprotection of vascular endothelial cells. I. Cell proliferation, DNA synthesis and damage

    SciTech Connect

    Rubin, D.B.; Drab, E.A.; Kang, H.J.; Baumann, F.E.; Blazek, E.R.

    1996-02-01

    Normal tissue toxicity limits radiation therapy and could depend on the extent of damage to the vascular endothelium. Aminothiols such as WR-1065 [N-(2-mercaptoethyl)-1,3-diaminopropane] provide radioprotection for normal tissues, but little is known about how the aminothiols specifically affect the endothelium. Bovine aortic endothelial cells in culture were exposed to WR-1065 for 2 h before irradiation ({sup 137}Cs {gamma} rays, 1 Gy/min). Alone, WR-1065 demonstrated an antiproliferative effect that was related to dose (0.5-4 mM) and was evident by lowered counts of adherent cells 48 h after exposure. WR-1065 was clearly radioprotective when assessed by colony formation and incorporation of [{sup 3}H]thymidine. However, when the number of adherent cells was evaluated, radioprotection appeared to be slight and evident only in logarithmically growing cells. WR-1065 at 2 mM suppressed single-strand DNA breaks after 3 Gy by 22% and double-strand breaks after 9 Gy by 47%. Also in the irradiated cells, WR-1065 more than doubled the rate of progression of cells from G{sub 1} to S phase. WR-1065 pretreatment elevated cellular glutathione (GSH) content more than twofold. Although pretreatment with buthionine sulfoximine inhibited the elevation of GSH, the radioprotective impact of WR-1065 on total DNA strand breaks and colony formation was unaffected. These results suggest that WR-1065 may enable tissue recovery from irradiation by promoting the replication of endothelial cells, possibly by mechanisms independent of GSH. 46 refs., 6 figs., 2 tabs.

  1. The Interaction between Fluid Wall Shear Stress and Solid Circumferential Strain Affects Endothelial Gene Expression.

    PubMed

    Amaya, Ronny; Pierides, Alexis; Tarbell, John M

    2015-01-01

    Endothelial cells lining the walls of blood vessels are exposed simultaneously to wall shear stress (WSS) and circumferential stress (CS) that can be characterized by the temporal phase angle between WSS and CS (stress phase angle - SPA). Regions of the circulation with highly asynchronous hemodynamics (SPA close to -180°) such as coronary arteries are associated with the development of pathological conditions such as atherosclerosis and intimal hyperplasia whereas more synchronous regions (SPA closer to 0°) are spared of disease. The present study evaluates endothelial cell gene expression of 42 atherosclerosis-related genes under asynchronous hemodynamics (SPA=-180 °) and synchronous hemodynamics (SPA=0 °). This study used a novel bioreactor to investigate the cellular response of bovine aortic endothelial cells (BAECS) exposed to a combination of pulsatile WSS and CS at SPA=0 or SPA=-180. Using a PCR array of 42 genes, we determined that BAECS exposed to non-reversing sinusoidal WSS (10±10 dyne/cm2) and CS (4 ± 4%) over a 7 hour testing period displayed 17 genes that were up regulated by SPA = -180 °, most of them pro-atherogenic, including NFκB and other NFκB target genes. The up regulation of NFκB p50/p105 and p65 by SPA =-180° was confirmed by Western blots and immunofluorescence staining demonstrating the nuclear translocation of NFκB p50/p105 and p65. These data suggest that asynchronous hemodynamics (SPA=-180 °) can elicit proatherogenic responses in endothelial cells compared to synchronous hemodynamics without shear stress reversal, indicating that SPA may be an important parameter characterizing arterial susceptibility to disease.

  2. The Interaction between Fluid Wall Shear Stress and Solid Circumferential Strain Affects Endothelial Gene Expression

    PubMed Central

    Amaya, Ronny; Pierides, Alexis; Tarbell, John M.

