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Sample records for human cerebral endothelial

  1. Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

    PubMed

    Musolino, Patricia L; Gong, Yi; Snyder, Juliet M T; Jimenez, Sandra; Lok, Josephine; Lo, Eng H; Moser, Ann B; Grabowski, Eric F; Frosch, Matthew P; Eichler, Florian S

    2015-11-01

    See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy. PMID:26377633

  2. Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

    PubMed

    Musolino, Patricia L; Gong, Yi; Snyder, Juliet M T; Jimenez, Sandra; Lok, Josephine; Lo, Eng H; Moser, Ann B; Grabowski, Eric F; Frosch, Matthew P; Eichler, Florian S

    2015-11-01

    See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy.

  3. Interleukin-6 triggers human cerebral endothelial cells proliferation and migration: The role for KDR and MMP-9

    SciTech Connect

    Yao, Jianhua S.; Zhai Wenwu; Young, William L.; Yang Guoyuan . E-mail: gyyang@anesthesia.ucsf.edu

    2006-04-21

    Interleukin-6 (IL-6) is involved in angiogenesis. However, the underlying mechanisms are unknown. Using human cerebral endothelial cell (HCEC), we report for First time that IL-6 triggers HCEC proliferation and migration in a dose-dependent manner, specifically associated with enhancement of VEGF expression, up-regulated and phosphorylated VEGF receptor-2 (KDR), and stimulated MMP-9 secretion. We investigated the signal pathway of IL-6/IL-6R responsible for KDR's regulation. Pharmacological inhibitor of PI3K failed to inhibit IL-6-mediated VEGF overexpression, while blocking ERK1/2 with PD98059 could abolish IL-6-induced KDR overexpression. Further, neutralizing endogenous VEGF attenuated KDR expression and phosphorylation, suggesting that IL-6-induced KDR activation is independent of VEGF stimulation. MMP-9 inhibitor GM6001 significantly decreases HCEC proliferation and migration (p < 0.05), indicating the crucial function of MMP-9 in promoting angiogenic changes in HCECs. We conclude that IL-6 triggers VEGF-induced angiogenic activity through increasing VEGF release, up-regulates KDR expression and phosphorylation through activating ERK1/2 signaling, and stimulates MMP-9 overexpression.

  4. Neuroprotective effects of systemic cerebral endothelial cell transplantation in a rat model of cerebral ischemia.

    PubMed

    Moon, Jong-Hyun; Na, Joo-Young; Lee, Min-Cheol; Choi, Kang-Ho; Lee, Jeong-Kil; Min, Jung-Joon; Kim, Kyung-Tae; Park, Jong-Tae; Park, Man-Seok; Kim, Hyung-Seok

    2016-01-01

    Human cerebral microvascular endothelial cell line (hCMEC)/D3 cells, which are from a stable clonal cell line of human immortalized cerebral endothelial cells, were intra-arterially transplanted through the common carotid artery in a rat model of photochemical-induced cerebral ischemia. Their therapeutic effects on infarct size, blood-brain barrier (BBB) breakdown, and outcome were examined. The hCMEC/D3 cells were genetically modified with the firefly luciferase gene for in vivo imaging post-transplantation. Transplanted hCMEC/D3 cells were identified in the infarcted brain by bioluminescence imaging at 1 day after transplantation. Compared with the control group, the hCMEC/D3-transplanted group showed reduced infarct size on day 3, reduced Evans blue dye leakage on day 1 indicating decreased BBB breakdown, and early recovery from Rotarod test neurological deficits. The hCMEC/D3-transplanted group also showed decreased levels of matrix metalloproteinase (MMP)-9, which were inversely correlated with TIMP-1 levels on post-transplantation days 1 and 3. The expression of tumor necrosis factor-α and interleukin-1β were markedly diminished in the hCMEC/D3-transplanted group compared with controls. The systemically transplanted cells selectively migrated and integrated into the ischemically lesioned area, which accelerated neurological recovery. This new cerebral endothelial cell-based therapy may hold promise for clinical trials in patients with ischemic stroke. PMID:27347342

  5. Neuroprotective effects of systemic cerebral endothelial cell transplantation in a rat model of cerebral ischemia.

    PubMed

    Moon, Jong-Hyun; Na, Joo-Young; Lee, Min-Cheol; Choi, Kang-Ho; Lee, Jeong-Kil; Min, Jung-Joon; Kim, Kyung-Tae; Park, Jong-Tae; Park, Man-Seok; Kim, Hyung-Seok

    2016-01-01

    Human cerebral microvascular endothelial cell line (hCMEC)/D3 cells, which are from a stable clonal cell line of human immortalized cerebral endothelial cells, were intra-arterially transplanted through the common carotid artery in a rat model of photochemical-induced cerebral ischemia. Their therapeutic effects on infarct size, blood-brain barrier (BBB) breakdown, and outcome were examined. The hCMEC/D3 cells were genetically modified with the firefly luciferase gene for in vivo imaging post-transplantation. Transplanted hCMEC/D3 cells were identified in the infarcted brain by bioluminescence imaging at 1 day after transplantation. Compared with the control group, the hCMEC/D3-transplanted group showed reduced infarct size on day 3, reduced Evans blue dye leakage on day 1 indicating decreased BBB breakdown, and early recovery from Rotarod test neurological deficits. The hCMEC/D3-transplanted group also showed decreased levels of matrix metalloproteinase (MMP)-9, which were inversely correlated with TIMP-1 levels on post-transplantation days 1 and 3. The expression of tumor necrosis factor-α and interleukin-1β were markedly diminished in the hCMEC/D3-transplanted group compared with controls. The systemically transplanted cells selectively migrated and integrated into the ischemically lesioned area, which accelerated neurological recovery. This new cerebral endothelial cell-based therapy may hold promise for clinical trials in patients with ischemic stroke.

  6. Immortalized human cerebral microvascular endothelial cells maintain the properties of primary cells in an in vitro model of immune migration across the blood brain barrier

    PubMed Central

    Daniels, Brian P.; Cruz-Orengo, Lillian; Pasieka, Tracy Jo; Couraud, Pierre-Olivier; Romero, Ignacio A.; Weksler, Babette; Cooper, John A.; Doering, Tamara L.; Klein, Robyn S.

    2012-01-01

    The immortalized human cerebral microvascular endothelial cell line HCMEC/D3 presents a less expensive and more logistically feasible alternative to primary human brain microvascular endothelial cells (HBMEC’s) for use in constructing in vitro models of the blood brain barrier (BBB). However, the fidelity of the HCMEC/D3 cell line to primary HBMEC’s in studies of immune transmigration has yet to be established. Flow cytometric analysis of primary human leukocyte migration across in vitro BBB’s generated with either HCMEC/D3 or primary HBMEC’s revealed that HCMEC/D3 maintains the immune barrier properties of primary HBMEC’s. Leukocyte migration responses and inflammatory cytokine production were statistically indistinguishable between both endothelial cell types, and both cell types responded similarly to astrocyte coculture, stimulation of leukocytes with phorbol myristate acetate (PMA) and ionomycin, and inflammatory cytokine treatment. This report is the first to validate the HCMEC/D3 cell line in a neuroimmunological experimental system via direct comparison to primary HBMEC’s, demonstrating remarkable fidelity in terms of barrier resistance, immune migration profiles, and responsiveness to inflammatory cytokines. Moreover, we report novel findings demonstrating that interaction effects between immune cells and resident CNS cells are preserved in HCMEC/D3, suggesting that important characteristics of neuroimmune interactions during CNS inflammation are preserved in systems utilizing this cell line. Together, these findings demonstrate that HCMEC/D3 is a valid and powerful tool for less expensive and higher throughput in vitro investigations of immune migration at the BBB. PMID:23068604

  7. 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.

  8. Endothelial Nitric Oxide Mediates Caffeine Antagonism of Alcohol-Induced Cerebral Artery Constriction.

    PubMed

    Chang, Jennifer; Fedinec, Alexander L; Kuntamallappanavar, Guruprasad; Leffler, Charles W; Bukiya, Anna N; Dopico, Alex M

    2016-01-01

    Despite preventive education, the combined consumption of alcohol and caffeine (particularly from "energy drinks") continues to rise. Physiologic perturbations by separate intake of ethanol and caffeine have been widely documented. However, the biologic actions of the alcohol-caffeine combination and their underlying subcellular mechanisms have been scarcely studied. Using intravital microscopy on a closed-cranial window and isolated, pressurized vessels, we investigated the in vivo and in vitro action of ethanol-caffeine mixtures on cerebral arteries from rats and mice, widely recognized models to address cerebrovascular pathophysiology and pharmacology. Caffeine at concentrations found in human circulation after ingestion of one to two cups of coffee (10 µM) antagonized the endothelium-independent constriction of cerebral arteries evoked by ethanol concentrations found in blood during moderate-heavy alcohol intoxication (40-70 mM). Caffeine antagonism against alcohol was similar whether evaluated in vivo or in vitro, suggesting independence of systemic factors and drug metabolism, but required a functional endothelium. Moreover, caffeine protection against alcohol increased nitric oxide (NO•) levels over those found in the presence of ethanol alone, disappeared upon blocking NO• synthase, and could not be detected in pressurized cerebral arteries from endothelial nitric-oxide synthase knockout (eNOS(-/-)) mice. Finally, incubation of de-endothelialized cerebral arteries with the NO• donor sodium nitroprusside (10 µM) fully restored the protective effect of caffeine. This study demonstrates for the first time that caffeine antagonizes ethanol-induced cerebral artery constriction and identifies endothelial NO• as the critical caffeine effector on smooth muscle targets. Conceivably, situations that perturb endothelial function and/or NO• availability will critically alter caffeine antagonism of alcohol-induced cerebrovascular constriction without

  9. Endothelial Nitric Oxide Mediates Caffeine Antagonism of Alcohol-Induced Cerebral Artery Constriction.

    PubMed

    Chang, Jennifer; Fedinec, Alexander L; Kuntamallappanavar, Guruprasad; Leffler, Charles W; Bukiya, Anna N; Dopico, Alex M

    2016-01-01

    Despite preventive education, the combined consumption of alcohol and caffeine (particularly from "energy drinks") continues to rise. Physiologic perturbations by separate intake of ethanol and caffeine have been widely documented. However, the biologic actions of the alcohol-caffeine combination and their underlying subcellular mechanisms have been scarcely studied. Using intravital microscopy on a closed-cranial window and isolated, pressurized vessels, we investigated the in vivo and in vitro action of ethanol-caffeine mixtures on cerebral arteries from rats and mice, widely recognized models to address cerebrovascular pathophysiology and pharmacology. Caffeine at concentrations found in human circulation after ingestion of one to two cups of coffee (10 µM) antagonized the endothelium-independent constriction of cerebral arteries evoked by ethanol concentrations found in blood during moderate-heavy alcohol intoxication (40-70 mM). Caffeine antagonism against alcohol was similar whether evaluated in vivo or in vitro, suggesting independence of systemic factors and drug metabolism, but required a functional endothelium. Moreover, caffeine protection against alcohol increased nitric oxide (NO•) levels over those found in the presence of ethanol alone, disappeared upon blocking NO• synthase, and could not be detected in pressurized cerebral arteries from endothelial nitric-oxide synthase knockout (eNOS(-/-)) mice. Finally, incubation of de-endothelialized cerebral arteries with the NO• donor sodium nitroprusside (10 µM) fully restored the protective effect of caffeine. This study demonstrates for the first time that caffeine antagonizes ethanol-induced cerebral artery constriction and identifies endothelial NO• as the critical caffeine effector on smooth muscle targets. Conceivably, situations that perturb endothelial function and/or NO• availability will critically alter caffeine antagonism of alcohol-induced cerebrovascular constriction without

  10. Pertussis Toxin Exploits Host Cell Signaling Pathways Induced by Meningitis-Causing E. coli K1-RS218 and Enhances Adherence of Monocytic THP-1 Cells to Human Cerebral Endothelial Cells

    PubMed Central

    Starost, Laura Julia; Karassek, Sascha; Sano, Yasuteru; Kanda, Takashi; Kim, Kwang Sik; Dobrindt, Ulrich; Rüter, Christian; Schmidt, Marcus Alexander

    2016-01-01

    Pertussis toxin (PTx), the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis, permeabilizes the blood–brain barrier (BBB) in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218’s effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB. PMID:27754355

  11. Reduction in cardiolipin decreases mitochondrial spare respiratory capacity and increases glucose transport into and across human brain cerebral microvascular endothelial cells.

    PubMed

    Nguyen, Hieu M; Mejia, Edgard M; Chang, Wenguang; Wang, Ying; Watson, Emily; On, Ngoc; Miller, Donald W; Hatch, Grant M

    2016-10-01

    Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. Cardiolipin is a mitochondrial phospholipid required for function of the electron transport chain and ATP generation. We examined the role of cardiolipin in maintaining mitochondrial function necessary to support barrier properties of brain microvessel endothelial cells. Knockdown of the terminal enzyme of cardiolipin synthesis, cardiolipin synthase, in hCMEC/D3 cells resulted in decreased cellular cardiolipin levels compared to controls. The reduction in cardiolipin resulted in decreased mitochondrial spare respiratory capacity, increased pyruvate kinase activity, and increased 2-deoxy-[(3) H]glucose uptake and glucose transporter-1 expression and localization to membranes in hCMEC/D3 cells compared to controls. The mechanism for the increase in glucose uptake was an increase in adenosine-5'-monophosphate kinase and protein kinase B activity and decreased glycogen synthase kinase 3 beta activity. Knockdown of cardiolipin synthase did not affect permeability of fluorescent dextran across confluent hCMEC/D3 monolayers grown on Transwell(®) inserts. In contrast, knockdown of cardiolipin synthase resulted in an increase in 2-deoxy-[(3) H]glucose transport across these monolayers compared to controls. The data indicate that in hCMEC/D3 cells, spare respiratory capacity is dependent on cardiolipin. In addition, reduction in cardiolipin in these cells alters their cellular energy status and this results in increased glucose transport into and across hCMEC/D3 monolayers. Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. In human adult brain endothelial cell hCMEC/D3 monolayers cultured on Transwell(®) plates, knockdown of cardiolipin synthase results in decrease in mitochondrial

  12. 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.

  13. Plasmodium falciparum Histidine-Rich Protein II Compromises Brain Endothelial Barriers and May Promote Cerebral Malaria Pathogenesis

    PubMed Central

    Pal, Priya; Daniels, Brian P.; Oskman, Anna; Diamond, Michael S.; Klein, Robyn S.

    2016-01-01

    ABSTRACT Cerebral malaria (CM) is a disease of the vascular endothelium caused by Plasmodium falciparum. It is characterized by parasite sequestration, inflammatory cytokine production, and vascular leakage. A distinguishing feature of P. falciparum infection is parasite production and secretion of histidine-rich protein II (HRPII). Plasma HRPII is a diagnostic and prognostic marker for falciparum malaria. We demonstrate that disruption of a human cerebral microvascular endothelial barrier by P. falciparum-infected erythrocytes depends on expression of HRPII. Purified recombinant or native HRPII can recapitulate these effects. HRPII action occurs via activation of the inflammasome, resulting in decreased integrity of tight junctions and increased endothelial permeability. We propose that HRPII is a virulence factor that may contribute to cerebral malaria by compromising endothelial barrier integrity within the central nervous system. PMID:27273825

  14. 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

  15. [Cerebral infarction in human immunodeficiency virus infection].

    PubMed

    Blanche, P; Toulon, P; de La Blanchardière, A; Sicard, D

    1995-06-01

    Patients infected with the human immunodeficiency virus (HIV) appear to have a high risk of ischaemic cerebral events. We observed two cases of cerebral infarction in patients with acquired immune deficiency syndrome (AIDS). In the first case, a 38-year-old homosexual with no cardiovascular risk other than smoking presented with rapidly progressive hemiparesia. Brain CT-scan visualized two infarcts in the territory of the right sylvian artery and the arteriography an occlusion of the internal carotid artery. In the second, a 37-year-old homosexual, hospitalization was required for a left-sided pure sensitive epilepsy seizure. There was no cardiovascular risk other than smoking. Magnetic resonance imaging showed parietal ischaemia and thrombus in the left atrium without atrial hypertrophy was seen at transoesophageal echocardiography. In both cases, there was no evidence of endocarditis, dissection of the neck vessels or disseminated intravascular coagulation nor of associated viral or bacterial infectious complication of AIDS. Angiographic findings eliminated cerebral vascularitis. Among the perturbed haemostasis factors previously reported in HIV+ patients, we observed free proteins S deficiency (68 and 43%) and heparin cofactor II deficiency (54 and 40%). Serum albumin was 33 and 32 g/l respectively. Outcome was favourable in both cases with anticoagulant therapy. These coagulation anomalies would not appear sufficient to explain cerebral infarction. Other mechanisms including immune complexed deposition, direct HIV toxicity for endothelial cells or the effect of cytokines on smooth muscles fibres and fibroblasts are probably more important causal factors. PMID:7638144

  16. Regulation of NOD-like receptors and inflammasome activation in cerebral endothelial cells.

    PubMed

    Nagyőszi, Péter; Nyúl-Tóth, Ádám; Fazakas, Csilla; Wilhelm, Imola; Kozma, Mihály; Molnár, Judit; Haskó, János; Krizbai, István A

    2015-11-01

    Cerebral endothelial cells (CECs) forming the blood-brain barrier are at the interface of the immune and the central nervous systems and thus may play an important role in the functional integration of the two systems. Here, we investigated how CECs recognize and respond to pathogen- and damage-associated molecular patterns to regulate the functions of the neurovascular unit. First we detected the expression of several NOD-like receptors (NLRs) - including NOD1, NOD2, NLRC4, NLRC5, NLRP1, NLRP3, NLRP5, NLRP9, NLRP10, NLRP12, NLRA, and NLRX - in human brain endothelial cells. Inflammatory cytokines, such as interferon-γ, tumor necrosis factor-α, and IL-1β had stimulatory effects on the transcription of many of these receptors. Expression of key inflammasome components (NOD2, NLRP3, and caspase 1) along with caspase-cleaved interleukins IL-1β and IL-33 could be induced by priming with lipopolysaccharide and activation with muramyl dipeptide. In addition, combined treatment with lipopolysaccharide and muramyl dipeptide resulted in IL-1β secretion in a caspase- and ERK1/2 kinase-dependent manner. Our findings demonstrate that NLRs and inflammasomes can be activated in cerebral endothelial cells, which may confer a yet unexplored role to the blood-brain barrier in neuroimmune and neuroinflammatory processes.