    2015-01-01

    Endothelial cells lining the walls of blood vessels are exposed simultaneously to wall shear stress (WSS) and circumferential stress (CS) that can be characterized by the temporal phase angle between WSS and CS (stress phase angle – SPA). Regions of the circulation with highly asynchronous hemodynamics (SPA close to -180°) such as coronary arteries are associated with the development of pathological conditions such as atherosclerosis and intimal hyperplasia whereas more synchronous regions (SPA closer to 0°) are spared of disease. The present study evaluates endothelial cell gene expression of 42 atherosclerosis-related genes under asynchronous hemodynamics (SPA=-180 °) and synchronous hemodynamics (SPA=0 °). This study used a novel bioreactor to investigate the cellular response of bovine aortic endothelial cells (BAECS) exposed to a combination of pulsatile WSS and CS at SPA=0 or SPA=-180. Using a PCR array of 42 genes, we determined that BAECS exposed to non-reversing sinusoidal WSS (10±10 dyne/cm2) and CS (4 ± 4 %) over a 7 hour testing period displayed 17 genes that were up regulated by SPA = -180 °, most of them pro-atherogenic, including NFκB and other NFκB target genes. The up regulation of NFκB p50/p105 and p65 by SPA =-180° was confirmed by Western blots and immunofluorescence staining demonstrating the nuclear translocation of NFκB p50/p105 and p65. These data suggest that asynchronous hemodynamics (SPA=-180 °) can elicit proatherogenic responses in endothelial cells compared to synchronous hemodynamics without shear stress reversal, indicating that SPA may be an important parameter characterizing arterial susceptibility to disease. PMID:26147292

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

    PubMed

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

    2015-01-01

    Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing. PMID:26436434

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. Overexpression of Ref-1 Inhibits Lead-induced Endothelial Cell Death via the Upregulation of Catalase.

    PubMed

    Lee, Kwon Ho; Lee, Sang Ki; Kim, Hyo Shin; Cho, Eun Jung; Joo, Hee Kyoung; Lee, Eun Ji; Lee, Ji Young; Park, Myoung Soo; Chang, Seok Jong; Cho, Chung-Hyun; Park, Jin Bong; Jeon, Byeong Hwa

    2009-12-01

    The role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the lead (Pb)-induced cellular response was investigated in the cultured endothelial cells. Pb caused progressive cellular death in endothelial cells, which occurred in a concentration- and time-dependent manner. However, Ref-1 overexpression with AdRef-1 significantly inhibited Pb-induced cell death in the endothelial cells. Also the overexpression of Ref-1 significantly suppressed Pb-induced superoxide and hydrogen peroxide elevation in the endothelial cells. Pb exposure induced the downregulation of catalase, it was inhibited by the Ref-1 overexpression in the endothelial cells. Taken together, our data suggests that the overexpression of Ref-1 inhibited Pb-induced cell death via the upregulation of catalase in the cultured endothelial cells.

  7. 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

  8. Overexpression of Ref-1 Inhibits Lead-induced Endothelial Cell Death via the Upregulation of Catalase

    PubMed Central

    Lee, Kwon Ho; Lee, Sang Ki; Kim, Hyo Shin; Cho, Eun Jung; Joo, Hee Kyoung; Lee, Eun Ji; Lee, Ji Young; Park, Myoung Soo; Chang, Seok Jong; Cho, Chung-Hyun; Park, Jin Bong

    2009-01-01

    The role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the lead (Pb)-induced cellular response was investigated in the cultured endothelial cells. Pb caused progressive cellular death in endothelial cells, which occurred in a concentration- and time-dependent manner. However, Ref-1 overexpression with AdRef-1 significantly inhibited Pb-induced cell death in the endothelial cells. Also the overexpression of Ref-1 significantly suppressed Pb-induced superoxide and hydrogen peroxide elevation in the endothelial cells. Pb exposure induced the downregulation of catalase, it was inhibited by the Ref-1 overexpression in the endothelial cells. Taken together, our data suggests that the overexpression of Ref-1 inhibited Pb-induced cell death via the upregulation of catalase in the cultured endothelial cells. PMID:20054488

  9. Effects of verteporfin-mediated photodynamic therapy on endothelial cells

    NASA Astrophysics Data System (ADS)