  17. 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

  18. 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

  19. Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.

    PubMed

    Zhou, Zinan; Tang, Alan T; Wong, Weng-Yew; Bamezai, Sharika; Goddard, Lauren M; Shenkar, Robert; Zhou, Su; Yang, Jisheng; Wright, Alexander C; Foley, Matthew; Arthur, J Simon C; Whitehead, Kevin J; Awad, Issam A; Li, Dean Y; Zheng, Xiangjian; Kahn, Mark L

    2016-04-01

    Cerebral cavernous malformations (CCMs) are common inherited and sporadic vascular malformations that cause strokes and seizures in younger individuals. CCMs arise from endothelial cell loss of KRIT1, CCM2 or PDCD10, non-homologous proteins that form an adaptor complex. How disruption of the CCM complex results in disease remains controversial, with numerous signalling pathways (including Rho, SMAD and Wnt/β-catenin) and processes such as endothelial-mesenchymal transition (EndMT) proposed to have causal roles. CCM2 binds to MEKK3 (refs 7, 8, 9, 10, 11), and we have recently shown that CCM complex regulation of MEKK3 is essential during vertebrate heart development. Here we investigate this mechanism in CCM disease pathogenesis. Using a neonatal mouse model of CCM disease, we show that expression of the MEKK3 target genes Klf2 and Klf4, as well as Rho and ADAMTS protease activity, are increased in the endothelial cells of early CCM lesions. By contrast, we find no evidence of EndMT or increased SMAD or Wnt signalling during early CCM formation. Endothelial-specific loss of Map3k3 (also known as Mekk3), Klf2 or Klf4 markedly prevents lesion formation, reverses the increase in Rho activity, and rescues lethality. Consistent with these findings in mice, we show that endothelial expression of KLF2 and KLF4 is increased in human familial and sporadic CCM lesions, and that a disease-causing human CCM2 mutation abrogates the MEKK3 interaction without affecting CCM complex formation. These studies identify gain of MEKK3 signalling and KLF2/4 function as causal mechanisms for CCM pathogenesis that may be targeted to develop new CCM therapeutics. PMID:27027284

  20. Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.

    PubMed

    Zhou, Zinan; Tang, Alan T; Wong, Weng-Yew; Bamezai, Sharika; Goddard, Lauren M; Shenkar, Robert; Zhou, Su; Yang, Jisheng; Wright, Alexander C; Foley, Matthew; Arthur, J Simon C; Whitehead, Kevin J; Awad, Issam A; Li, Dean Y; Zheng, Xiangjian; Kahn, Mark L

    2016-04-01

    Cerebral cavernous malformations (CCMs) are common inherited and sporadic vascular malformations that cause strokes and seizures in younger individuals. CCMs arise from endothelial cell loss of KRIT1, CCM2 or PDCD10, non-homologous proteins that form an adaptor complex. How disruption of the CCM complex results in disease remains controversial, with numerous signalling pathways (including Rho, SMAD and Wnt/β-catenin) and processes such as endothelial-mesenchymal transition (EndMT) proposed to have causal roles. CCM2 binds to MEKK3 (refs 7, 8, 9, 10, 11), and we have recently shown that CCM complex regulation of MEKK3 is essential during vertebrate heart development. Here we investigate this mechanism in CCM disease pathogenesis. Using a neonatal mouse model of CCM disease, we show that expression of the MEKK3 target genes Klf2 and Klf4, as well as Rho and ADAMTS protease activity, are increased in the endothelial cells of early CCM lesions. By contrast, we find no evidence of EndMT or increased SMAD or Wnt signalling during early CCM formation. Endothelial-specific loss of Map3k3 (also known as Mekk3), Klf2 or Klf4 markedly prevents lesion formation, reverses the increase in Rho activity, and rescues lethality. Consistent with these findings in mice, we show that endothelial expression of KLF2 and KLF4 is increased in human familial and sporadic CCM lesions, and that a disease-causing human CCM2 mutation abrogates the MEKK3 interaction without affecting CCM complex formation. These studies identify gain of MEKK3 signalling and KLF2/4 function as causal mechanisms for CCM pathogenesis that may be targeted to develop new CCM therapeutics.

  1. Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signaling

    PubMed Central

    Zhou, Zinan; Tang, Alan T.; Wong, Weng-Yew; Bamezai, Sharika; Goddard, Lauren M.; Shenkar, Robert; Zhou, Su; Yang, Jisheng; Wright, Alexander C.; Foley, Matthew; Arthur, J. Simon C.; Whitehead, Kevin J.; Awad, Issam A.; Li, Dean Y.; Zheng, Xiangjian; Kahn, Mark L.

    2016-01-01

    Cerebral cavernous malformations (CCMs) are common inherited and sporadic vascular malformations that cause stroke and seizures in younger individuals1. CCMs arise from endothelial cell loss of KRIT1, CCM2, or PDCD10, non-homologous proteins that form an adaptor complex2. How disruption of the CCM complex results in disease remains controversial, with numerous signaling pathways (including Rho3,4, SMAD5 and Wnt/β-catenin6) and processes such as endothelial-mesenchymal transition (EndMT)5 proposed to play causal roles. CCM2 binds MEKK37–11, and we have recently demonstrated that CCM complex regulation of MEKK3 is essential during vertebrate heart development12. Here, we investigate this mechanism in CCM disease pathogenesis. Using a neonatal mouse model of CCM disease, we find that expression of the MEKK3 target genes KLF2 and KLF4, as well as Rho and ADAMTS protease activity, are increased in the endothelial cells of early CCM lesions. In contrast, we find no evidence of EndMT or increased SMAD or Wnt signaling during early CCM formation. Endothelial-specific loss of Mekk3, Klf2, or Klf4 dramatically prevents lesion formation, reverses the increase in Rho activity, and rescues lethality. Consistent with these findings in mice, we demonstrate that endothelial expression of KLF2 and KLF4 is elevated in human familial and sporadic CCM lesions, and that a disease-causing human CCM2 mutation abrogates MEKK3 interaction without affecting CCM complex formation. These studies identify gain of MEKK3 signaling and KLF2/4 function as causal mechanisms for CCM pathogenesis that may be targeted to develop new CCM therapeutics. PMID:27027284

  2. A primate model for human cerebral malaria: Plasmodium coatneyi-infected rhesus monkeys.

    PubMed

    Aikawa, M; Brown, A; Smith, C D; Tegoshi, T; Howard, R J; Hasler, T H; Ito, Y; Perry, G; Collins, W E; Webster, K

    1992-04-01

    A major factor in the pathogenesis of human cerebral malaria is blockage of cerebral microvessels by the sequestration of parasitized human red blood cells (PRBC). In vitro studies indicate that sequestration of PRBC in the microvessels is mediated by the attachment of knobs on PRBC to receptors on the endothelial cell surface such as CD36, thrombospondin (TSP), and intercellular adhesion molecule-1 (ICAM-1). However, it is difficult to test this theory in vivo because fresh human brain tissues from cerebral malarial autopsy cases are not easy to obtain. Although several animal models for human cerebral malaria have been proposed, none have shown pathologic findings that are similar to those seen in humans. In order to develop an animal model for human cerebral malaria, we studied brains of rhesus monkeys infected with the primate malaria parasite, Plasmodium coatneyi. Our study demonstrated PRBC sequestration and cytoadherence of knobs on PRBC to endothelial cells in the cerebral microvessels of these monkeys. Cerebral microvessels with sequestered PRBC were shown by immunohistochemical analysis to possess CD36, TSP, and ICAM-1. These proteins were not evident in the cerebral microvessels of uninfected control monkeys. Thus, our study indicates, for the first time, that rhesus monkeys infected with P. coatneyi can be used as a primate model to study human cerebral malaria. By using this animal model, we may be able to evaluate strategies for the development of vaccines to prevent human cerebral malaria. PMID:1374220

  3. Non-coding RNAs in cerebral endothelial pathophysiology: emerging roles in stroke.

    PubMed

    Yin, Ke-Jie; Hamblin, Milton; Chen, Y Eugene

    2014-11-01

    Cerebral vascular endothelial cells form the major element of the blood-brain barrier (BBB) and constitute the primary interface between circulating blood and brain parenchyma. The structural and functional changes in cerebral endothelium during cerebral ischemia are well known to result in BBB disruption, vascular inflammation, edema, and angiogenesis. These complex pathological processes directly contribute to brain infarction, neurological deficits, and post-stroke neurovascular remodeling. Ischemic endothelial dysfunction appears to be tightly controlled by multiple gene signaling networks. Non-coding RNAs (ncRNAs) are functional RNA molecules that are generally not translated into proteins but can actively regulate the expression and function of many thousands of protein-coding genes by different mechanisms. Various classes of ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), small nucleolar RNAs (snoRNAs) and piwi-interacting RNAs (piRNAs), are highly expressed in the cerebrovascular endothelium where they serve as critical mediators to maintain normal cerebral vascular functions. Dysregulation of ncRNA activities has been closely linked to the pathophysiology of cerebral vascular endothelium and neurologic functional disorders in the brain's response to ischemic stimuli. In this review, we summarize recent advancements of these ncRNA mediators in the brain vasculature, highlighting the specific roles of endothelial miRNAs in stroke.

  4. Isolation, characterization, and long-term cultivation of porcine and murine cerebral capillary endothelial cells.

    PubMed

    Tontsch, U; Bauer, H C

    1989-03-01

    We present a simple method for isolation and long-term cultivation of porcine and murine cerebral capillary endothelial cells (cEC). Two major points are made. First, that the "characteristic" morphology of the endothelial cells depends mainly on the presence of endothelial cell growth factors in the culture medium and second, that the identification of the cells as endothelial cells requires a special lectin instead of criteria used for large vessel endothelial cells, such as factor VIII staining or LDL uptake. Pure cerebral capillaries were isolated by means of a series of centrifugation steps; endothelial cells were released by collagenase treatment and cultivated on plastic petri dishes, which proved to be better for cell attachment than collagen or gelatin coating. The microvascular cells were cultivated in either the presence or absence of growth factors. Medium 199 + 10% FCS produced mainly spindle-shaped cells, growing in the "hills and valleys" pattern, which, if not passaged for weeks, showed three dimensional tubular structures. Cells of the "cobblestone" phenotype were promoted in medium 199 + 10% FCS, enriched with endothelial cell growth supplement (ECGS) and heparin (referred to as complete medium). These cells retained their phenotype for months and could be passaged up to 35 times till now. If ECGS and heparin were omitted from these cultures, the cells became elongated and resembled smooth muscle cells. This effect was reversible when the cells were transferred to complete medium. With cEC, cloned by limiting dilution, we noticed this reversal phenomenon as well. We used several markers to characterize the microvascular cells and could show that the lectin of Bandeiraea simplicifolia is a highly reliable marker for endothelial cells and that the monoclonal antibody alpha-sm-1 (anti-smooth muscle cell actin) is excellent for determining smooth muscle cells.

  5. Glutathione in Cerebral Microvascular Endothelial Biology and Pathobiology: Implications for Brain Homeostasis

    PubMed Central

    Li, Wei; Busu, Carmina; Circu, Magdalena L.; Aw, Tak Yee

    2012-01-01

    The integrity of the vascular endothelium of the blood-brain barrier (BBB) is central to cerebrovascular homeostasis. Given the function of the BBB as a physical and metabolic barrier that buffers the systemic environment, oxidative damage to the endothelial monolayer will have significant deleterious impact on the metabolic, immunological, and neurological functions of the brain. Glutathione (GSH) is a ubiquitous major thiol within mammalian cells that plays important roles in antioxidant defense, oxidation-reduction reactions in metabolic pathways, and redox signaling. The existence of distinct GSH pools within the subcellular organelles supports an elegant mode for independent redox regulation of metabolic processes, including those that control cell fate. GSH-dependent homeostatic control of neurovascular function is relatively unexplored. Significantly, GSH regulation of two aspects of endothelial function is paramount to barrier preservation, namely, GSH protection against oxidative endothelial cell injury and GSH control of postdamage cell proliferation in endothelial repair and/or wound healing. This paper highlights our current insights and hypotheses into the role of GSH in cerebral microvascular biology and pathobiology with special focus on endothelial GSH and vascular integrity, oxidative disruption of endothelial barrier function, GSH regulation of endothelial cell proliferation, and the pathological implications of GSH disruption in oxidative stress-associated neurovascular disorders, such as diabetes and stroke. PMID:22745639

  6. 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

  7. Mechanical Injury Induces Brain Endothelial-Derived Microvesicle Release: Implications for Cerebral Vascular Injury during Traumatic Brain Injury.

    PubMed

    Andrews, Allison M; Lutton, Evan M; Merkel, Steven F; Razmpour, Roshanak; Ramirez, Servio H

    2016-01-01

    It is well established that the endothelium responds to mechanical forces induced by changes in shear stress and strain. However, our understanding of vascular remodeling following traumatic brain injury (TBI) remains incomplete. Recently published studies have revealed that lung and umbilical endothelial cells produce extracellular microvesicles (eMVs), such as microparticles, in response to changes in mechanical forces (blood flow and mechanical injury). Yet, to date, no studies have shown whether brain endothelial cells produce eMVs following TBI. The brain endothelium is highly specialized and forms the blood-brain barrier (BBB), which regulates diffusion and transport of solutes into the brain. This specialization is largely due to the presence of tight junction proteins (TJPs) between neighboring endothelial cells. Following TBI, a breakdown in tight junction complexes at the BBB leads to increased permeability, which greatly contributes to the secondary phase of injury. We have therefore tested the hypothesis that brain endothelium responds to mechanical injury, by producing eMVs that contain brain endothelial proteins, specifically TJPs. In our study, primary human adult brain microvascular endothelial cells (BMVEC) were subjected to rapid mechanical injury to simulate the abrupt endothelial disruption that can occur in the primary injury phase of TBI. eMVs were isolated from the media following injury at 2, 6, 24, and 48 h. Western blot analysis of eMVs demonstrated a time-dependent increase in TJP occludin, PECAM-1 and ICAM-1 following mechanical injury. In addition, activation of ARF6, a small GTPase linked to extracellular vesicle production, was increased after injury. To confirm these results in vivo, mice were subjected to sham surgery or TBI and blood plasma was collected 24 h post-injury. Isolation and analysis of eMVs from blood plasma using cryo-EM and flow cytometry revealed elevated levels of vesicles containing occludin following brain trauma

  8. Mechanical Injury Induces Brain Endothelial-Derived Microvesicle Release: Implications for Cerebral Vascular Injury during Traumatic Brain Injury

    PubMed Central

    Andrews, Allison M.; Lutton, Evan M.; Merkel, Steven F.; Razmpour, Roshanak; Ramirez, Servio H.

    2016-01-01

    It is well established that the endothelium responds to mechanical forces induced by changes in shear stress and strain. However, our understanding of vascular remodeling following traumatic brain injury (TBI) remains incomplete. Recently published studies have revealed that lung and umbilical endothelial cells produce extracellular microvesicles (eMVs), such as microparticles, in response to changes in mechanical forces (blood flow and mechanical injury). Yet, to date, no studies have shown whether brain endothelial cells produce eMVs following TBI. The brain endothelium is highly specialized and forms the blood-brain barrier (BBB), which regulates diffusion and transport of solutes into the brain. This specialization is largely due to the presence of tight junction proteins (TJPs) between neighboring endothelial cells. Following TBI, a breakdown in tight junction complexes at the BBB leads to increased permeability, which greatly contributes to the secondary phase of injury. We have therefore tested the hypothesis that brain endothelium responds to mechanical injury, by producing eMVs that contain brain endothelial proteins, specifically TJPs. In our study, primary human adult brain microvascular endothelial cells (BMVEC) were subjected to rapid mechanical injury to simulate the abrupt endothelial disruption that can occur in the primary injury phase of TBI. eMVs were isolated from the media following injury at 2, 6, 24, and 48 h. Western blot analysis of eMVs demonstrated a time-dependent increase in TJP occludin, PECAM-1 and ICAM-1 following mechanical injury. In addition, activation of ARF6, a small GTPase linked to extracellular vesicle production, was increased after injury. To confirm these results in vivo, mice were subjected to sham surgery or TBI and blood plasma was collected 24 h post-injury. Isolation and analysis of eMVs from blood plasma using cryo-EM and flow cytometry revealed elevated levels of vesicles containing occludin following brain trauma

  9. Modulation of cerebral microvascular permeability by endothelial nicotinic acetylcholine receptors.

    PubMed

    Hawkins, Brian T; Egleton, Richard D; Davis, Thomas P

    2005-07-01

    Nicotine increases the permeability of the blood-brain barrier in vivo. This implies a possible role for nicotinic acetylcholine receptors in the regulation of cerebral microvascular permeability. Expression of nicotinic acetylcholine receptor subunits in cerebral microvessels was investigated with immunofluorescence microscopy. Positive immunoreactivity was found for receptor subunits alpha3, alpha5, alpha7, and beta2, but not subunits alpha4, beta3, or beta4. Blood-brain barrier permeability was assessed via in situ brain perfusion with [14C]sucrose. Nicotine increased the rate of sucrose entry into the brain from 0.3 +/- 0.1 to 1.1 +/- 0.2 microl.g(-1).min(-1), as previously described. This nicotine-induced increase in blood-brain barrier permeability was significantly attenuated by both the blood-brain barrier-permeant nicotinic antagonist mecamylamine and the blood-brain barrier-impermeant nicotinic antagonist hexamethonium to 0.5 +/- 0.2 and 0.3 +/- 0.2 microl.g(-1).min(-1), respectively. These data suggest that nicotinic acetylcholine receptors expressed on the cerebral microvascular endothelium mediate nicotine-induced changes in blood-brain barrier permeability.