    Kraus, Daniel; Chen, Bin

    2015-03-01

    Photodynamic therapy (PDT) is a treatment modality in which cytotoxic reactive oxygen species are generated from oxygen and other biological molecules when a photosensitizer is activated by light. PDT has been approved for the treatment of cancers and age-related macular degeneration (AMD) due to its effectiveness in cell killing and manageable normal tissue complications. In this study, we characterized the effects of verteporfin-PDT on SVEC mouse endothelial cells and determined its underlying cell death mechanisms. We found that verteporfin was primarily localized in mitochondria and endoplasmic reticulum (ER) in SVEC cells. Light treatment of photosensitized SVEC cells induced a rapid onset of cell apoptosis. In addition to significant structural damages to mitochondria and ER, verteporfin-PDT caused substantial degradation of ER signaling molecules, suggesting ER stress. These results demonstrate that verteporfin-PDT triggered SVEC cell apoptosis by both mitochondrial and ER stress pathways. Results from this study may lead to novel therapeutic approaches to enhance PDT outcome.

  10. Endothelialization of Magnetic Graft Materials using SPION-labeled Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Newman, Brant R.; Dragomir-Daescu, Dan; Harbuzariu, Adriana; McIntosh, Malcolm; Harburn, J. Jonathan; Parakka, Anthony; Kalra, Manju; Holmes, David; Simari, Robert D.; Sandhu, Gurpreet S.

    2010-12-01

    Seeding vascular grafts with autologous endothelial cells (EC) has been shown to improve in vivo patency, but high cost and development time have prevented widespread clinical use. A technique for loading EC with superparamagnetic iron-oxide nanospheres (SPIONs) was recently described. SPION-loaded EC experience magnetic attractive forces in the presence of sufficient magnetic field gradients. Using a multi-factorial design of experiments approach, the quantity and spatial distribution of magnetizable metal particles within a poly (ether urethane) matrix were systematically varied to produce unique material specimens. Specimens were seeded with SPION-loaded ECs, and cell coverage was quantified at various post-seeding time intervals using micrographic image analysis. The effects of changing design parameters on cell capture and sustained cell viability on magnetic substrates were statistically examined. Magnetized ferrites and samarium cobalt demonstrated cell capture, though cytotoxicity prevented sustained cell growth. Cobalt chromium substrates showed effective cell capture and growth to near complete confluence for up to one month.

  11. MicroRNA profiling of diverse endothelial cell types

    PubMed Central

    2011-01-01

    Background MicroRNAs are ~22-nt long regulatory RNAs that serve as critical modulators of post-transcriptional gene regulation. The diversity of miRNAs in endothelial cells (ECs) and the relationship of this diversity to epithelial and hematologic cells is unknown. We investigated the baseline miRNA signature of human ECs cultured from the aorta (HAEC), coronary artery (HCEC), umbilical vein (HUVEC), pulmonary artery (HPAEC), pulmonary microvasculature (HPMVEC), dermal microvasculature (HDMVEC), and brain microvasculature (HBMVEC) to understand the diversity of miRNA expression in ECs. Results We identified 166 expressed miRNAs, of which 3 miRNAs (miR-99b, miR-20b and let-7b) differed significantly between EC types and predicted EC clustering. We confirmed the significance of these miRNAs by RT-PCR analysis and in a second data set by Sylamer analysis. We found wide diversity of miRNAs between endothelial, epithelial and hematologic cells with 99 miRNAs shared across cell types and 31 miRNAs unique to ECs. We show polycistronic miRNA chromosomal clusters have common expression levels within a given cell type. Conclusions EC miRNA expression levels are generally consistent across EC types. Three microRNAs were variable within the dataset indicating potential regulatory changes that could impact on EC phenotypic differences. MiRNA expression in endothelial, epithelial and hematologic cells differentiate these cell types. This data establishes a valuable resource characterizing the diverse miRNA signature of ECs. PMID:22047531

  12. Atrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells

    PubMed Central

    Nojiri, Takashi; Hosoda, Hiroshi; Tokudome, Takeshi; Miura, Koichi; Ishikane, Shin; Otani, Kentaro; Kishimoto, Ichiro; Shintani, Yasushi; Inoue, Masayoshi; Kimura, Toru; Sawabata, Noriyoshi; Minami, Masato; Nakagiri, Tomoyuki; Funaki, Soichiro; Takeuchi, Yukiyasu; Maeda, Hajime; Kidoya, Hiroyasu; Kiyonari, Hiroshi; Shioi, Go; Arai, Yuji; Hasegawa, Takeshi; Takakura, Nobuyuki; Hori, Megumi; Ohno, Yuko; Miyazato, Mikiya; Mochizuki, Naoki; Okumura, Meinoshin; Kangawa, Kenji

    2015-01-01

    Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients; however, surgery is often accompanied with trauma, which can promote early recurrence by provoking detachment of tumor cells into the blood stream or inducing systemic inflammation or both. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to NPR1 [also called guanylyl cyclase-A (GC-A) receptor]. In mouse models, we found that metastasis of GC-A–nonexpressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specific GC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. We examined the effect of ANP on tumor metastasis in mice treated with lipopolysaccharide, which mimics systemic inflammation induced by surgical stress. ANP inhibited the adhesion of cancer cells to pulmonary arterial and micro-vascular endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells. PMID:25775533

  13. An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.

    PubMed

    Martinelli, Roberta; Carman, Christopher V

    2015-12-24

    Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells ('APCs') referred to as 'immunological synapses'. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of 'semi-professional APCs'. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic 'planar cellular APC model' for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

  14. Cytotoxicity of voriconazole on cultured human corneal endothelial cells.

    PubMed

    Han, Sang Beom; Shin, Young Joo; Hyon, Joon Young; Wee, Won Ryang

    2011-10-01

    The purpose of the present study was to evaluate the toxicity of voriconazole on cultured human corneal endothelial cells (HCECs). HCECs were cultured and exposed to various concentrations of voriconazole (5.0 to 1,000 μg/ml). Cell viability was measured using a Cell Counting Kit-8 (CCK-8) and live/dead viability/cytotoxicity assays. Cell damage was assessed using phase-contrast microscopy after 24 h of exposure to voriconazole. To analyze the effect of voriconazole on the intercellular barrier, immunolocalization of zonula occludens 1 (ZO1) was performed. A flow cytometric assay was performed to evaluate the apoptotic and necrotic effects of voriconazole on HCECs. Cytotoxicity tests demonstrated the dose-dependent toxic effect of voriconazole on HCECs. Voriconazole concentrations of ≥100 μg/ml led to a significant reduction in cell viability. The morphological characteristics of HCECs also changed in a dose-dependent manner. Increasing concentrations of voriconazole resulted in fading staining for ZO1. Higher concentrations of voriconazole resulted in an increased number of propidium iodide (PI)-positive cells, indicating activation of the proapoptotic pathway. In conclusion, voriconazole may have a dose-dependent toxic effect on cultured HCECs. The results of this study suggest that although voriconazole concentrations of up to 50 μg/ml do not decrease cell viability, intracameral voriconazole concentrations of ≥100 μg/ml may increase the risk of corneal endothelial damage.

  15. Thermal Pretreatment Improves Viability of Cryopreserved Human Endothelial Cells.

    PubMed

    Hofmann, Nicola; Sun, Huan; Chatterjee, Anamika; Saha, Debapriya; Glasmacher, Birgit

    2015-10-01

    A high survival rate of cryopreserved cells requires optimal cooling and thawing rates in the presence of a cryoprotective agent (CPA) or a combination of CPAs in adequate concentrations. One of the most widely used CPAs, dimethyl sulfoxide (Me2SO), however is toxic at high concentrations and has detrimental effects on cellular functions. Additional processing steps are necessary to remove the CPA after thawing, which make the process expensive and time consuming. Therefore it is of great interest to develop new cryoprotective strategies to replace the currently used CPAs or to reduce their concentration. The aim of this study was to investigate if thermal activation of human pulmonary microvascular endothelial cells (HPMEC ST-1.6R), prior to cryopreservation, could improve their post-thaw viability since the resulting heat shock protein expression acts as an intrinsic cellular protection mechanism. The results of this study suggest that both heat and cold shock pretreatments improve cryopreservation outcome of the HPMEC ST-1.6R cells. By re-cultivating cells after heat shock treatment before cryopreservation, a significant increase in cellular membrane integrity and adherence capacity could be achieved. However a combination of thermal activation and cryopreservation with alternative CPAs such as ectoine and L-proline could not further enhance the cell viability. The results of this study showed that pretreatment of endothelial cells with thermal activation could be used to reduce the Me2SO concentration required in order to preserve cell viability after cryopreservation. PMID:26419006