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

    PubMed

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

    2013-05-01

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

  11. Interendothelial claudin-5 expression depends on cerebral endothelial cell–matrix adhesion by β1-integrins

    PubMed Central

    Osada, Takashi; Gu, Yu-Huan; Kanazawa, Masato; Tsubota, Yoshiaki; Hawkins, Brian T; Spatz, Maria; Milner, Richard; del Zoppo, Gregory J

    2011-01-01

    The hypothesis tested by these studies states that in addition to interendothelial cell tight junction proteins, matrix adhesion by β1-integrin receptors expressed by endothelial cells have an important role in maintaining the cerebral microvessel permeability barrier. Primary brain endothelial cells from C57 BL/6 mice were incubated with β1-integrin function-blocking antibody (Ha2/5) or isotype control and the impacts on claudin-5 expression and microvessel permeability were quantified. Both flow cytometry and immunofluorescence studies demonstrated that the interendothelial claudin-5 expression by confluent endothelial cells was significantly decreased in a time-dependent manner by Ha2/5 exposure relative to isotype. Furthermore, to assess the barrier properties, transendothelial electrical resistance and permeability measurements of the monolayer, and stereotaxic injection into the striatum of mice were performed. Ha2/5 incubation reduced the resistance of endothelial cell monolayers significantly, and significantly increased permeability to 40 and 150 kDa dextrans. Ha2/5 injection into mouse striatum produced significantly greater IgG extravasation than the isotype or the control injections. This study demonstrates that blockade of β1-integrin function changes interendothelial claudin-5 expression and increases microvessel permeability. Hence, endothelial cell–matrix interactions via β1-integrin directly affect interendothelial cell tight junction claudin-5 expression and brain microvascular permeability. PMID:21772312

  12. Haptoglobin phenotype may alter endothelial progenitor cell cluster formation in cerebral small vessel disease.

    PubMed

    Rouhl, R P W; van Oostenbrugge, R J; Damoiseaux, J G M C; Debrus-Palmans, L L; Theunissen, R O M F I H; Knottnerus, I L H; Staals, J E A; Delanghe, J R; Tervaert, J W Cohen; Lodder, J

    2009-02-01

    Cerebral small vessel disease results in silent ischemic lesions (SIL) among which is leukoaraiosis. In this process, endothelial damage is probably involved. Endothelial progenitor cells (EPC), are involved in endothelial repair. By restoring the damaged endothelium, EPC could mitigate SIL and cerebral small vessel disease. Haptoglobin 1-1, one of three phenotypes of haptoglobin, relates to SIL and may therefore attenuate the endothelial repair by EPC. Our aim was to quantify EPC number and function and to assess haptoglobin phenotype and its effect on EPC function in patients with a high prevalence of SIL: lacunar stroke patients. We assessed EPC In 42 lacunar stroke patients and 18 controls by flow cytometry and culture with fetal calf serum, patient and control serum. We determined haptoglobin phenotype and cultured EPC with the three different haptoglobin phenotypes. We found that EPC cluster counts were lower in patients (96.9 clusters/well +/- 83.4 (mean +/- SD)), especially in those with SIL (85.0 +/- 64.3), than in controls (174.4 +/- 112.2). Cluster formation was inhibited by patient serum, especially by SIL patient serum, but not by control serum. Patients with haptoglobin 1-1 had less clusters in culture, and when haptoglobin 1-1 was added to EPC cultures, cluster numbers were lower than with the other haptoglobin phenotypes. We conclude that lacunar stroke patients, especially those with SIL, have impaired EPC cluster formation, which may point at decreased endothelial repair potential. The haptoglobin 1-1 phenotype is likely a causative factor in this impairment. PMID:19355924

  13. Real-time estimation of paracellular permeability of cerebral endothelial cells by capacitance sensor array

    NASA Astrophysics Data System (ADS)

    Hyun Jo, Dong; Lee, Rimi; Hyoung Kim, Jin; Oh Jun, Hyoung; Geol Lee, Tae; Hun Kim, Jeong

    2015-06-01

    Vascular integrity is important in maintaining homeostasis of brain microenvironments. In various brain diseases including Alzheimer’s disease, stroke, and multiple sclerosis, increased paracellular permeability due to breakdown of blood-brain barrier is linked with initiation and progression of pathological conditions. We developed a capacitance sensor array to monitor dielectric responses of cerebral endothelial cell monolayer, which could be utilized to evaluate the integrity of brain microvasculature. Our system measured real-time capacitance values which demonstrated frequency- and time-dependent variations. With the measurement of capacitance at the frequency of 100 Hz, we could differentiate the effects of vascular endothelial growth factor (VEGF), a representative permeability-inducing factor, on endothelial cells and quantitatively analyse the normalized values. Interestingly, we showed differential capacitance values according to the status of endothelial cell monolayer, confluent or sparse, evidencing that the integrity of monolayer was associated with capacitance values. Another notable feature was that we could evaluate the expression of molecules in samples in our system with the reference of real-time capacitance values. We suggest that this dielectric spectroscopy system could be successfully implanted as a novel in vitro assay in the investigation of the roles of paracellular permeability in various brain diseases.

  14. Adhesion and stress relaxation forces between melanoma and cerebral endothelial cells.

    PubMed

    Végh, Attila G; Fazakas, Csilla; Nagy, Krisztina; Wilhelm, Imola; Molnár, Judit; Krizbai, István A; Szegletes, Zsolt; Váró, György

    2012-02-01

    Mechanical parameters play a crucial role in proper cellular functions. This article examines the process of the appearance and breaking of adhesion forces during contact between the confluent cerebral endothelial cell layer and a melanoma cell attached to a tipless cantilever. This adhesion is the initial phase of melanoma transmigration through the endothelial cell layer. Taking the force measurement, if the contact was prolonged for several seconds, a decrease in the load force was observed, which corresponds to stress relaxation of the cells. The dependence of adhesion force and stress relaxation on dwell time showed a saturation-like behavior. These stress relaxation curves could be fitted with the sum of two exponentials, suggesting that two independent processes take place simultaneously. The breakup of the adhesion during the retraction of the cantilever with the attached melanoma cell is not continuous but shows jumps. Between living endothelial and melanoma cells, a minimum jump size of about 20 pN could be determined. The minimum jump is independent of the dwell time and load force. It seems to be the elementary binding force between these two cell types. In case of fixed endothelial cells, the adhesion force was strongly decreased and the jumps disappeared, whereas the stress relaxation did not show considerable change upon fixation. PMID:22038122

  15. Modulation of cerebral endothelial cell function by TGF-β in glioblastoma: VEGF-dependent angiogenesis versus endothelial mesenchymal transition.

    PubMed

    Krishnan, Shanmugarajan; Szabo, Emese; Burghardt, Isabel; Frei, Karl; Tabatabai, Ghazaleh; Weller, Michael

    2015-09-01

    Glioblastoma are among the most angiogenic tumors. The molecular mechanisms that control blood vessel formation by endothelial cells (EC) in glioblastoma remain incompletely understood. Transforming growth factor-β (TGF-β) is a key regulatory cytokine that has proinvasive and stemness-maintaining autocrine properties in glioblastoma and confers immunosuppression to the tumor microenvironment. Here we characterize potential pro- and anti-angiogenic activities of TGF-β in the context of glioblastoma in vitro, using human brain-derived microvascular endothelial cells (hCMEC/D3) and glioblastoma-derived endothelial cells (GMEC) as model systems. We find that TGF-β induces vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) mRNA expression and protein release in a TGF-β receptor (TβR) II / activin-like kinase (ALK)-5-dependent manner under normoxia and hypoxia, defining potential indirect proangiogenic activity of TGF-β in glioblastoma. In parallel, exogenous TGF-β has also inhibitory effects on EC properties and induces endothelial-mesenchymal transition (EndMT) in hCMEC and GMEC. Accordingly, direct inhibition of endogenous TGF-β/ALK-5 signalling increases EC properties such as tube formation, von-Willebrand factor (vWF) and claudin (CLDN) 5 expression. Yet, the supernatant of TGF-β-stimulated hCMEC and GMEC strongly promotes EC-related gene expression and tube formation in a cediranib-sensitive manner. These observations shed light on the complex pro- and anti-angiogenic pathways involving the cross-talk between TGF-β and VEGF/PLGF signalling in glioblastoma which may involve parallel stimulation of angiogenesis and EndMT in distinct target cell populations.

  16. 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.

  17. The Age of Human Cerebral Cortex Neurons

    SciTech Connect

    Bhardwaj, R D; Curtis, M A; Spalding, K L; Buchholz, B A; Fink, D; Bjork-Eriksson, T; Nordborg, C; Gage, F H; Druid, H; Eriksson, P S; Frisen, J

    2006-04-06

    The traditional static view of the adult mammalian brain has been challenged by the realization of continuous generation of neurons from stem cells. Based mainly on studies in experimental animals, adult neurogenesis may contribute to recovery after brain insults and decreased neurogenesis has been implicated in the pathogenesis of neurological and psychiatric diseases in man. The extent of neurogenesis in the adult human brain has, however, been difficult to establish. We have taken advantage of the integration of {sup 14}C, generated by nuclear bomb tests during the Cold War, in DNA to establish the age of neurons in the major areas of the human cerebral cortex. Together with the analysis of the cortex from patients who received BrdU, which integrates in the DNA of dividing cells, our results demonstrate that whereas non-neuronal cells turn over, neurons in the human cerebral cortex are not generated postnatally at detectable levels, but are as old as the individual.

  18. Modeling human brain development with cerebral organoids.

    PubMed

    Muzio, Luca; Consalez, G Giacomo

    2013-01-01

    The recent discovery of a new three-dimensional culture system for the derivation of cerebral organoids from human induced pluripotent stem cells provides developmental neurobiologists with the first example of a three-dimensional framework for the study of human brain development. This innovative approach permits the in vitro assembly of a human embryonic brain rudiment that recapitulates the developing human cerebrum. Organoids contain progenitor populations that develop to yield mature cortical neuron subtypes, potentially allowing investigators to study complex brain diseases that lack appropriate animal models. PMID:24367992

  19. 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

  20. Amyloid-β peptide on sialyl-Lewis(X)-selectin-mediated membrane tether mechanics at the cerebral endothelial cell surface.

    PubMed

    Askarova, Sholpan; Sun, Zhe; Sun, Grace Y; Meininger, Gerald A; Lee, James C-M

    2013-01-01

    Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer's disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewis(x) (sLe(x)) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLe(x) and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf ), and produced a bimodal population of Fmtf , suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf . In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB.

  1. Amyloid-β Peptide on Sialyl-LewisX-Selectin-Mediated Membrane Tether Mechanics at the Cerebral Endothelial Cell Surface

    PubMed Central

    Askarova, Sholpan; Sun, Zhe; Sun, Grace Y.; Meininger, Gerald A.; Lee, James C-M.

    2013-01-01

    Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer's disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewisx (sLex) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLex and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf), and produced a bimodal population of Fmtf, suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf. In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB. PMID:23593361

  2. Amyloid-β peptide on sialyl-Lewis(X)-selectin-mediated membrane tether mechanics at the cerebral endothelial cell surface.

    PubMed

    Askarova, Sholpan; Sun, Zhe; Sun, Grace Y; Meininger, Gerald A; Lee, James C-M

    2013-01-01

    Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer's disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewis(x) (sLe(x)) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLe(x) and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf ), and produced a bimodal population of Fmtf , suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf . In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB. PMID:23593361

  3. Microparticles generated during chronic cerebral ischemia deliver proapoptotic signals to cultured endothelial cells

    SciTech Connect

    Schock, Sarah C.; Edrissi, Hamidreza; Burger, Dylan; Cadonic, Robert; Hakim, Antoine; Thompson, Charlie

    2014-07-18

    Highlights: • Microparticles are elevated in the plasma in a rodent model of chronic cerebral ischemia. • These microparticles initiate apoptosis in cultured cells. • Microparticles contain caspase 3 and they activate receptors for TNF-α and TRAIL. - Abstract: Circulating microparticles (MPs) are involved in many physiological processes and numbers are increased in a variety of cardiovascular disorders. The present aims were to characterize levels of MPs in a rodent model of chronic cerebral hypoperfusion (CCH) and to determine their signaling properties. MPs were isolated from the plasma of rats exposed to CCH and quantified by flow cytometry. When MPs were added to cultured endothelial cells or normal rat kidney cells they induced cell death in a time and dose dependent manner. Analysis of pellets by electron microscopy indicates that cell death signals are carried by particles in the range of 400 nm in diameter or less. Cell death involved the activation of caspase 3 and was not a consequence of oxidative stress. Inhibition of the Fas/FasL signaling pathway also did not improve cell survival. MPs were found to contain caspase 3 and treating the MPs with a caspase 3 inhibitor significantly reduced cell death. A TNF-α receptor blocker and a TRAIL neutralizing antibody also significantly reduced cell death. Levels of circulating MPs are elevated in a rodent model of chronic cerebral ischemia. MPs with a diameter of 400 nm or less activate the TNF-α and TRAIL signaling pathways and may deliver caspase 3 to cultured cells.

  4. 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.

  5. 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

  6. Regulation of endothelial nitric oxide synthase by agmatine after transient global cerebral ischemia in rat brain.

    PubMed

    Mun, Chin Hee; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

    2010-09-01

    Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) plays a protective role in cerebral ischemia by maintaining vascular permeability, whereas NO derived from neuronal and inducible NOS is neurotoxic and can participate in neuronal damage occurring in ischemia. Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane if brain vessels to promote cell death and tissue injury. We previously reported that agmatine, synthesized from L-arginine by arginine decarboxylase (ADC) which is expressed in endothelial cells, has shown a direct increased eNOS expression and decreased MMPs expression in bEnd3 cells. But, there are few reports about the regulation of eNOS by agmatine in ischemic animal model. In the present study, we examined the expression of eNOS and MMPs by agmatine treatment after transient global ischemia in vivo. Global ischemia was induced with four vessel occlusion (4-VO) and agmatine (100 mg/kg) was administered intraperitoneally at the onset of reperfusion. The animals were euthanized at 6 and 24 hours after global ischemia and prepared for other analysis. Global ischemia led severe neuronal damage in the rat hippocampus and cerebral cortex, but agmatine treatment protected neurons from ischemic injury. Moreover, the level and expression of eNOS was increased by agmatine treatment, whereas inducible NOS (iNOS) and MMP-9 protein expressions were decreased in the brain. These results suggest that agmatine protects microvessels in the brain by activation eNOS as well as reduces extracellular matrix degradation during the early phase of ischemic insult.

  7. Regulation of endothelial nitric oxide synthase by agmatine after transient global cerebral ischemia in rat brain.

    PubMed

    Mun, Chin Hee; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

    2010-09-01

    Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) plays a protective role in cerebral ischemia by maintaining vascular permeability, whereas NO derived from neuronal and inducible NOS is neurotoxic and can participate in neuronal damage occurring in ischemia. Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane if brain vessels to promote cell death and tissue injury. We previously reported that agmatine, synthesized from L-arginine by arginine decarboxylase (ADC) which is expressed in endothelial cells, has shown a direct increased eNOS expression and decreased MMPs expression in bEnd3 cells. But, there are few reports about the regulation of eNOS by agmatine in ischemic animal model. In the present study, we examined the expression of eNOS and MMPs by agmatine treatment after transient global ischemia in vivo. Global ischemia was induced with four vessel occlusion (4-VO) and agmatine (100 mg/kg) was administered intraperitoneally at the onset of reperfusion. The animals were euthanized at 6 and 24 hours after global ischemia and prepared for other analysis. Global ischemia led severe neuronal damage in the rat hippocampus and cerebral cortex, but agmatine treatment protected neurons from ischemic injury. Moreover, the level and expression of eNOS was increased by agmatine treatment, whereas inducible NOS (iNOS) and MMP-9 protein expressions were decreased in the brain. These results suggest that agmatine protects microvessels in the brain by activation eNOS as well as reduces extracellular matrix degradation during the early phase of ischemic insult. PMID:21212863

  8. Microparticles generated during chronic cerebral ischemia deliver proapoptotic signals to cultured endothelial cells.

    PubMed

    Schock, Sarah C; Edrissi, Hamidreza; Burger, Dylan; Cadonic, Robert; Hakim, Antoine; Thompson, Charlie

    2014-07-18

    Circulating microparticles (MPs) are involved in many physiological processes and numbers are increased in a variety of cardiovascular disorders. The present aims were to characterize levels of MPs in a rodent model of chronic cerebral hypoperfusion (CCH) and to determine their signaling properties. MPs were isolated from the plasma of rats exposed to CCH and quantified by flow cytometry. When MPs were added to cultured endothelial cells or normal rat kidney cells they induced cell death in a time and dose dependent manner. Analysis of pellets by electron microscopy indicates that cell death signals are carried by particles in the range of 400 nm in diameter or less. Cell death involved the activation of caspase 3 and was not a consequence of oxidative stress. Inhibition of the Fas/FasL signaling pathway also did not improve cell survival. MPs were found to contain caspase 3 and treating the MPs with a caspase 3 inhibitor significantly reduced cell death. A TNF-α receptor blocker and a TRAIL neutralizing antibody also significantly reduced cell death. Levels of circulating MPs are elevated in a rodent model of chronic cerebral ischemia. MPs with a diameter of 400 nm or less activate the TNF-α and TRAIL signaling pathways and may deliver caspase 3 to cultured cells. PMID:24976400

  9. Effect of surface charge of immortalized mouse cerebral endothelial cell monolayer on transport of charged solutes.

    PubMed

    Yuan, Wei; Li, Guanglei; Gil, Eun Seok; Lowe, Tao Lu; Fu, Bingmei M

    2010-04-01

    Charge carried by the surface glycocalyx layer (SGL) of the cerebral endothelium has been shown to significantly modulate the permeability of the blood-brain barrier (BBB) to charged solutes in vivo. The cultured monolayer of bEnd3, an immortalized mouse cerebral endothelial cell line, is becoming a popular in vitro BBB model due to its easy growth and maintenance of many BBB characteristics over repeated passages. To test whether the SGL of bEnd3 monolayer carries similar charge as that in the intact BBB and quantify this charge, which can be characterized by the SGL thickness (L(f)) and charge density (C(mf)), we measured the solute permeability of bEnd3 monolayer to neutral solutes and to solutes with similar size but opposite charges: negatively charged alpha-lactalbumin (-11) and positively charged ribonuclease (+3). Combining the measured permeability data with a transport model across the cell monolayer, we predicted the L(f) and the C(mf) of bEnd3 monolayer, which is approximately 160 nm and approximately 25 mEq/L, respectively. We also investigated whether orosomucoid, a plasma glycoprotein modulating the charge of the intact BBB, alters the charge of bEnd3 monolayer. We found that 1 mg/mL orosomucoid would increase SGL charge density of bEnd3 monolayer to approximately 2-fold of its control value.

  10. Effect of surface charge of immortalized mouse cerebral endothelial cell monolayer on transport of charged solutes.

    PubMed

    Yuan, Wei; Li, Guanglei; Gil, Eun Seok; Lowe, Tao Lu; Fu, Bingmei M

    2010-04-01

    Charge carried by the surface glycocalyx layer (SGL) of the cerebral endothelium has been shown to significantly modulate the permeability of the blood-brain barrier (BBB) to charged solutes in vivo. The cultured monolayer of bEnd3, an immortalized mouse cerebral endothelial cell line, is becoming a popular in vitro BBB model due to its easy growth and maintenance of many BBB characteristics over repeated passages. To test whether the SGL of bEnd3 monolayer carries similar charge as that in the intact BBB and quantify this charge, which can be characterized by the SGL thickness (L(f)) and charge density (C(mf)), we measured the solute permeability of bEnd3 monolayer to neutral solutes and to solutes with similar size but opposite charges: negatively charged alpha-lactalbumin (-11) and positively charged ribonuclease (+3). Combining the measured permeability data with a transport model across the cell monolayer, we predicted the L(f) and the C(mf) of bEnd3 monolayer, which is approximately 160 nm and approximately 25 mEq/L, respectively. We also investigated whether orosomucoid, a plasma glycoprotein modulating the charge of the intact BBB, alters the charge of bEnd3 monolayer. We found that 1 mg/mL orosomucoid would increase SGL charge density of bEnd3 monolayer to approximately 2-fold of its control value. PMID:20087768

  11. Transcranial laser stimulation improves human cerebral oxygenation

    PubMed Central

    Tian, Fenghua; Hase, Snehal N.

    2016-01-01

    Background and Objective Transcranial laser stimulation of the brain with near‐infrared light is a novel form of non‐invasive photobiomodulation or low‐level laser therapy (LLLT) that has shown therapeutic potential in a variety of neurological and psychological conditions. Understanding of its neurophysiological effects is essential for mechanistic study and treatment evaluation. This study investigated how transcranial laser stimulation influences cerebral hemodynamics and oxygenation in the human brain in vivo using functional near‐infrared spectroscopy (fNIRS). Materials and Methods Two separate experiments were conducted in which 1,064‐nm laser stimulation was administered at (1) the center and (2) the right side of the forehead, respectively. The laser emitted at a power of 3.4 W and in an area of 13.6 cm2, corresponding to 0.25 W/cm2 irradiance. Stimulation duration was 10 minutes. Nine healthy male and female human participants of any ethnic background, in an age range of 18–40 years old were included in each experiment. Results In both experiments, transcranial laser stimulation induced an increase of oxygenated hemoglobin concentration (Δ[HbO2]) and a decrease of deoxygenated hemoglobin concentration (Δ[Hb]) in both cerebral hemispheres. Improvements in cerebral oxygenation were indicated by a significant increase of differential hemoglobin concentration (Δ[HbD] = Δ[HbO2] − Δ[Hb]). These effects increased in a dose‐dependent manner over time during laser stimulation (10 minutes) and persisted after laser stimulation (6 minutes). The total hemoglobin concentration (Δ[HbT] = Δ[HbO2] + Δ[Hb]) remained nearly unchanged in most cases. Conclusion Near‐infrared laser stimulation applied to the forehead can transcranially improve cerebral oxygenation in healthy humans. Lasers Surg. Med. 48:343–349, 2016. © 2016 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc. PMID:26817446

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

    PubMed Central

    Chou, Chung-Hsing; Modo, Michel

    2016-01-01

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

  14. LPS Induces Occludin Dysregulation in Cerebral Microvascular Endothelial Cells via MAPK Signaling and Augmenting MMP-2 Levels

    PubMed Central

    Qin, Lan-hui; Huang, Wen; Mo, Xue-an; Chen, Yan-lan; Wu, Xiang-hong

    2015-01-01

    Disrupted blood-brain barrier (BBB) integrity contributes to cerebral edema during central nervous system infection. The current study explored the mechanism of lipopolysaccharide- (LPS-) induced dysregulation of tight junction (TJ) proteins. Human cerebral microvascular endothelial cells (hCMEC/D3) were exposed to LPS, SB203580 (p38MAPK inhibitor), or SP600125 (JNK inhibitor), and cell vitality was determined by MTT assay. The proteins expressions of p38MAPK, JNK, and TJs (occludin and zonula occludens- (ZO-) 1) were determined by western blot. The mRNA levels of TJ components and MMP-2 were measured with quantitative real-time polymerase chain reaction (qRT-PCR), and MMP-2 protein levels were determined by enzyme-linked immunosorbent assay (ELISA). LPS, SB203580, and SP600125 under respective concentrations of 10, 7.69, or 0.22 µg/mL had no effects on cell vitality. Treatment with LPS decreased mRNA and protein levels of occludin and ZO-1 and enhanced p38MAPK and JNK phosphorylation and MMP-2 expression. These effects were attenuated by pretreatment with SB203580 or SP600125, but not in ZO-1 expression. Both doxycycline hyclate (a total MMP inhibitor) and SB-3CT (a specific MMP-2 inhibitor) partially attenuated the LPS-induced downregulation of occludin. These data suggest that MMP-2 overexpression and p38MAPK/JNK pathways are involved in the LPS-mediated alterations of occludin in hCMEC/D3; however, ZO-1 levels are not influenced by p38MAPK/JNK. PMID:26290681

  15. 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.

  16. Loss of endothelial protein C receptors links coagulation and inflammation to parasite sequestration in cerebral malaria in African children

    PubMed Central

    Wassmer, Samuel C.; Milner, Danny A.; Chisala, Ngawina V.; Taylor, Terrie E.; Seydel, Karl B.; Molyneux, Malcolm E.; Faragher, Brian; Esmon, Charles T.; Downey, Colin; Toh, Cheng-Hock; Craig, Alister G.; Heyderman, Robert S.

    2013-01-01

    Cerebral malaria (CM) is a major cause of mortality in African children and the mechanisms underlying its development, namely how malaria-infected erythrocytes (IEs) cause disease and why the brain is preferentially affected, remain unclear. Brain microhemorrhages in CM suggest a clotting disorder, but whether this phenomenon is important in pathogenesis is debated. We hypothesized that localized cerebral microvascular thrombosis in CM is caused by a decreased expression of the anticoagulant and protective receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR) and that low constitutive expression of these regulatory molecules in the brain make it particularly vulnerable. Autopsies from Malawian children with CM showed cerebral fibrin clots and loss of EPCR, colocalized with sequestered IEs. Using a novel assay to examine endothelial phenotype ex vivo using subcutaneous microvessels, we demonstrated that loss of EPCR and TM at sites of IE cytoadherence is detectible in nonfatal CM. In contrast, although clotting factor activation was seen in the blood of CM patients, this was compensated and did not disseminate. Because of the pleiotropic nature of EPCR and TM, these data implicate disruption of the endothelial protective properties at vulnerable sites and particularly in the brain, linking coagulation and inflammation with IE sequestration. PMID:23741007

  17. Recombinant tissue-type plasminogen activator transiently enhances blood-brain barrier permeability during cerebral ischemia through vascular endothelial growth factor-mediated endothelial endocytosis in mice.

    PubMed

    Suzuki, Yasuhiro; Nagai, Nobuo; Yamakawa, Kasumi; Muranaka, Yoshinori; Hokamura, Kazuya; Umemura, Kazuo

    2015-12-01

    Recombinant tissue-type plasminogen activator (rt-PA) modulates cerebrovascular permeability and exacerbates brain injury in ischemic stroke, but its mechanisms remain unclear. We studied the involvement of vascular endothelial growth factor (VEGF)-mediated endocytosis in the increase of blood-brain barrier (BBB) permeability potentiated by rt-PA after ischemic stroke. The rt-PA treatment at 4 hours after middle cerebral artery occlusion induced a transient increase in BBB permeability after ischemic stroke in mice, which was suppressed by antagonists of either low-density lipoprotein receptor families (LDLRs) or VEGF receptor-2 (VEGFR-2). In immortalized bEnd.3 endothelial cells, rt-PA treatment upregulated VEGF expression and VEGFR-2 phosphorylation under ischemic conditions in an LDLR-dependent manner. In addition, rt-PA treatment increased endocytosis and transcellular transport in bEnd.3 monolayers under ischemic conditions, which were suppressed by the inhibition of LDLRs, VEGF, or VEGFR-2. The rt-PA treatment also increased the endocytosis of endothelial cells in the ischemic brain region after stroke in mice. These findings indicate that rt-PA increased BBB permeability via induction of VEGF, which at least partially mediates subsequent increase in endothelial endocytosis. Therefore, inhibition of VEGF induction may have beneficial effects after thrombolytic therapy with rt-PA treatment after stroke.

  18. 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.

  19. Modest Visceral Fat Gain Causes Endothelial Dysfunction In Healthy Humans

    PubMed Central

    Romero-Corral, Abel; Sert-Kuniyoshi, Fatima H.; Sierra-Johnson, Justo; Orban, Marek; Gami, Apoor; Davison, Diane; Singh, Prachi; Pusalavidyasagar, Snigdha; Huyber, Christine; Votruba, Susanne; Lopez-Jimenez, Francisco; Jensen, Michael D.; Somers, Virend K.

    2014-01-01

    Objective This study sought to determine the impact of fat gain and its distribution on endothelial function in lean healthy humans. Background Endothelial dysfunction has been identified as an independent predictor of cardiovascular events. Whether fat gain impairs endothelial function is unknown. Methods A randomized controlled study to assess the effects of fat gain on endothelial function. We recruited 43 normal weight healthy volunteers (mean age 29 years; 18 women). Subjects were assigned to gain weight (approximately 4 kg) (n=35) or to maintain weight (n=8). Endothelial function (brachial artery flow mediated dilation -FMD) was measured at baseline, after fat gain (8 weeks) and after weight loss (16 weeks) for fat-gainers and at baseline and follow-up (8 weeks) for weight-maintainers. Body composition was measured by DXA and abdominal CT scans. Results After an average weight gain of 4.1 kg, fat-gainers significantly increased their total, visceral and subcutaneous fat. Blood pressure and overnight polysomnography did not change after fat gain or loss. FMD remained unchanged in weight-maintainers. FMD decreased in fat-gainers (9.1 ± 3% vs. 7.8 ± 3.2%, p =0.003), but recovered to baseline when subjects shed the gained weight. There was a significant correlation between the decrease in FMD and the increase in visceral fat gain (rho = −0.42, p=0.004), but not with subcutaneous fat gain (rho = −0.22, p=0.15). Conclusions In normal weight healthy young subjects, modest fat gain results in impaired endothelial function, even in the absence of changes in blood pressure. Endothelial function recovers after weight loss. Increased visceral rather than subcutaneous fat predicts endothelial dysfunction. PMID:20705223

  20. 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

  1. Pharmacological neutropenia prevents endothelial dysfunction but not smooth muscle functions impairment induced by middle cerebral artery occlusion

    PubMed Central

    Pétrault, Olivier; Ouk, Thavarak; Gautier, Sophie; Laprais, Maud; Gelé, Patrick; Bastide, Michèle; Bordet, Régis

    2005-01-01

    The polymorphonuclear neutrophils (PMN) activation and mobilization observed in acute cerebral infarction contribute to the brain tissue damage, but PMN could also be involved in postischemic functional injury of ischemied blood vessel. This study was undertaken to investigate whether pharmacological neutropenia could modify the postischemic endothelial dysfunction in comparison to smooth muscle whose impairment is likely more related to reperfusion and oxidative stress. A cerebral ischemia–reperfusion by endoluminal occlusion of right middle cerebral artery (MCA) was performed 4 days after intravenous administration of vinblastine or 12 h after RP-3 anti-rat neutrophils monoclonal antibody (mAb RP-3) injection into the peritoneal cavity, on male Wistar rats with 1-h ischemia then followed by 24-h reperfusion period. Brain infarct volume was measured by histomorphometric analysis and vascular endothelial and smooth muscle reactivity of MCA was analysed using Halpern myograph. Neutropenia induced a neuroprotective effect as demonstrated by a significant decrease of brain infarct size. In parallel to neuroprotection, neutropenia prevented postischemic impairment of endothelium-dependent relaxing response to acetylcholine. In contrast, smooth muscle functional alterations were not prevented by neutropenia. Ischemia–reperfusion-induced myogenic tone impairment remained unchanged in vinblastine and mAb RP-3-treated rats. Postischemic Kir2.x-dependent relaxation impairment was not prevented in neutropenic conditions. The fully relaxation of smooth muscle response to sodium nitroprusside was similar in all groups. Our results evidenced the dissociate prevention of pharmacologically induced neutropenia on postischemic vascular endothelial and smooth muscle impairment. The selective endothelial protection by neutropenia is parallel to a neuroprotective effect suggesting a possible relationship between the two phenomena. PMID:15700030

  2. 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.

  3. The human cerebral cortex flattens during adolescence.

    PubMed

    Alemán-Gómez, Yasser; Janssen, Joost; Schnack, Hugo; Balaban, Evan; Pina-Camacho, Laura; Alfaro-Almagro, Fidel; Castro-Fornieles, Josefina; Otero, Soraya; Baeza, Immaculada; Moreno, Dolores; Bargalló, Nuria; Parellada, Mara; Arango, Celso; Desco, Manuel

    2013-09-18

    The human cerebral cortex appears to shrink during adolescence. To delineate the dynamic morphological changes involved in this process, 52 healthy male and female adolescents (11-17 years old) were neuroimaged twice using magnetic resonance imaging, approximately 2 years apart. Using a novel morphometric analysis procedure combining the FreeSurfer and BrainVisa image software suites, we quantified global and lobar change in cortical thickness, outer surface area, the gyrification index, the average Euclidean distance between opposing sides of the white matter surface (gyral white matter thickness), the convex ("exposed") part of the outer cortical surface (hull surface area), sulcal length, depth, and width. We found that the cortical surface flattens during adolescence. Flattening was strongest in the frontal and occipital cortices, in which significant sulcal widening and decreased sulcal depth co-occurred. Globally, sulcal widening was associated with cortical thinning and, for the frontal cortex, with loss of surface area. For the other cortical lobes, thinning was related to gyral white matter expansion. The overall flattening of the macrostructural three-dimensional architecture of the human cortex during adolescence thus involves changes in gray matter and effects of the maturation of white matter.

  4. The human cerebral cortex flattens during adolescence.

    PubMed

    Alemán-Gómez, Yasser; Janssen, Joost; Schnack, Hugo; Balaban, Evan; Pina-Camacho, Laura; Alfaro-Almagro, Fidel; Castro-Fornieles, Josefina; Otero, Soraya; Baeza, Immaculada; Moreno, Dolores; Bargalló, Nuria; Parellada, Mara; Arango, Celso; Desco, Manuel

    2013-09-18

    The human cerebral cortex appears to shrink during adolescence. To delineate the dynamic morphological changes involved in this process, 52 healthy male and female adolescents (11-17 years old) were neuroimaged twice using magnetic resonance imaging, approximately 2 years apart. Using a novel morphometric analysis procedure combining the FreeSurfer and BrainVisa image software suites, we quantified global and lobar change in cortical thickness, outer surface area, the gyrification index, the average Euclidean distance between opposing sides of the white matter surface (gyral white matter thickness), the convex ("exposed") part of the outer cortical surface (hull surface area), sulcal length, depth, and width. We found that the cortical surface flattens during adolescence. Flattening was strongest in the frontal and occipital cortices, in which significant sulcal widening and decreased sulcal depth co-occurred. Globally, sulcal widening was associated with cortical thinning and, for the frontal cortex, with loss of surface area. For the other cortical lobes, thinning was related to gyral white matter expansion. The overall flattening of the macrostructural three-dimensional architecture of the human cortex during adolescence thus involves changes in gray matter and effects of the maturation of white matter. PMID:24048830

  5. 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

  6. 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

  7. 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.