  16. Thermal Pretreatment Improves Viability of Cryopreserved Human Endothelial Cells.

    PubMed

    Hofmann, Nicola; Sun, Huan; Chatterjee, Anamika; Saha, Debapriya; Glasmacher, Birgit

    2015-10-01

    A high survival rate of cryopreserved cells requires optimal cooling and thawing rates in the presence of a cryoprotective agent (CPA) or a combination of CPAs in adequate concentrations. One of the most widely used CPAs, dimethyl sulfoxide (Me2SO), however is toxic at high concentrations and has detrimental effects on cellular functions. Additional processing steps are necessary to remove the CPA after thawing, which make the process expensive and time consuming. Therefore it is of great interest to develop new cryoprotective strategies to replace the currently used CPAs or to reduce their concentration. The aim of this study was to investigate if thermal activation of human pulmonary microvascular endothelial cells (HPMEC ST-1.6R), prior to cryopreservation, could improve their post-thaw viability since the resulting heat shock protein expression acts as an intrinsic cellular protection mechanism. The results of this study suggest that both heat and cold shock pretreatments improve cryopreservation outcome of the HPMEC ST-1.6R cells. By re-cultivating cells after heat shock treatment before cryopreservation, a significant increase in cellular membrane integrity and adherence capacity could be achieved. However a combination of thermal activation and cryopreservation with alternative CPAs such as ectoine and L-proline could not further enhance the cell viability. The results of this study showed that pretreatment of endothelial cells with thermal activation could be used to reduce the Me2SO concentration required in order to preserve cell viability after cryopreservation.

  17. A Comparison of Endothelial Cell Loss in Combined Cataract and MIGS (Hydrus) Procedure to Phacoemulsification Alone: 6-Month Results

    PubMed Central

    Fea, Antonio M.; Consolandi, Giulia; Pignata, Giulia; Cannizzo, Paola Maria Loredana; Lavia, Carlo; Billia, Filippo; Rolle, Teresa; Grignolo, Federico M.

    2015-01-01

    Purpose. To compare the corneal endothelial cell loss after phacoemulsification, alone or combined with microinvasive glaucoma surgery (MIGS), in nonglaucomatous versus primary open angle glaucoma (POAG) eyes affected by age-related cataract. Methods. 62 eyes of 62 patients were divided into group 1 (n = 25, affected by age-related cataract) and group 2 (n = 37, affected by age-related cataract and POAG). All patients underwent cataract surgery. Group 2 was divided into subgroups A (n = 19, cataract surgery alone) and B (n = 18, cataract surgery and MIGS). Prior to and 6 months after surgery the patients' endothelium was studied. Main outcomes were CD (cell density), SD (standard deviation), CV (coefficient of variation), and 6A (hexagonality coefficient) variations after surgeries. Results. There were no significant differences among the groups concerning preoperative endothelial parameters. The differences in CD before and after surgery were significant in all groups: 9.1% in group 1, 17.24% in group 2A, and 11.71% in group 2B. All endothelial parameters did not significantly change after surgery. Conclusions. Phacoemulsification determined a loss of endothelial cells in all groups. After surgery the change in endothelial parameters after MIGS was comparable to the ones of patients who underwent cataract surgery alone. PMID:26664740

  18. A Comparison of Endothelial Cell Loss in Combined Cataract and MIGS (Hydrus) Procedure to Phacoemulsification Alone: 6-Month Results.

    PubMed

    Fea, Antonio M; Consolandi, Giulia; Pignata, Giulia; Cannizzo, Paola Maria Loredana; Lavia, Carlo; Billia, Filippo; Rolle, Teresa; Grignolo, Federico M

    2015-01-01

    Purpose. To compare the corneal endothelial cell loss after phacoemulsification, alone or combined with microinvasive glaucoma surgery (MIGS), in nonglaucomatous versus primary open angle glaucoma (POAG) eyes affected by age-related cataract. Methods. 62 eyes of 62 patients were divided into group 1 (n = 25, affected by age-related cataract) and group 2 (n = 37, affected by age-related cataract and POAG). All patients underwent cataract surgery. Group 2 was divided into subgroups A (n = 19, cataract surgery alone) and B (n = 18, cataract surgery and MIGS). Prior to and 6 months after surgery the patients' endothelium was studied. Main outcomes were CD (cell density), SD (standard deviation), CV (coefficient of variation), and 6A (hexagonality coefficient) variations after surgeries. Results. There were no significant differences among the groups concerning preoperative endothelial parameters. The differences in CD before and after surgery were significant in all groups: 9.1% in group 1, 17.24% in group 2A, and 11.71% in group 2B. All endothelial parameters did not significantly change after surgery. Conclusions. Phacoemulsification determined a loss of endothelial cells in all groups. After surgery the change in endothelial parameters after MIGS was comparable to the ones of patients who underwent cataract surgery alone.

  19. PC12 Cells Differentiate into Chromaffin Cell-Like Phenotype in Coculture with Adrenal Medullary Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Mizrachi, Yaffa; Naranjo, Jose R.; Levi, Ben-Zion; Pollard, Harvey B.; Lelkes, Peter I.

    1990-08-01

    Previously we described specific in vitro interactions between PC12 cells, a cloned, catecholamine-secreting pheochromocytoma cell line derived from the rat adrenal medulla, and bovine adrenal medullary endothelial cells. We now demonstrate that these interactions induce the PC12 cells to acquire physical and biochemical characteristics reminiscent of chromaffin cells. Under coculture conditions involving direct cell-cell contact, the endothelial cells and the PC12 cells reduced their rates of proliferation; upon prolonged coculture PC12 cells clustered into nests of cells similar to the organization of chromaffin cells seen in vivo. Within 3 days in coculture with endothelial cells, but not with unrelated control cells, PC12 cells synthesized increased levels of [Met]enkephalin. In addition, PC12 cells, growing on confluent endothelial monolayers, failed to extend neurites in response to nerve growth factor. Neither medium conditioned by endothelial cells nor fixed endothelial cells could by themselves induce all of these different phenomena in the PC12 cells. These results suggest that under coculture conditions PC12 cells change their state of differentiation toward a chromaffin cell-like phenotype. The rapid, transient increase in the expression of the protooncogene c-fos suggests that the mechanism(s) inducing the change in the state of differentiation in PC12 cells in coculture with the endothelial cells may be distinct from that described for the differentiation of PC12 cells--e.g., by glucocorticoids. We propose that similar interactions between endothelial cells and chromaffin cell precursors may occur during embryonic development and that these interactions might be instrumental for the organ-specific differentiation of the adrenal medulla in vivo.

  20. Three-dimensional culture conditions differentially affect astrocyte modulation of brain endothelial barrier function in response to transforming growth factor β1.

    PubMed

    Hawkins, Brian T; Grego, Sonia; Sellgren, Katelyn L

    2015-05-22

    Blood-brain barrier (BBB) function is regulated by dynamic interactions among cell types within the neurovascular unit, including astrocytes and endothelial cells. Co-culture models of the BBB typically involve astrocytes seeded on two-dimensional (2D) surfaces, which recent studies indicate cause astrocytes to express a phenotype similar to that of reactive astrocytes in situ. We hypothesized that the culture conditions of astrocytes would differentially affect their ability to modulate BBB function in vitro. Brain endothelial cells were grown alone or in co-culture with astrocytes. Astrocytes were grown either as conventional (2D) monolayers, or in a collagen-based gel which allows them to grow in a three-dimensional (3D) construct. Astrocytes were viable in 3D conditions, and displayed a marked reduction in their expression of glial fibrillary acidic protein (GFAP), suggesting reduced activation. Stimulation of astrocytes with transforming growth factor (TGF)β1 decreased transendothelial electrical resistance (TEER) and reduced expression of claudin-5 in co-cultures, whereas treatment of endothelial cells in the absence of astrocytes was without effect. The effect of TGFβ1 on TEER was significantly more pronounced in endothelial cells cultured with 3D astrocytes compared to 2D astrocytes. These results demonstrate that astrocyte culture conditions differentially affect their ability to modulate brain endothelial barrier function, and suggest a direct relationship between reactive gliosis and BBB permeability. Moreover, these studies demonstrate the potential importance of physiologically relevant culture conditions to in vitro modeling of disease processes that affect the neurovascular unit.