  8. Subtractive transcriptomics : establishing polarity drives human endothelial morphogenesis

    SciTech Connect

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

    2006-04-15

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

  9. Design and physicochemical characterization of poly(amidoamine) nanoparticles and the toxicological evaluation in human endothelial cells: applications to peptide delivery to the brain.

    PubMed

    Coué, Grégory; Freese, Christian; Unger, Ronald E; Kirkpatrick, C James; Pickl, Karin E; Sinner, Frank M; Engbersen, Johan F J

    2013-01-01

    In this study, we investigated nanoparticles formulated by self-assembly of a biodegradable poly(amidoamine) (PAA) and a fluorescently labeled peptide, in their capacity to internalize in endothelial cells and deliver the peptide, with possible applications for brain drug delivery. The nanoparticles were characterized in terms of size, surface charge, and loading efficiency, and were applied on human cerebral microvascular endothelial cells (hCMEC/D3) and human umbilical vein endothelial cells (Huvec) cells. Cell-internalization and cytotoxicity experiments showed that the PAA-based nanocomplexes were essentially nontoxic, and the peptide was successfully internalized into cells. The results indicate that these PAAs have an excellent property as nontoxic carriers for intracellular protein and peptide delivery, and provide opportunities for novel applications in the delivery of peptides to endothelial cells of the brain.

  10. 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

  11. Mineralocorticoid Receptors Modulate Vascular Endothelial Function in Human Obesity

    PubMed Central

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

    2015-01-01

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

  12. Correlations between endothelial function in the systemic and cerebral circulation and insulin resistance in type 2 diabetes mellitus.

    PubMed

    Prakash, Kiran; Chandran, Dinu S; Khadgawat, Rajesh; Jaryal, Ashok Kumar; Deepak, Kishore K

    2016-01-01

    Insulin resistance is associated with endothelial dysfunction in type 2 diabetes mellitus, which can lead to impaired vascular reactivities of both systemic and cerebral circulations. Appropriate 'correction' of vascular reactivity results for non-endothelium-dependent systemic effects avoids misinterpretation of endothelial function. Therefore, we 'corrected' vascular reactivity results and explored the potential correlations between systemic vascular reactivity, cerebrovascular reactivity and insulin resistance. In 34 patients, 'systemic vascular reactivity' was assessed by quantifying reactive hyperaemia. Cerebrovascular reactivity was assessed by quantifying changes in cerebral blood flow velocity during hypercapnia. To minimize the influence of non-endothelium-dependent systemic effects on vascular reactivity results, 'corrected systemic vascular reactivity' was calculated by normalizing systemic vascular reactivity using the measurements from the contralateral side; and cerebrovascular reactivity results were corrected by calculating percentage and absolute changes in cerebrovascular conductance index ('percent cerebrovascular conductance index' and 'delta cerebrovascular conductance index', respectively). Insulin resistance was estimated by homeostatic model assessment. Correlation between conventional cerebrovascular reactivity and systemic vascular reactivity was not significant. But correlations between 'corrected systemic vascular reactivity' and 'percent cerebrovascular conductance index' (r = 0.51; p = 0.002) and 'corrected systemic vascular reactivity' and 'delta cerebrovascular conductance index' (r = 0.50; p = 0.003) were significant. Among all vascular reactivity parameters, only 'delta cerebrovascular conductance index' was significantly correlated with homeostatic model assessment of insulin resistance (r = -0.38; p = 0.029). In conclusion, endothelial function in the systemic and cerebral circulations is moderately

  13. Influence of Hypoxia on Cerebral Blood Flow Regulation in Humans.

    PubMed

    Steinback, Craig D; Poulin, Marc J

    2016-01-01

    The brain is a vital organ that relies on a constant and adequate supply of blood to match oxygen and glucose delivery with the local metabolic demands of active neurones. It is well established that cerebral blood flow is altered in response to both neural activity and humoral stimuli. Thus, augmented neural activation (e.g. visual stimulation) leads to locally increased cerebral blood flow via functional hyperaemia, whereas humoral stimuli (i.e. alterations in arterial PO2 and PCO2) produce global increases in cerebral blood flow. Perhaps not surprisingly, cerebrovascular responses to neural activity and humoral stimuli may not be highly correlated because they reflect different physiological mechanisms for vasodilation. Exquisite regulation of cerebral blood flow is particularly important under hypoxic conditions when cerebral PO2 can be reduced substantially. Indeed, cerebrovascular reactivity to hypoxia determines the capacity of cerebral vessels to respond and compensate for a reduced oxygen supply. This reactivity is dynamic, changing with prolonged exposure to hypoxic environments, and in patients and healthy individuals exposed to chronic intermittent periods of hypoxia. More recently, a number of animal studies have provided evidence that glial cells (i.e. astrocytes) play an important role in regulating cerebral blood flow under normoxic and hypoxic conditions. This review aims to summarize our current understanding of cerebral blood flow control during hypoxia in humans and put into context the underlying neurovascular mechanisms that may contribute to this regulation. PMID:27343093

  14. Analysis of the endothelial nitric oxide synthase gene as a modifier of the cerebral response to ischemia.

    PubMed

    Dutra, Ana Virginia; Lin, Hsiu-Fen; Juo, Suh-Hang Hank; Boyadjis, Melanie; Moussouttas, Michael; Reddy, P Leema; Grewal, Raji Paul

    2006-01-01

    We studied the endothelial nitric oxide synthase (eNOS or NOS-3) gene as a potential modifier of the cerebral response to ischemia by investigating the association of two common polymorphisms with ischemic stroke volume. We genotyped an intronic variable number tandem repeat and a single nucleotide polymorphism, G894T, in 132 patients with nonlacunar ischemic strokes in whom clinical data and stroke lesion volume were recorded. Our results show that all genotypes are in Hardy-Weinberg equilibrium. After adjustment of covariates, neither of the NOS-3 polymorphisms showed significant differences comparing the genotypes and mean stroke volume (analysis of variance). Our results do not suggest a major gene effect of the NOS-3 gene as a modifier of the cerebral response to ischemia. PMID:17904064

  15. 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.

  16. 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

  17. 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

  18. Cerebral organoids model human brain development and microcephaly.

    PubMed

    Lancaster, Madeline A; Renner, Magdalena; Martin, Carol-Anne; Wenzel, Daniel; Bicknell, Louise S; Hurles, Matthew E; Homfray, Tessa; Penninger, Josef M; Jackson, Andrew P; Knoblich, Juergen A

    2013-09-19

    The complexity of the human brain has made it difficult to study many brain disorders in model organisms, highlighting the need for an in vitro model of human brain development. Here we have developed a human pluripotent stem cell-derived three-dimensional organoid culture system, termed cerebral organoids, that develop various discrete, although interdependent, brain regions. These include a cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes. Furthermore, cerebral organoids are shown to recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells. Finally, we use RNA interference and patient-specific induced pluripotent stem cells to model microcephaly, a disorder that has been difficult to recapitulate in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could help to explain the disease phenotype. Together, these data show that three-dimensional organoids can recapitulate development and disease even in this most complex human tissue.

  19. Cerebral organoids model human brain development and microcephaly

    PubMed Central

    Lancaster, Madeline A.; Renner, Magdalena; Martin, Carol-Anne; Wenzel, Daniel; Bicknell, Louise S.; Hurles, Matthew E.; Homfray, Tessa; Penninger, Josef M.; Jackson, Andrew P.; Knoblich, Juergen A.

    2013-01-01

    The complexity of the human brain has made it difficult to study many brain disorders in model organisms, and highlights the need for an in vitro model of human brain development. We have developed a human pluripotent stem cell-derived 3D organoid culture system, termed cerebral organoid, which develops various discrete though interdependent brain regions. These include cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes. Furthermore, cerebral organoids recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells. Finally, we use RNAi and patient-specific iPS cells to model microcephaly, a disorder that has been difficult to recapitulate in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could explain the disease phenotype. Our data demonstrate that 3D organoids can recapitulate development and disease of even this most complex human tissue. PMID:23995685

  20. Infection of human endothelial cells by human T-lymphotropic virus type I.

    PubMed Central

    Ho, D D; Rota, T R; Hirsch, M S

    1984-01-01

    We studied the effects of human T-lymphotropic virus type I (HTLV-I) on human endothelial cells in vitro. During cocultivation with an HTLV-I producer cell line (C91/PL), endothelial cells formed characteristic multinucleated syncytial giant cells. Inoculation with concentrated cell-free supernatant fluid from C91/PL cultures produced similar cytopathic effects, which were neutralized by pretreatment with HTLV-I specific human serum. HTLV-I antigens were detected in the cytoplasm of the multinucleated cells by indirect immunofluorescence. When endothelial cells showed maximal cytopathic changes, reverse transcriptase activity was demonstrated in the supernatant fluid and HTLV-I was isolated by cocultivation with peripheral blood mononuclear cells. This study demonstrates that HTLV-I tropism is not limited to lymphoid cells but extends to human endothelial cells as well. Images PMID:6095308

  1. 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.

  2. 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.

  3. Autonomic neural control of dynamic cerebral autoregulation in humans

    NASA Technical Reports Server (NTRS)

    Zhang, Rong; Zuckerman, Julie H.; Iwasaki, Kenichi; Wilson, Thad E.; Crandall, Craig G.; Levine, Benjamin D.

    2002-01-01

    BACKGROUND: The purpose of the present study was to determine the role of autonomic neural control of dynamic cerebral autoregulation in humans. METHODS AND RESULTS: We measured arterial pressure and cerebral blood flow (CBF) velocity in 12 healthy subjects (aged 29+/-6 years) before and after ganglion blockade with trimethaphan. CBF velocity was measured in the middle cerebral artery using transcranial Doppler. The magnitude of spontaneous changes in mean blood pressure and CBF velocity were quantified by spectral analysis. The transfer function gain, phase, and coherence between these variables were estimated to quantify dynamic cerebral autoregulation. After ganglion blockade, systolic and pulse pressure decreased significantly by 13% and 26%, respectively. CBF velocity decreased by 6% (P<0.05). In the very low frequency range (0.02 to 0.07 Hz), mean blood pressure variability decreased significantly (by 82%), while CBF velocity variability persisted. Thus, transfer function gain increased by 81%. In addition, the phase lead of CBF velocity to arterial pressure diminished. These changes in transfer function gain and phase persisted despite restoration of arterial pressure by infusion of phenylephrine and normalization of mean blood pressure variability by oscillatory lower body negative pressure. CONCLUSIONS: These data suggest that dynamic cerebral autoregulation is altered by ganglion blockade. We speculate that autonomic neural control of the cerebral circulation is tonically active and likely plays a significant role in the regulation of beat-to-beat CBF in humans.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. Rapid nontranscriptional activation of endothelial nitric oxide synthase mediates increased cerebral blood flow and stroke protection by corticosteroids

    PubMed Central

    Limbourg, Florian P.; Huang, Zhihong; Plumier, Jean-Christophe; Simoncini, Tommaso; Fujioka, Masayuki; Tuckermann, Jan; Schütz, Günther; Moskowitz, Michael A.; Liao, James K.

    2002-01-01

    Many cellular responses to corticosteroids involve the transcriptional modulation of target genes by the glucocorticoid receptor (GR). A rapid, non-nuclear effect of GR was found to mediate neuroprotection. High-dose corticosteroids (20 mg/kg intraperitoneally), given within 2 hours of transient cerebral ischemia, acutely increased endothelial nitric oxide synthase (eNOS) activity, augmented regional cerebral blood flow (CBF) by 40% to 50%, and reduced cerebral infarct size by 32%. These neuroprotective effects of corticosteroids were abolished by the GR antagonist RU486 and by inhibition of phosphatidylinositol 3-kinase (PI3K), and were absent in eNOS–/– mice. To determine the mechanism by which GR activated eNOS, we measured the effect of corticosteroids on PI3K and the protein kinase Akt. In a ligand-dependent manner, GR activated PI3K and Akt in vitro and in vivo caused NO-dependent vasodilation, which was blocked by cotreatment with RU486 or the PI3K inhibitor LY294002 but not by transcriptional inhibitors. Indeed, a mutant GR, which cannot dimerize and bind to DNA, still activated PI3K and Akt in response to corticosteroids. These findings indicate that non-nuclear GR rapidly activates eNOS through the PI3K/Akt pathway and suggest that this mechanism mediates the acute neuroprotective effects of corticosteroids through augmentation of CBF. PMID:12464678

  9. 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

  10. 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.

  11. 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

  12. Experimental Sleep Restriction Causes Endothelial Dysfunction in Healthy Humans

    PubMed Central

    Calvin, Andrew D.; Covassin, Naima; Kremers, Walter K.; Adachi, Taro; Macedo, Paula; Albuquerque, Felipe N.; Bukartyk, Jan; Davison, Diane E.; Levine, James A.; Singh, Prachi; Wang, Shihan; Somers, Virend K.

    2014-01-01

    Background Epidemiologic evidence suggests a link between short sleep duration and cardiovascular risk, although the nature of any relationship and mechanisms remain unclear. Short sleep duration has also been linked to an increase in cardiovascular events. Endothelial dysfunction has itself been implicated as a mediator of heightened cardiovascular risk. We sought to determine the effect of 8 days/8 nights of partial sleep restriction on endothelial function in healthy humans. Methods and Results Sixteen healthy volunteers underwent a randomized study of usual sleep versus sleep restriction of two‐thirds normal sleep time for 8 days/8 nights in a hospital‐based clinical research unit. The main outcome was endothelial function measured by flow‐mediated brachial artery vasodilatation (FMD). Those randomized to sleep restriction slept 5.1 hours/night during the experimental period compared with 6.9 hours/night in the control group. Sleep restriction was associated with significant impairment in FMD (8.6±4.6% during the initial pre‐randomization acclimation phase versus 5.2±3.4% during the randomized experimental phase, P=0.01) whereas no change was seen in the control group (5.0±3.0 during the acclimation phase versus 6.73±2.9% during the experimental phase, P=0.10) for a between‐groups difference of −4.40% (95% CI −7.00 to −1.81%, P=0.003). No change was seen in non‐flow mediated vasodilatation (NFMD) in either group. Conclusion In healthy individuals, moderate sleep restriction causes endothelial dysfunction. Clinical Trial Registration URL: ClinicalTrials.gov. Unique identifier: NCT01334788. PMID:25424573

  13. 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

  14. Coenzyme Q10 Protects Human Endothelial Cells from β-Amyloid Uptake and Oxidative Stress-Induced Injury

    PubMed Central

    Durán-Prado, Mario; Frontiñán, Javier; Santiago-Mora, Raquel; Peinado, Juan Ramón; Parrado-Fernández, Cristina; Gómez-Almagro, María Victoria; Moreno, María; López-Domínguez, José Alberto; Villalba, José Manuel; Alcaín, Francisco J.

    2014-01-01

    Neuropathological symptoms of Alzheimer's disease appear in advances stages, once neuronal damage arises. Nevertheless, recent studies demonstrate that in early asymptomatic stages, ß-amyloid peptide damages the cerebral microvasculature through mechanisms that involve an increase in reactive oxygen species and calcium, which induces necrosis and apoptosis of endothelial cells, leading to cerebrovascular dysfunction. The goal of our work is to study the potential preventive effect of the lipophilic antioxidant coenzyme Q (CoQ) against ß-amyloid-induced damage on human endothelial cells. We analyzed the protective effect of CoQ against Aβ-induced injury in human umbilical vein endothelial cells (HUVECs) using fluorescence and confocal microscopy, biochemical techniques and RMN-based metabolomics. Our results show that CoQ pretreatment of HUVECs delayed Aβ incorporation into the plasma membrane and mitochondria. Moreover, CoQ reduced the influx of extracellular Ca2+, and Ca2+ release from mitochondria due to opening the mitochondrial transition pore after β-amyloid administration, in addition to decreasing O2.− and H2O2 levels. Pretreatment with CoQ also prevented ß-amyloid-induced HUVECs necrosis and apoptosis, restored their ability to proliferate, migrate and form tube-like structures in vitro, which is mirrored by a restoration of the cell metabolic profile to control levels. CoQ protected endothelial cells from Aβ-induced injury at physiological concentrations in human plasma after oral CoQ supplementation and thus could be a promising molecule to protect endothelial cells against amyloid angiopathy. PMID:25272163

  15. First Case of Human Cerebral Taenia martis Cysticercosis

    PubMed Central

    Benoilid, Aurélien; Kremer, Stéphane; Dalvit, Constanza; Lefebvre, Nicolas; Hansmann, Yves; Chenard, Marie-Pierre; Mathieu, Bruno; Grimm, Felix; Deplazes, Peter; Pfaff, Alexander W.; Abou-Bacar, Ahmed; Marescaux, Christian; Candolfi, Ermanno

    2015-01-01

    Taenia martis is a tapeworm affecting mustelids, with rodents serving as intermediate hosts. The larval stage (cysticercus) has been found before only rarely in humans or primates. We hereby describe a case of cerebral T. martis cysticercosis in a French immunocompetent patient, confirmed by DNA analyses of biopsy material. PMID:26019196

  16. Impaired vascular remodeling after endothelial progenitor cell transplantation in MMP9-deficient mice suffering cortical cerebral ischemia

    PubMed Central

    Morancho, Anna; Ma, Feifei; Barceló, Verónica; Giralt, Dolors; Montaner, Joan; Rosell, Anna

    2015-01-01

    Endothelial progenitor cells (EPCs) are being investigated for advanced therapies, and matrix metalloproteinase 9 (MMP9) has an important role in stroke recovery. Our aim was to determine whether tissue MMP9 influences the EPC-induced angiogenesis after ischemia. Wild-type (WT) and MMP9-deficient mice (MMP9/KO) were subjected to cerebral ischemia and treated with vehicle or outgrowth EPCs. After 3 weeks, we observed an increase in the peri-infarct vessel density in WT animals but not in MMP9/KO mice; no differences were found in the vehicle-treated groups. Our data suggest that tissue MMP9 has a crucial role in EPC-induced vascular remodeling after stroke. PMID:26219597

  17. Bacterial programmed cell death of cerebral endothelial cells involves dual death pathways

    PubMed Central

    Bermpohl, Daniela; Halle, Annett; Freyer, Dorette; Dagand, Emilie; Braun, Johann S.; Bechmann, Ingo; Schröder, Nicolas W.J.; Weber, Joerg R.