  1. Endothelial cell injury in cardiovascular surgery: the procoagulant response.

    PubMed

    Boyle, E M; Verrier, E D; Spiess, B D

    1996-11-01

    The vascular endothelium plays a critical role in the regulation of coagulation through the constitutive expression and release of anticoagulants and the inducible expression of procoagulant substances. Cardiopulmonary bypass dysregulates this process by activating endothelial cells, initially promoting bleeding and then thrombosis. Endothelial cell activation in response to circulating inflammatory mediators leads to the initiation of coagulation when tissue factor is expressed throughout the intravascular space. This results in the widespread consumption of coagulation factors. Additionally, there is a cardiopulmonary bypass-related qualitative platelet defect that is exacerbated by thrombocytopenia as platelets are consumed from the circulation by clot and adherence to the cardiopulmonary bypass circuit. Finally, cardiopulmonary bypass results in the endothelial release of plasminogen activators, which lead to an increase in systemic fibrinolysis. The diffuse generation of thrombin, driven by the inducible intravascular expression of tissue factor, plays a major role in all of these processes. Efforts to understand the critical role of the endothelium in coagulation may lead to novel therapies to prevent bleeding or thrombosis in cardiovascular surgery patients.

  2. Antioxidant Effects of Sheep Whey Protein on Endothelial Cells.

    PubMed

    Kerasioti, Efthalia; Stagos, Dimitrios; Georgatzi, Vasiliki; Bregou, Erinda; Priftis, Alexandros; Kafantaris, Ioannis; Kouretas, Dimitrios

    2016-01-01

    Excessive production of reactive oxygen species (ROS) may cause endothelial dysfunction and consequently vascular disease. In the present study, the possible protective effects of sheep whey protein (SWP) from tert-butyl hydroperoxide- (tBHP-) induced oxidative stress in endothelial cells (EA.hy926) were assessed using oxidative stress biomarkers. These oxidative stress biomarkers were glutathione (GSH) and ROS levels determined by flow cytometry. Moreover, thiobarbituric acid-reactive substances (TBARS), protein carbonyls (CARB), and oxidized glutathione (GSSG) were determined spectrophotometrically. The results showed that SWP at 0.78, 1.56, 3.12, and 6.24 mg of protein mL(-1) increased GSH up to 141%, while it decreased GSSG to 46.7%, ROS to 58.5%, TBARS to 52.5%, and CARB to 49.0%. In conclusion, the present study demonstrated for the first time that SWP protected endothelial cells from oxidative stress. Thus, SWP may be used for developing food supplements or biofunctional foods to attenuate vascular disturbances associated with oxidative stress. PMID:27127549

  3. Antioxidant Effects of Sheep Whey Protein on Endothelial Cells

    PubMed Central

    Kerasioti, Efthalia; Stagos, Dimitrios; Georgatzi, Vasiliki; Bregou, Erinda; Priftis, Alexandros; Kafantaris, Ioannis; Kouretas, Dimitrios

    2016-01-01

    Excessive production of reactive oxygen species (ROS) may cause endothelial dysfunction and consequently vascular disease. In the present study, the possible protective effects of sheep whey protein (SWP) from tert-butyl hydroperoxide- (tBHP-) induced oxidative stress in endothelial cells (EA.hy926) were assessed using oxidative stress biomarkers. These oxidative stress biomarkers were glutathione (GSH) and ROS levels determined by flow cytometry. Moreover, thiobarbituric acid-reactive substances (TBARS), protein carbonyls (CARB), and oxidized glutathione (GSSG) were determined spectrophotometrically. The results showed that SWP at 0.78, 1.56, 3.12, and 6.24 mg of protein mL−1 increased GSH up to 141%, while it decreased GSSG to 46.7%, ROS to 58.5%, TBARS to 52.5%, and CARB to 49.0%. In conclusion, the present study demonstrated for the first time that SWP protected endothelial cells from oxidative stress. Thus, SWP may be used for developing food supplements or biofunctional foods to attenuate vascular disturbances associated with oxidative stress. PMID:27127549