    2005-01-01

    Major barriers separating the blood from tissue compartments in the body are composed of endothelial cells. Interaction of bacteria with such barriers defines the course of invasive infections, and meningitis has served as a model system to study endothelial cell injury. Here we report the impressive ability of Streptococcus pneumoniae, clinically one of the most important pathogens, to induce 2 morphologically distinct forms of programmed cell death (PCD) in brain-derived endothelial cells. Pneumococci and the major cytotoxins H202 and pneumolysin induce apoptosis-like PCD independent of TLR2 and TLR4. On the other hand, pneumococcal cell wall, a major proinflammatory component, causes caspase-driven classical apoptosis that is mediated through TLR2. These findings broaden the scope of bacterial-induced PCD, link these effects to innate immune TLRs, and provide insight into the acute and persistent phases of damage during meningitis. PMID:15902310

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  19. Cerebral versus systemic hemodynamics during graded orthostatic stress in humans

    NASA Technical Reports Server (NTRS)

    Levine, B. D.; Giller, C. A.; Lane, L. D.; Buckey, J. C.; Blomqvist, C. G.

    1994-01-01

    BACKGROUND: Orthostatic syncope is usually attributed to cerebral hypoperfusion secondary to systemic hemodynamic collapse. Recent research in patients with neurocardiogenic syncope has suggested that cerebral vasoconstriction may occur during orthostatic hypotension, compromising cerebral autoregulation and possibly contributing to the loss of consciousness. However, the regulation of cerebral blood flow (CBF) in such patients may be quite different from that of healthy individuals, particularly when assessed during the rapidly changing hemodynamic conditions associated with neurocardiogenic syncope. To be able to interpret the pathophysiological significance of these observations, a clear understanding of the normal responses of the cerebral circulation to orthostatic stress must be obtained, particularly in the context of the known changes in systemic and regional distributions of blood flow and vascular resistance during orthostasis. Therefore, the specific aim of this study was to examine the changes that occur in the cerebral circulation during graded reductions in central blood volume in the absence of systemic hypotension in healthy humans. We hypothesized that cerebral vasoconstriction would occur and CBF would decrease due to activation of the sympathetic nervous system. We further hypothesized, however, that the magnitude of this change would be small compared with changes in systemic or skeletal muscle vascular resistance in healthy subjects with intact autoregulation and would be unlikely to cause syncope without concomitant hypotension. METHODS AND RESULTS: To test this hypothesis, we studied 13 healthy men (age, 27 +/- 7 years) during progressive lower body negative pressure (LBNP). We measured systemic flow (Qc is cardiac output; C2H2 rebreathing), regional forearm flow (FBF; venous occlusion plethysmography), and blood pressure (BP; Finapres) and calculated systemic (SVR) and forearm (FVR) vascular resistances. Changes in brain blood flow were

  20. 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.

  1. High glucose causes dysfunction of the human glomerular endothelial glycocalyx.

    PubMed

    Singh, A; Fridén, V; Dasgupta, I; Foster, R R; Welsh, G I; Tooke, J E; Haraldsson, B; Mathieson, P W; Satchell, S C

    2011-01-01

    The endothelial glycocalyx is a gel-like layer which covers the luminal side of blood vessels. The glomerular endothelial cell (GEnC) glycocalyx is composed of proteoglycan core proteins, glycosaminoglycan (GAG) chains, and sialoglycoproteins and has been shown to contribute to the selective sieving action of the glomerular capillary wall. Damage to the systemic endothelial glycocalyx has recently been associated with the onset of albuminuria in diabetics. In this study, we analyze the effects of high glucose on the biochemical structure of the GEnC glycocalyx and quantify functional changes in its protein-restrictive action. We used conditionally immortalized human GEnC. Proteoglycans were analyzed by Western blotting and indirect immunofluorescence. Biosynthesis of GAG was analyzed by radiolabeling and quantified by anion exchange chromatography. FITC-albumin was used to analyze macromolecular passage across GEnC monolayers using an established in vitro model. We observed a marked reduction in the biosynthesis of GAG by the GEnC under high-glucose conditions. Further analysis confirmed specific reduction in heparan sulfate GAG. Expression of proteoglycan core proteins remained unchanged. There was also a significant increase in the passage of albumin across GEnC monolayers under high-glucose conditions without affecting interendothelial junctions. These results reproduce changes in GEnC barrier properties caused by enzymatic removal of heparan sulfate from the GEnC glycocalyx. They provide direct evidence of high glucose-induced alterations in the GEnC glycocalyx and demonstrate changes to its function as a protein-restrictive layer, thus implicating glycocalyx damage in the pathogenesis of proteinuria in diabetes.

  2. Recent insights into cerebral cavernous malformations: animal models of CCM and the human phenotype

    PubMed Central

    Chan, Aubrey C.; Li, Dean Y.; Berg, Michel J.; Whitehead, Kevin J.

    2010-01-01

    Cerebral cavernous malformations are common vascular lesions of the central nervous system that predispose to seizures, focal neurologic deficits and potentially fatal hemorrhagic stroke. Human genetic studies have identified three genes associated with the disease and biochemical studies of these proteins have identified interaction partners and possible signaling pathways. A variety of animal models of CCM have been described to help translate the cellular and biochemical insights into a better understanding of disease mechanism. In this minireview, we discuss the contributions of animal models to our growing understanding of the biology of cavernous malformations, including the elucidation of the cellular context of CCM protein actions and the in vivo confirmation of abnormal endothelial cell–cell interactions. Challenges and progress towards developing a faithful model of CCM biology are reviewed. PMID:20096037

  3. The seeding of human aortic endothelial cells on the extra-cellular matrix of human umbilical vein endothelial cells.

    PubMed Central

    Solomon, D. E.

    1992-01-01

    A post confluent layer (6th passage) of human umbilical vein endothelial cells (HUVECs) was treated with 3 mM ethylene diamine tetra-acetic acid (EDTA) to expose the subendothelial extra-cellular matrix (ECM). Normal human aortic endothelial cells (HAECs) harvested by mechanical scraping were seeded onto the ECM of the HUVECs. The cells quickly attached and proliferated with normal morphology. To ensure confluency the HAECs were pooled after a brief trypsin/EDTA incubation and seeded onto the ECM of the same HUVECs (6th passage) cell line. They attached within 2 hours, and the cells grew to confluence displaying cobblestone morphology characteristic of phenotypic endothelium. HUVECs (11th passage) were seeded onto (6th passage) HUVECs ECM. The cells attached, proliferated to confluence within the normal time interval (7-8 days) and were positively characterized. A Corvita 6mm graft supplied with a gelatin/heparin matrix was densely seeded with HUVECs (6th passage). These cells also proliferated to confluence. The implications for improving the design of arterial grafts are discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:1390196

  4. 3D map of the human corneal endothelial cell.

    PubMed

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

    2016-01-01

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

  5. 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.

  6. 3D map of the human corneal endothelial cell

    PubMed Central

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

    2016-01-01

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

  7. 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

  8. 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.

  9. Cerebral organoids model human brain development and microcephaly.

    PubMed

    Lancaster, Madeline A; Renner, Magdalena; Martin, Carol-Anne; Wenzel, Daniel; Bicknell, Louise S; Hurles, Matthew E; Homfray, Tessa; Penninger, Josef M; Jackson, Andrew P; Knoblich, Juergen A

    2013-09-19

    The complexity of the human brain has made it difficult to study many brain disorders in model organisms, highlighting the need for an in vitro model of human brain development. Here we have developed a human pluripotent stem cell-derived three-dimensional organoid culture system, termed cerebral organoids, that develop various discrete, although interdependent, brain regions. These include a cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes. Furthermore, cerebral organoids are shown to recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells. Finally, we use RNA interference and patient-specific induced pluripotent stem cells to model microcephaly, a disorder that has been difficult to recapitulate in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could help to explain the disease phenotype. Together, these data show that three-dimensional organoids can recapitulate development and disease even in this most complex human tissue. PMID:23995685

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    1978-06-01

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

  12. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells

    PubMed Central

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru

    2016-01-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca2+-dependent using the Ca2+ chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  13. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    PubMed

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  14. 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

  15. Assessment of endothelial and neurovascular function in human skin microcirculation.

    PubMed

    Roustit, Matthieu; Cracowski, Jean-Luc

    2013-07-01

    Peripheral microvascular dysfunction has been described in many physiological and pathological conditions. Owing to its accessibility, the cutaneous microcirculation provides a unique index of microvascular function. Skin microvascular function has therefore been proposed as a prognostic marker or for evaluating the effect of drugs on the microcirculation. Various reactivity tests, coupled with techniques measuring skin blood flux, are used to non-invasively explore both endothelial and neurovascular microvascular functioning in humans. We review the advantages and limitations of the main reactivity tests, including post-occlusive reactive hyperemia, local thermal hyperemia, pressure-induced vasodilation, and iontophoresis of vasodilators, combined with measurement techniques such as laser Doppler and laser speckle contrast imaging. Recent advances in our comprehension of the physiological pathways underlying these reactivity tests, as well as technological developments in microcirculation imaging, have provided reliable and reproducible tools for studying the microcirculation.

  16. Preservation of Cellular Glutathione Status and Mitochondrial Membrane Potential by N-Acetylcysteine and Insulin Sensitizers Prevent Carbonyl Stress-Induced Human Brain Endothelial Cell Apoptosis

    PubMed Central

    Okouchi, Masahiro; Okayama, Naotsuka; Aw, Tak Yee

    2011-01-01

    Oxidative stress-induced cerebral endothelial cell dysfunction is associated with cerebral microvascular complication of primary diabetic encephaolopathy, a neurodegenerative disorder of long-standing diabetes, but the injury mechanisms are poorly understood. This study sought to determine the contribution of carbonyl (methylglyoxal, MG) stress to human brain endothelial cell (IHEC) apoptosis, the relationship to cellular redox status and mitochondrial membrane potential, and the protection by thiol antioxidant and insulin sensitizers. MG exposure induced IHEC apoptosis in association with perturbed cellular glutathione (GSH) redox status, decreased mitochondrial membrane potential (Δψm), activation of caspase-9 and -3, and cleavage of polyADP-ribose polymerase. Insulin sensitizers such as biguanides or AMP-activated protein kinase activator, but not glitazones, afforded cytoprotection through preventing Δψm collapse and activation of caspase-9 that was independent of cellular GSH. Similarly, cyclosporine A prevented Δψm collapse, while N-acetylcysteine (NAC) mediated the recovery of cellular GSH redox balance that secondarily preserved Δψm. Collectively, these results provide mechanistic insights into the role of GSH redox status and mitochondrial potential in carbonyl stress-induced apoptosis of brain endothelial cells, with implications for cerebral microvascular complications associated with primary diabetic encephalopathy. The findings that thiol antioxidant and insulin sensitizers afforded cytoprotection suggest potential therapeutic approaches. PMID:19807652

  17. Human growth hormone stimulates proliferation of human retinal microvascular endothelial cells in vitro

    SciTech Connect

    Rymaszewski, Z.; Cohen, R.M.; Chomczynski, P. )

    1991-01-15

    Growth hormone (GH) has been implicated in the pathogenesis of proliferative diabetic retinopathy. The authors sought to determine whether this could be mediated by an effect of GH on proliferation of endothelial cells, and, for this purpose, established long-term cultures of human retinal microvascular endothelial cells (hREC) from normal postmortem human eyes. High-purity hREC preparations were selected for experiments, based on immunogluorescence with acetylated low density lipoprotein (LDL) and anti-factor VIII-related antigen. Growth requirements for these cells were complex, including serum for maintenance at slow growth rates and additional mitogens for more rapid proliferation. Exposure of hREC to physiologic doses of human GH (hGH) resulted in 100% greater cell number vs. control but could be elicited only in the presence of serum. When differing serum conditions were compared, hGH stimulated ({sup 3}H)thymidine incorporation up to 1.6- to 2.2-fold under each condition and increased DNA content significantly in the presence of human, horse, and fetal calf serum. In summary, hREC respond to physiologic concentrations of hGH in vitro with enhanced proliferation. This specific effect of GH on retinal microvascular endothelial cells supports the hypothesis of role for GH in endothelial cell biology.

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

    NASA Astrophysics Data System (ADS)

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

    1998-04-01

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

  19. 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.

  20. [Changes of cerebral blood flow during diving reactions in humans].

    PubMed

    Baranova, T I; Berlov, D N; Ianvareva, I N

    2014-05-01

    The characteristics of human cerebral blood flow were estimated during the implementation of the diving response, simulated by complex cold-hypoxic-hypercapnic exposure (CHHE), and under the influence of separate cold, hypercapnic and hypoxic stimuli. Was studied 18 people aged 18-22 years who had no special training. Cerebral blood flow was recorded by transcranial Doppler. It is shown that in the CHHE with the respect initial state to observe a marked increase in cerebral blood flow linear velocity (BFV) to 82.3 ± 15.2%, as well as reducing characterizing the tone of resistance vessels of the brain pulsatility index (PI) to 77.2 ± 13.1%. During cold and tactile stimulation of facial skin BFV and PI did not change significantly, with a single breath hold (Genchi test) BFV increased by 52.3 ± 12.5%, PI at 64.5 ± 15%. The latent period of cerebral blood flow (14-43) allow suppose metabolic (chemical) nature of regulatory influences, which provide changes of considered indicators.

  1. Quercetin protects human brain microvascular endothelial cells from fibrillar β-amyloid1–40-induced toxicity

    PubMed Central

    Li, Yongjie; Zhou, Sibai; Li, Jinze; Sun, Yuhua; Hasimu, Hamlati; Liu, Rui; Zhang, Tiantai

    2015-01-01

    Amyloid beta-peptides (Aβ) are known to undergo active transport across the blood-brain barrier, and cerebral amyloid angiopathy has been shown to be a prominent feature in the majority of Alzheimer׳s disease. Quercetin is a natural flavonoid molecule and has been demonstrated to have potent neuroprotective effects, but its protective effect on endothelial cells under Aβ-damaged condition is unclear. In the present study, the protective effects of quercetin on brain microvascular endothelial cells injured by fibrillar Aβ1–40 (fAβ1–40) were observed. The results show that fAβ1–40-induced cytotoxicity in human brain microvascular endothelial cells (hBMECs) can be relieved by quercetin treatment. Quercetin increases cell viability, reduces the release of lactate dehydrogenase, and relieves nuclear condensation. Quercetin also alleviates intracellular reactive oxygen species generation and increases superoxide dismutase activity. Moreover, it strengthens the barrier integrity through the preservation of the transendothelial electrical resistance value, the relief of aggravated permeability, and the increase of characteristic enzyme levels after being exposed to fAβ1–40. In conclusion, quercetin protects hBMECs from fAβ1–40-induced toxicity. PMID:26579424

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. Pentosan polysulfate regulates scavenger receptor-mediated, but not fluid-phase, endocytosis in immortalized cerebral endothelial cells.

    PubMed

    Deli, M A; Abrahám, C S; Takahata, H; Katamine, S; Niwa, M

    2000-12-01

    1. Effects of pentosan polysulfate (PPS) and the structurally related sulfated polyanions dextran sulfate, fucoidan, and heparin on the scavenger receptor-mediated and fluidphase endocytosis in GP8 immortalized rat brain endothelial cells were investigated. 2. Using 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarboxyamine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL), we found a binding site with high affinity and low binding capacity, and another one with low affinity and high binding capacity. Increasing ligand concentrations could not saturate DiI-AcLDL uptake. DiI-AcLDL uptake, but not binding, was sensitive to pretreatment with filipin, an inhibitor of caveola formation. 3. PPS (20-200 microg/ml) significantly reduced the binding of DiI-AcLDL after coincubation for 3 hr, though this effect was less expressed after 18 hr. Among other polyanions, only fucoidan decreased the DiI-AcLDL binding after 3 hr, whereas dextran sulfate significantly increased it after 18 hr. PPS treatment induced an increase in DiI-AcLDL uptake, whereas other polysulfated compounds caused a significant reduction. 4. Fluid-phase endocytosis determined by the accumulation of Lucifer yellow was concentration and time dependent in GP8 cells. Coincubation with PPS or other sulfated polyanions could not significantly alter the rate of Lucifer yellow uptake. 5. In conclusion. PPS decreased the binding and increased the uptake of DiI-AcLDL in cerebral endothelial cells, an effect not mimicked by the other polyanions investigated.