  4. Adhesion of Fusobacterium necrophorum to bovine endothelial cells is mediated by outer membrane proteins.

    PubMed

    Kumar, Amit; Gart, Elena; Nagaraja, T G; Narayanan, Sanjeev

    2013-03-23

    Fusobacterium necrophorum, a Gram-negative anaerobe, is frequently associated with suppurative and necrotic infections of animals and humans. The organism is a major bovine pathogen, and in cattle, the common fusobacterial infections are hepatic abscesses, foot rot, and necrotic laryngitis. The species comprises two subspecies: F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme. Bacterial adhesion to the host cell surface is a critical initial step in the pathogenesis, and outer membrane proteins (OMP) play an important role in adhesion and establishment of certain Gram-negative bacterial infections. The means by which F. necrophorum attaches to epithelial or endothelial cells has not been determined. We evaluated whether OMP of F. necrophorum, isolated from a liver abscess, mediated adhesion to bovine endothelial cells (adrenal gland capillary endothelial cell line). The extent of binding of subsp. necrophorum to the endothelial cells was higher than that of F. necrophorum subsp. funduliforme. Trypsin treatment of bacterial cells decreased their binding to endothelial cells indicating the protein nature of adhesins. Preincubation of endothelial cells with OMP extracted from F. necrophorum decreased the binding of bacterial cells. In addition, binding of each subspecies to endothelial cells was inhibited by polyclonal antibodies raised against respective OMP and the antibody-mediated inhibition was subspecies specific. The western blot analysis of OMP bound to endothelial cells with anti-OMP antibodies showed four OMP of 17, 24, 40 and 74 kDa. We conclude that OMP of F. necrophorum play a role in adhesion of bacterial cells to the endothelial cells.

  5. A microfluidic cell culture system for monitoring of sequential changes in endothelial cells after heat stress.

    PubMed

    Tazawa, Hidekatsu; Sato, Kenjiro; Tsutiya, Atsuhiro; Tokeshi, Manabu; Ohtani-Kaneko, Ritsuko

    2015-08-01

    Endothelial damage induced by a highly elevated body temperature is crucial in some diseases including viral hemorrhagic fevers. Here, we report the heat-induced sequential changes of endothelial cells under shear stress, which were determined with a microfluidic culture system. Although live cell imaging showed only minor changes in the appearance of heat-treated cells, Hsp70 mRNA expression analysis demonstrated that the endothelial cells in channels of the system responded well to heat treatment. F-actin staining also revealed clear changes in the bundles of actin filaments after heat treatment. Well-organized bundles of actin filaments in control cells disappeared in heat-treated cells cultured in the channel. Furthermore, the system enabled detection of sequential changes in plasminogen activator inhibitor-1 (PAI-1) secretion from endothelial cells. PAI-1 concentration in the effluent solution was significantly elevated for the first 15min after initiation of heat treatment, and then decreased subsequently. This study provides fundamental information on heat-induced endothelial changes under shear stress and introduces a potent tool for analyzing endothelial secretions. PMID:26044666

  6. A microfluidic cell culture system for monitoring of sequential changes in endothelial cells after heat stress.

    PubMed

    Tazawa, Hidekatsu; Sato, Kenjiro; Tsutiya, Atsuhiro; Tokeshi, Manabu; Ohtani-Kaneko, Ritsuko

    2015-08-01

    Endothelial damage induced by a highly elevated body temperature is crucial in some diseases including viral hemorrhagic fevers. Here, we report the heat-induced sequential changes of endothelial cells under shear stress, which were determined with a microfluidic culture system. Although live cell imaging showed only minor changes in the appearance of heat-treated cells, Hsp70 mRNA expression analysis demonstrated that the endothelial cells in channels of the system responded well to heat treatment. F-actin staining also revealed clear changes in the bundles of actin filaments after heat treatment. Well-organized bundles of actin filaments in control cells disappeared in heat-treated cells cultured in the channel. Furthermore, the system enabled detection of sequential changes in plasminogen activator inhibitor-1 (PAI-1) secretion from endothelial cells. PAI-1 concentration in the effluent solution was significantly elevated for the first 15min after initiation of heat treatment, and then decreased subsequently. This study provides fundamental information on heat-induced endothelial changes under shear stress and introduces a potent tool for analyzing endothelial secretions.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  8. WR-1065 and radioprotection of vascular endothelial cells. II. Morphology