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

    PubMed Central

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

    2016-01-01

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

  6. Adherence of human basophils to cultured umbilical vein endothelial cells.

    PubMed Central

    Bochner, B S; Peachell, P T; Brown, K E; Schleimer, R P

    1988-01-01

    The mechanism by which circulating human basophils adhere to vascular endothelium and migrate to sites of allergic reactions is unknown. Agents have been identified which stimulate the adherence of purified basophils to cultured human umbilical vein vascular endothelial cells (HuVEC). Treatment of HuVEC with interleukin 1, tumor necrosis factor (TNF), bacterial endotoxin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in time and dose-dependent increases of adhesiveness for basophils. Coincubation of basophils and HuVEC for 10 min with C5a, formyl-methionyl-leucyl-phenylalanine, the calcium ionophore A23187, platelet-activating factor, TNF, and TPA also resulted in significant dose-dependent increases in basophil adherence; this effect resulted from activation of the basophil. Adherence of basophils to HuVEC was time and temperature dependent, required divalent cations, and was unaffected by glucocorticoids. Monoclonal antibody 60.3, directed against the beta-subunit of the leukocyte adherence complex CD18, inhibited the binding of basophils to HuVEC. Adherence of basophils to vascular endothelium may be important in initiating basophil infiltrates in vivo. PMID:3130394

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

    PubMed

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

    2011-12-01

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

  8. Human Urinary Kallidinogenase Promotes Angiogenesis and Cerebral Perfusion in Experimental Stroke

    PubMed Central

    Chen, Yanting; Zhang, Meijuan; Qian, Lai; Chen, Yan; Wu, Zhengzheng; Xu, Yun; Li, Jingwei

    2015-01-01

    Angiogenesisis a key restorative mechanism in response to ischemia, and pro-angiogenic therapy could be beneficial in stroke. Accumulating experimental and clinical evidence suggest that human urinary kallidinogenase (HUK) improves stroke outcome, but the underlying mechanisms are not clear. The aim of current study was to verify roles of HUK in post-ischemic angiogenesis and identify relevant mediators. In rat middle cerebral artery occlusion (MCAO) model, we confirmed that HUK treatment could improve stroke outcome, indicated by reduced infarct size and improved neurological function. Notably, the 18F-FDG micro-PET scan indicated that HUK enhanced cerebral perfusion in rats after MCAO treatment. In addition, HUK promotespost-ischemic angiogenesis, with increased vessel density as well as up-regulated VEGF andapelin/APJ expression in HUK-treated MCAO mice. In endothelial cell cultures, induction of VEGF and apelin/APJ expression, and ERK1/2 phosphorylation by HUK was further confirmed. These changes were abrogated by U0126, a selective ERK1/2 inhibitor. Moreover, F13A, a competitive antagonist of APJ receptor, significantly suppressed HUK-induced VEGF expression. Furthermore, angiogenic functions of HUK were inhibited in the presence of selective bradykinin B1 or B2 receptor antagonist both in vitro and in vivo. Our findings indicate that HUK treatment promotes post-ischemic angiogenesis and cerebral perfusion via activation of bradykinin B1 and B2 receptors, which is potentially due to enhancement expression of VEGF and apelin/APJ in ERK1/2 dependent way. PMID:26222055

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

    SciTech Connect

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

    2006-04-01

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

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

    PubMed

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

    2016-04-01

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

  11. 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.

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

    PubMed Central

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

    2015-01-01

    Aim To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. Materials and Methods Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression. Results hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPaseα1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet’s membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis. Conclusion hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium. PMID:26689688

  13. Dynamic analysis of the human brain with complex cerebral sulci.

    PubMed

    Tseng, Jung-Ge; Huang, Bo-Wun; Ou, Yi-Wen; Yen, Ke-Tien; Wu, Yi-Te

    2016-07-01

    The brain is one of the most vulnerable organs inside the human body. Head accidents often appear in daily life and are easy to cause different level of brain damage inside the skull. Once the brain suffered intense locomotive impact, external injuries, falls, or other accidents, it will result in different degrees of concussion. This study employs finite element analysis to compare the dynamic characteristics between the geometric models of an assumed simple brain tissue and a brain tissue with complex cerebral sulci. It is aimed to understand the free vibration of the internal brain tissue and then to protect the brain from injury caused by external influences. Reverse engineering method is used for a Classic 5-Part Brain (C18) model produced by 3B Scientific Corporation. 3D optical scanner is employed to scan the human brain structure model with complex cerebral sulci and imported into 3D graphics software to construct a solid brain model to simulate the real complex brain tissue. Obtaining the normal mode analysis by inputting the material properties of the true human brain into finite element analysis software, and then to compare the simplified and the complex of brain models. PMID:27459595

  14. A multi-modal parcellation of human cerebral cortex.

    PubMed

    Glasser, Matthew F; Coalson, Timothy S; Robinson, Emma C; Hacker, Carl D; Harwell, John; Yacoub, Essa; Ugurbil, Kamil; Andersson, Jesper; Beckmann, Christian F; Jenkinson, Mark; Smith, Stephen M; Van Essen, David C

    2016-08-11

    Understanding the amazingly complex human cerebral cortex requires a map (or parcellation) of its major subdivisions, known as cortical areas. Making an accurate areal map has been a century-old objective in neuroscience. Using multi-modal magnetic resonance images from the Human Connectome Project (HCP) and an objective semi-automated neuroanatomical approach, we delineated 180 areas per hemisphere bounded by sharp changes in cortical architecture, function, connectivity, and/or topography in a precisely aligned group average of 210 healthy young adults. We characterized 97 new areas and 83 areas previously reported using post-mortem microscopy or other specialized study-specific approaches. To enable automated delineation and identification of these areas in new HCP subjects and in future studies, we trained a machine-learning classifier to recognize the multi-modal 'fingerprint' of each cortical area. This classifier detected the presence of 96.6% of the cortical areas in new subjects, replicated the group parcellation, and could correctly locate areas in individuals with atypical parcellations. The freely available parcellation and classifier will enable substantially improved neuroanatomical precision for studies of the structural and functional organization of human cerebral cortex and its variation across individuals and in development, aging, and disease. PMID:27437579

  15. L-arginine normalizes endothelial function in cerebral vessels from hypercholesterolemic rabbits.

    PubMed Central

    Rossitch, E; Alexander, E; Black, P M; Cooke, J P

    1991-01-01

    We hypothesized that normal vascular reactivity could be restored in vessels from hypercholesterolemic animals by exposing them to L-arginine, the precursor of endothelium-derived relaxing factor (EDRF). Basilar arteries were harvested from New Zealand white rabbits fed normal chow or that supplemented with 2% cholesterol for 10 wk. Vessels were cannulated for perfusion at physiologic pressure. Changes in vessel diameter were monitored by videomicroscopy. In comparison to normal vessels, those from hypercholesterolemic animals vasoconstricted more to KCl, endothelin (E), and 5-hydroxytryptamine (5-HT). Conversely, vasodilation to acetylcholine (ACh) (but not that to verapamil) was significantly impaired in the hypercholesterolemic animals. In vitro administration of L-arginine (3 mM) for 45 min normalized vasodilation to ACh and vasoconstriction to E, 5-HT, and KCl in the isolated vessels from hypercholesterolemic animals. This effect was stereospecific, since D-arginine had no effect. To conclude, these data confirm that hypercholesterolemia attenuates endothelium-derived relaxation, and enhances the sensitivity of these vessels to vasoconstrictors. In vitro administration of L-arginine normalized vascular reactivity of isolated vessels from hypercholesterolemic animals. Thus, hypercholesterolemia induces a reversible endothelial dysfunction that may be corrected by supplying the precursor of EDRF, L-arginine. PMID:2010542

  16. Endothelial Differentiation of Human Adipose-Derived Stem Cells on Polyglycolic Acid/Polylactic Acid Mesh.

    PubMed

    Deng, Meng; Gu, Yunpeng; Liu, Zhenjun; Qi, Yue; Ma, Gui E; Kang, Ning

    2015-01-01

    Adipose-derived stem cell (ADSC) is considered as a cell source potentially useful for angiogenesis in tissue engineering and regenerative medicine. This study investigated the growth and endothelial differentiation of human ADSCs on polyglycolic acid/polylactic acid (PGA/PLA) mesh compared to 2D plastic. Cell adhesion, viability, and distribution of hADSCs on PGA/PLA mesh were observed by CM-Dil labeling, live/dead staining, and SEM examination while endothelial differentiation was evaluated by flow cytometry, Ac-LDL/UEA-1 uptake assay, immunofluorescence stainings, and gene expression analysis of endothelial related markers. Results showed hADSCs gained a mature endothelial phenotype with a positive ratio of 21.4 ± 3.7% for CD31+/CD34- when induced in 3D mesh after 21 days, which was further verified by the expressions of a comprehensive range of endothelial related markers, whereas hADSCs in 2D induced and 2D/3D noninduced groups all failed to differentiate into endothelial cells. Moreover, compared to 2D groups, the expression for α-SMA was markedly suppressed in 3D cultured hADSCs. This study first demonstrated the endothelial differentiation of hADSCs on the PGA/PLA mesh and pointed out the synergistic effect of PGA/PLA 3D culture and growth factors on the acquisition of mature characteristic endothelial phenotype. We believed this study would be the initial step towards the generation of prevascularized tissue engineered constructs.

  17. Cerebral Microcirculation and Oxygen Tension in the Human Secondary Cortex

    PubMed Central

    Linninger, A. A.; Gould, I. G.; Marinnan, T.; Hsu, C.-Y.; Chojecki, M.; Alaraj, A.

    2013-01-01

    The three-dimensional spatial arrangement of the cortical microcirculatory system is critical for understanding oxygen exchange between blood vessels and brain cells. A three-dimensional computer model of a 3 × 3 × 3 mm3 subsection of the human secondary cortex was constructed to quantify oxygen advection in the microcirculation, tissue oxygen perfusion, and consumption in the human cortex. This computer model accounts for all arterial, capillary and venous blood vessels of the cerebral microvascular bed as well as brain tissue occupying the extravascular space. Microvessels were assembled with optimization algorithms emulating angiogenic growth; a realistic capillary bed was built with space filling procedures. The extravascular tissue was modeled as a porous medium supplied with oxygen by advection–diffusion to match normal metabolic oxygen demand. The resulting synthetic computer generated network matches prior measured morphometrics and fractal patterns of the cortical microvasculature. This morphologically accurate, physiologically consistent, multi-scale computer network of the cerebral microcirculation predicts the oxygen exchange of cortical blood vessels with the surrounding gray matter. Oxygen tension subject to blood pressure and flow conditions were computed and validated for the blood as well as brain tissue. Oxygen gradients along arterioles, capillaries and veins agreed with in vivo trends observed recently in imaging studies within experimental tolerances and uncertainty. PMID:23842693

  18. Ultrastructural characteristics of human adult and infant cerebral cortical neurons.

    PubMed Central

    Ong, W Y; Garey, L J

    1991-01-01

    Biopsy specimens of human cerebral cortex from three adults and two infants were studied by correlating their light microscopic features in semithin sections with their ultrastructural characteristics. There was good tissue preservation, due to a minimum delay between obtaining the specimens and fixation. Pyramidal cells had a prominent apical dendrite, fine heterochromatin clumps in the nucleus and generally small numbers of cytoplasmic organelles, except for numerous free ribosomes in some of the large pyramids of Layers III to VI. Non-pyramidal cells lacked an apical dendrite and were further classified, on size and ultrastructure, into small, medium and large types. Large numbers of asymmetrical and symmetrical synapses were present in the neuropil but very few axosomatic synapses were found in the human cerebral cortex compared with subhuman primates and other mammals. Some symmetrical synapses were characterised by the presence of wide pre- and postsynaptic densities. The same general features of the adult cortex were also encountered in the infant, with certain exceptions. Many of the infant neurons had less densely packed heterochromatin, but greater numbers of free ribosomes, compared with the adult, and lipofuscin was absent. There was a total absence of myelinated fibres from the infant cortex; more large diameter dendrites were present than in the adult and axosomatic synapses were commoner. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 PMID:2050578

  19. Systemic Expression of Vascular Endothelial Growth Factor in Patients with Cerebral Cavernous Malformation Treated by Stereotactic Radiosurgery

    PubMed Central

    Park, Sang-Jin

    2016-01-01

    Objective Increased expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), is associated with the pathogenesis of cerebral cavernous malformations (CCMs). The purpose of this study was to investigate plasma levels of VEGF in normal subjects and in patients with CCM and to evaluate change in these levels following stereotactic radiosurgery (SRS). Methods Peripheral venous blood was collected from 6 patients with CCM before SRS using Gamma Knife and at the 1 week, 1 month, 3month, and 6 month follow-up visits. Plasma VEGF levels were measured using commercially available enzyme-linked immunosorbent assay kits. Peripheral blood samples were obtained from 10 healthy volunteers as controls. Results Mean plasma VEGF level of 41.9 pg/mL (range, 11.7–114.9 pg/mL) in patients with CCM at baseline was higher than that of the healthy controls (29.3 pg/mL, range, 9.2–64.3 pg/mL), without significant differences between CCM patients and controls (p=0.828). Plasma VEGF level following SRS dropped to 24.6 pg/mL after 1 week, and decreased to 18.5 pg/mL after 1 month, then increased to 24.3 pg/mL after 3 months, and 32.6 pg/mL after 6 months. Two patients suffering from rebleeding after SRS showed a higher level of VEGF at 6 months after SRS than their pretreatment level. Conclusion Plasma VEGF levels in patients with CCM were elevated over controls at baseline, and decreased from baseline to 1 month after SRS and increased further for up to 6 months. Theses results indicated that anti-angiogenic effect of SRS might play a role in the treatment of CCMs. PMID:27651861

  20. Systemic Expression of Vascular Endothelial Growth Factor in Patients with Cerebral Cavernous Malformation Treated by Stereotactic Radiosurgery

    PubMed Central

    Park, Sang-Jin

    2016-01-01

    Objective Increased expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), is associated with the pathogenesis of cerebral cavernous malformations (CCMs). The purpose of this study was to investigate plasma levels of VEGF in normal subjects and in patients with CCM and to evaluate change in these levels following stereotactic radiosurgery (SRS). Methods Peripheral venous blood was collected from 6 patients with CCM before SRS using Gamma Knife and at the 1 week, 1 month, 3month, and 6 month follow-up visits. Plasma VEGF levels were measured using commercially available enzyme-linked immunosorbent assay kits. Peripheral blood samples were obtained from 10 healthy volunteers as controls. Results Mean plasma VEGF level of 41.9 pg/mL (range, 11.7–114.9 pg/mL) in patients with CCM at baseline was higher than that of the healthy controls (29.3 pg/mL, range, 9.2–64.3 pg/mL), without significant differences between CCM patients and controls (p=0.828). Plasma VEGF level following SRS dropped to 24.6 pg/mL after 1 week, and decreased to 18.5 pg/mL after 1 month, then increased to 24.3 pg/mL after 3 months, and 32.6 pg/mL after 6 months. Two patients suffering from rebleeding after SRS showed a higher level of VEGF at 6 months after SRS than their pretreatment level. Conclusion Plasma VEGF levels in patients with CCM were elevated over controls at baseline, and decreased from baseline to 1 month after SRS and increased further for up to 6 months. Theses results indicated that anti-angiogenic effect of SRS might play a role in the treatment of CCMs.

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

    PubMed Central

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

    2015-01-01

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

  2. 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

  3. A 90-Kilodalton Endothelial Cell Molecule Mediating Lymphocyte Binding in Humans

    NASA Astrophysics Data System (ADS)

    Salmi, Marko; Jalkanen, Sirpa

    1992-09-01

    Interactions between leukocyte surface receptors and their ligands on vascular endothelial cells control lymphocyte traffic between the blood and various lymphoid organs, as well as extravasation of leukocytes into sites of inflammation. A heretofore undescribed 90-kilodalton human endothelial cell adhesion molecule (VAP-1) defined by a monoclonal antibody 1B2 is described. The expression pattern, molecular mass, functional properties, and an amino-terminal amino acid sequence define VAP-1 as an endothelial ligand for lymphocytes. VAP-1 helps to elucidate the complex heterotypic cell interactions that direct tissue-selective lymphocyte migration in man.

  4. Human endothelial progenitor cells internalize high-density lipoprotein.

    PubMed

    Srisen, Kaemisa; Röhrl, Clemens; Meisslitzer-Ruppitsch, Claudia; Ranftler, Carmen; Ellinger, Adolf; Pavelka, Margit; Neumüller, Josef

    2013-01-01

    Endothelial progenitor cells (EPCs) originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL), and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate), cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal intraellular

  5. The future of human cerebral cartography: a novel approach.

    PubMed

    Frackowiak, Richard; Markram, Henry

    2015-05-19

    Cerebral cartography can be understood in a limited, static, neuroanatomical sense. Temporal information from electrical recordings contributes information on regional interactions adding a functional dimension. Selective tagging and imaging of molecules adds biochemical contributions. Cartographic detail can also be correlated with normal or abnormal psychological or behavioural data. Modern cerebral cartography is assimilating all these elements. Cartographers continue to collect ever more precise data in the hope that general principles of organization will emerge. However, even detailed cartographic data cannot generate knowledge without a multi-scale framework making it possible to relate individual observations and discoveries. We propose that, in the next quarter century, advances in cartography will result in progressively more accurate drafts of a data-led, multi-scale model of human brain structure and function. These blueprints will result from analysis of large volumes of neuroscientific and clinical data, by a process of reconstruction, modelling and simulation. This strategy will capitalize on remarkable recent developments in informatics and computer science and on the existence of much existing, addressable data and prior, though fragmented, knowledge. The models will instantiate principles that govern how the brain is organized at different levels and how different spatio-temporal scales relate to each other in an organ-centred context.

  6. The future of human cerebral cartography: a novel approach.

    PubMed

    Frackowiak, Richard; Markram, Henry

    2015-05-19

    Cerebral cartography can be understood in a limited, static, neuroanatomical sense. Temporal information from electrical recordings contributes information on regional interactions adding a functional dimension. Selective tagging and imaging of molecules adds biochemical contributions. Cartographic detail can also be correlated with normal or abnormal psychological or behavioural data. Modern cerebral cartography is assimilating all these elements. Cartographers continue to collect ever more precise data in the hope that general principles of organization will emerge. However, even detailed cartographic data cannot generate knowledge without a multi-scale framework making it possible to relate individual observations and discoveries. We propose that, in the next quarter century, advances in cartography will result in progressively more accurate drafts of a data-led, multi-scale model of human brain structure and function. These blueprints will result from analysis of large volumes of neuroscientific and clinical data, by a process of reconstruction, modelling and simulation. This strategy will capitalize on remarkable recent developments in informatics and computer science and on the existence of much existing, addressable data and prior, though fragmented, knowledge. The models will instantiate principles that govern how the brain is organized at different levels and how different spatio-temporal scales relate to each other in an organ-centred context. PMID:25823868

  7. Mapping the human cerebral cortex using 3-D medial manifolds

    NASA Astrophysics Data System (ADS)

    Szekely, Gabor; Brechbuehler, Christian; Kuebler, Olaf; Ogniewicz, Robert; Budinger, Thomas F.

    1992-09-01

    Novel imaging technologies provide a detailed look at structure and function of the tremendously complex and variable human brain. Optimal exploitation of the information stored in the rapidly growing collection of acquired and segmented MRI data calls for robust and reliable descriptions of the individual geometry of the cerebral cortex. A mathematical description and representation of 3-D shape, capable of dealing with form of variable appearance, is at the focus of this paper. We base our development on the Medial Axis Transformation (MAT) customarily defined in 2-D although the concept generalizes to any number of dimensions. Our implementation of the 3-D MAT combines full 3-D Voronoitesselation generated by the set of all border points with regularization procedures to obtain geometrically and topologically correct medial manifolds. The proposed algorithm was tested on synthetic objects and has been applied to 3-D MRI data of 1 mm isotropic resolution to obtain a description of the sulci in the cerebral cortex. Description and representation of the cortical anatomy is significant in clinical applications, medical research, and instrumentation developments.

  8. The future of human cerebral cartography: a novel approach

    PubMed Central

    Frackowiak, Richard; Markram, Henry

    2015-01-01

    Cerebral cartography can be understood in a limited, static, neuroanatomical sense. Temporal information from electrical recordings contributes information on regional interactions adding a functional dimension. Selective tagging and imaging of molecules adds biochemical contributions. Cartographic detail can also be correlated with normal or abnormal psychological or behavioural data. Modern cerebral cartography is assimilating all these elements. Cartographers continue to collect ever more precise data in the hope that general principles of organization will emerge. However, even detailed cartographic data cannot generate knowledge without a multi-scale framework making it possible to relate individual observations and discoveries. We propose that, in the next quarter century, advances in cartography will result in progressively more accurate drafts of a data-led, multi-scale model of human brain structure and function. These blueprints will result from analysis of large volumes of neuroscientific and clinical data, by a process of reconstruction, modelling and simulation. This strategy will capitalize on remarkable recent developments in informatics and computer science and on the existence of much existing, addressable data and prior, though fragmented, knowledge. The models will instantiate principles that govern how the brain is organized at different levels and how different spatio-temporal scales relate to each other in an organ-centred context. PMID:25823868

  9. Association of Aortic Compliance and Brachial Endothelial Function with Cerebral Small Vessel Disease in Type 2 Diabetes Mellitus Patients: Assessment with High-Resolution MRI

    PubMed Central

    Shan, Yan; Zeng, Mengsu; Lin, Huandong; Yan, Hongmei

    2016-01-01

    Objective. To assess the possible association of aortic compliance and brachial endothelial function with cerebral small vessel disease in type 2 diabetes mellitus (DM2) patients by using 3.0 T high-resolution magnetic resonance imaging. Methods. Sixty-two clinically confirmed DM2 patients (25 women and 37 men; mean age: 56.8 ± 7.5 years) were prospectively enrolled for noninvasive MR examinations of the aorta, brachial artery, and brain. Aortic arch pulse wave velocity (PWV), flow-mediated dilation (FMD) of brachial artery, lacunar brain infarcts, and periventricular and deep white matter hyperintensities (WMHs) were assessed. Pearson and Spearman correlation analysis were performed to analyze the association between PWV and FMD with clinical data and biochemical test results. Univariable logistic regression analyses were used to analyze the association between PWV and FMD with cerebral small vessel disease. Multiple logistic regression analyses were used to find out the independent predictive factors of cerebral small vessel disease. Results. Mean PWV was 6.73 ± 2.00 m/s and FMD was 16.67 ± 9.11%. After adjustment for compounding factors, PWV was found significantly associated with lacunar brain infarcts (OR = 2.00; 95% CI: 1.14–3.2; P < 0.05) and FMD was significantly associated with periventricular WMHs (OR = 0.82; 95% CI: 0.71–0.95; P < 0.05). Conclusions. Quantitative evaluation of aortic compliance and endothelial function by using high-resolution MRI may be potentially useful to stratify DM2 patients with risk of cerebral small vessel disease. PMID:27525261

  10. Association of Aortic Compliance and Brachial Endothelial Function with Cerebral Small Vessel Disease in Type 2 Diabetes Mellitus Patients: Assessment with High-Resolution MRI.

    PubMed

    Shan, Yan; Lin, Jiang; Xu, Pengju; Zeng, Mengsu; Lin, Huandong; Yan, Hongmei

    2016-01-01

    Objective. To assess the possible association of aortic compliance and brachial endothelial function with cerebral small vessel disease in type 2 diabetes mellitus (DM2) patients by using 3.0 T high-resolution magnetic resonance imaging. Methods. Sixty-two clinically confirmed DM2 patients (25 women and 37 men; mean age: 56.8 ± 7.5 years) were prospectively enrolled for noninvasive MR examinations of the aorta, brachial artery, and brain. Aortic arch pulse wave velocity (PWV), flow-mediated dilation (FMD) of brachial artery, lacunar brain infarcts, and periventricular and deep white matter hyperintensities (WMHs) were assessed. Pearson and Spearman correlation analysis were performed to analyze the association between PWV and FMD with clinical data and biochemical test results. Univariable logistic regression analyses were used to analyze the association between PWV and FMD with cerebral small vessel disease. Multiple logistic regression analyses were used to find out the independent predictive factors of cerebral small vessel disease. Results. Mean PWV was 6.73 ± 2.00 m/s and FMD was 16.67 ± 9.11%. After adjustment for compounding factors, PWV was found significantly associated with lacunar brain infarcts (OR = 2.00; 95% CI: 1.14-3.2; P < 0.05) and FMD was significantly associated with periventricular WMHs (OR = 0.82; 95% CI: 0.71-0.95; P < 0.05). Conclusions. Quantitative evaluation of aortic compliance and endothelial function by using high-resolution MRI may be potentially useful to stratify DM2 patients with risk of cerebral small vessel disease. PMID:27525261

  11. Expression of endothelial selectin ligands on human leukocytes following dive.

    PubMed

    Glavas, Duska; Markotic, Anita; Valic, Zoran; Kovacic, Natasa; Palada, Ivan; Martinic, Roko; Breskovic, Toni; Bakovic, Darija; Brubakk, Alf O; Dujic, Zeljko

    2008-09-01

    The fact that impaired endothelial-dependent vasodilatation after scuba diving often occurs without visible changes in the endothelial layer implies its biochemical origin. Since Lewisx(CD15) and sialyl-Lewisx(CD15s) are granulocyte and monocyte carbohydrate antigens recognized as ligands by endothelial selectins, we assumed that they could be sensitive markers for impaired vasodilatation following diving. Using flow cytometry, we determined the CD15 and CD15s peripheral blood mononuclear cells of eight divers, 30 mins before and 50 mins after a single dive to 54 m for 20 mins bottom time. The number of gas bubbles in the right heart was monitored by ultrasound. Gas bubbles were seen in all eight divers, with the average number of bubbles/cm2 1.9+/-1.9. The proportion of CD15+monocytes increased 2-fold after the dive as well as the subpopulation of monocytes highly expressing CD15s. The absolute number of monocytes was slightly, but not significantly, increased after the dive, whereas the absolute number of granulocytes was markedly elevated (up to 61%). There were no significant correlations between bubble formation and CD15+monocyte expression (r=-0.56; P=0.17), as well as with monocytes highly expressing CD15s (r=0.43; P=0.29). This study suggests that biochemical changes induced by scuba diving primarily activate existing monocytes rather than increase the number of monocytes at a time of acute arterial endothelial dysfunction.

  12. 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

  13. Cerebral blood flow in humans following resuscitation from cardiac arrest

    SciTech Connect

    Cohan, S.L.; Mun, S.K.; Petite, J.; Correia, J.; Tavelra Da Silva, A.T.; Waldhorn, R.E.

    1989-06-01

    Cerebral blood flow was measured by xenon-133 washout in 13 patients 6-46 hours after being resuscitated from cardiac arrest. Patients regaining consciousness had relatively normal cerebral blood flow before regaining consciousness, but all patients who died without regaining consciousness had increased cerebral blood flow that appeared within 24 hours after resuscitation (except in one patient in whom the first measurement was delayed until 28 hours after resuscitation, by which time cerebral blood flow was increased). The cause of the delayed-onset increase in cerebral blood flow is not known, but the increase may have adverse effects on brain function and may indicate the onset of irreversible brain damage.

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

    SciTech Connect

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

    1987-05-01

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

  15. 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

  16. 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.

  17. Nicotine induced changes in gene expression by human coronary artery endothelial cells.

    PubMed

    Zhang, S; Day, I; Ye, S

    2001-02-01

    The primary role of cigarette smoking in the development of coronary heart disease is to cause damage to the vascular endothelium, leading to endothelial cell dysfunction and initiating the pathogenesis of coronary atherosclerosis. We studied the response of human coronary artery endothelial cells to nicotine exposure by examining the expression of a panel of genes encoding molecules that have been shown to be involved in atherogenesis. Treatment of primary human coronary artery endothelial cells with nicotine for 24 h at concentrations (10(-5) and 10(-7) M) similar to those in the blood of smokers resulted in increased mRNA levels of endothelial nitric oxide synthase, angiotensin-I converting enzyme, tissue-type plasminogen activator, plasminogen activator inhibitor-1, von Willebrand factor, and vascular cell adhesion molecule-1. No change was detected in the expression levels of the genes encoding basic fibroblast growth factor, endothelin-1, endothelial leukocyte adhesion molecule-1 and matrix metalloproteinase-2 under these conditions. These data indicate that nicotine alters the expression of a number of endothelial genes whose products play major roles in regulating the vascular tone and thrombogenicity, making a contribution to the understanding of the effects of cigarette smoking on the development of coronary atherosclerosis. PMID:11166759

  18. Endotoxin and tumor necrosis factor induce interleukin-1 gene expression in adult human vascular endothelial cells.

    PubMed

    Libby, P; Ordovas, J M; Auger, K R; Robbins, A H; Birinyi, L K; Dinarello, C A

    1986-08-01

    Interleukin 1 (IL-1) can induce potentially pathogenic functions of vascular endothelial cells. This mediator was formerly thought to be produced primarily by activated macrophages. We report here that bacterial endotoxin and recombinant human tumor necrosis factor cause accumulation of IL-1 beta mRNA in adult human vascular endothelial cells. IL-1 alpha mRNA was also detected when endothelial cells were exposed to endotoxin under "superinduction" conditions in the presence of cycloheximide. Metabolic labeling of these cells during endotoxin stimulation demonstrated increased synthesis and secretion of immunoprecipitable IL-1 protein that comigrated electrophoretically with the predominant monocyte species. In parallel with increased IL-1 mRNA and protein, endothelial cells exposed to endotoxin also release biologically active IL-1 that was neutralized by anti-IL-1-antibody. Because bloodborne agents must traverse the endothelium before entering tissues, endothelial IL-1 production induced by microbial products or other injurious stimuli could initiate local responses to invasion. Endothelial cells are both a source of and target for IL-1; accordingly, this novel autocrine mechanism might play an early role in the pathogenesis of vasculitis, allograft rejection, and arteriosclerosis.

  19. Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation

    PubMed Central

    Zhang, Yuan; Wang, Ting; Yang, Ke; Xu, Ji; Ren, Lijie; Li, Weiping; Liu, Wenlan

    2016-01-01

    Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1’s downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke.

  20. Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

    PubMed

    Zhang, Yuan; Wang, Ting; Yang, Ke; Xu, Ji; Ren, Lijie; Li, Weiping; Liu, Wenlan

    2016-01-01

    Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1's downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke.

  1. Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation

    PubMed Central

    Zhang, Yuan; Wang, Ting; Yang, Ke; Xu, Ji; Ren, Lijie; Li, Weiping; Liu, Wenlan

    2016-01-01

    Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1’s downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke. PMID:27630541

  2. Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

    PubMed

    Zhang, Yuan; Wang, Ting; Yang, Ke; Xu, Ji; Ren, Lijie; Li, Weiping; Liu, Wenlan

    2016-01-01

    Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1's downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke. PMID:27630541

  3. Vascular endothelial growth factor in human preterm lung.

    PubMed

    Lassus, P; Ristimäki, A; Ylikorkala, O; Viinikka, L; Andersson, S

    1999-05-01

    Endothelial cell damage is characteristic for respiratory distress syndrome and development of chronic lung disease. Vascular endothelial growth factor (VEGF) is an endothelial mitogen that takes part in the growth and repair of vascular endothelial cells. We measured VEGF in 189 tracheal aspirate samples (TAF), and in 24 plasma samples from 44 intubated preterm infants (gestational age, 27.3 +/- 2.0 wk; birth weight, 962 +/- 319 g) during their first postnatal week. VEGF in TAF increased from 25 +/- 12 pg/ml (mean +/- SEM) on Day 1 to 526 +/- 120 pg/ml on Day 7 (mean concentrations, 106 +/- 25 pg/ml on Days 1 to 3 and 342 +/- 36 pg/ml on Days 4 to 7). In plasma, mean concentration of VEGF during the first week was 48 +/- 6 pg/ml, with no increase observed. In TAF, higher VEGF was found in patients born to mothers with premature rupture of the membranes, or chorionamnionitis, whereas preeclampsia of the mother was associated with lower VEGF (all p < 0.05). In TAF, no correlations existed between VEGF and gestational age or birth weight, but a correlation existed between lecithin/sphengomyelin ratio and VEGF (p < 0.05). During Days 4 to 7 patients developing bronchopulmonary dysplasia (BPD) had lower VEGF in TAF than did those surviving without BPD (235 +/- 31 versus 383 +/- 50; p < 0.05). VEGF increased rapidly in the lungs of the preterm infant during the first days of life. VEGF may be indicative of pulmonary maturity and may participate in pulmonary repair after acute lung injury.

  4. Understanding the Dorsal and Ventral Systems of the Human Cerebral Cortex: Beyond Dichotomies

    ERIC Educational Resources Information Center

    Borst, Gregoire; Thompson, William L.; Kosslyn, Stephen M.

    2011-01-01

    Traditionally, characterizations of the macrolevel functional organization of the human cerebral cortex have focused on the left and right cerebral hemispheres. However, the idea of left brain versus right brain functions has been shown to be an oversimplification. We argue here that a top-bottom divide, rather than a left-right divide, is a more…

  5. Honokiol suppresses TNF-α-induced neutrophil adhesion on cerebral endothelial cells by disrupting polyubiquitination and degradation of IκBα

    PubMed Central

    Chen, Po-Jen; Wang, Yu-Ling; Kuo, Liang-Mou; Lin, Chwan-Fwu; Chen, Chun-Yu; Tsai, Yung-Fong; Shen, Jiann-Jong; Hwang, Tsong-Long

    2016-01-01

    Adhesion molecules expressed on cerebral endothelial cells (ECs) mediate leukocyte recruitment and play a significant role in cerebral inflammation. Increased levels of adhesion molecules on the EC surface induce leukocyte infiltration into inflammatory areas and are thus hallmarkers of inflammation. Honokiol, isolated from the Chinese medicinal herb Magnolia officinalis, has various pharmacological activities, including anti-inflammatory effects, yet the nature of honokiol targeting molecules remains to be revealed. Here, we investigated the inhibitory effect of honokiol on neutrophil adhesion and vascular cell adhesion molecule-1 (VCAM-1) expression, which underlie its molecular target, and mechanisms for inactivating nuclear factor κ enhancer binding protein (NF-κB) in mouse cerebral ECs. Honokiol inhibited tumour necrosis factor-α (TNF-α)-induced neutrophil adhesion and VCAM-1 gene expression in cerebral ECs. The inflammatory transcription factor NF-κB was downregulated by honokiol. Honokiol significantly blocked TNF-α–induced NF-κB p65 nuclear translocation and degradation of the proteasome-dependent inhibitor of NF-κB α (IκBα). From docking model prediction, honokiol directly targeted the ubiquitin–ubiquitin interface of Lys48-linked polychains. Moreover, honokiol prevented the TNF-α-induced Lys48-linked polyubiquitination, including IκBα-polyubiquitin interaction. Honokiol has protective anti-inflammatory effects on TNF-α-induced neutrophil adhesion and VCAM-1 gene expression in cerebral ECs, at least in part by directly inhibiting ubiquitination-mediated IκBα degradation and then preventing NF-κB nuclear translocation. PMID:27212040

  6. Patterns of human local cerebral glucose metabolism during epileptic seizures

    SciTech Connect

    Engel, J. Jr.; Kuhl, D.E.; Phelps, M.E.

    1982-10-01

    Ictal patterns of local cerebral metabolic rate have been studied in epileptic patients by positron computed tomography with /sup 18/F-labeled 2-fluoro-2-deoxy-D-glucose. Partial seizures were associated with activation of anatomic structures unique to each patient studied. Ictal increases and decreases in local cerebral metabolism were observed. Scans performed during generalized convulsions induced by electroshock demonstrated a diffuse ictal increase and postictal decrease in cerebral metabolism. Petit mal absences were associated with a diffuse increase in cerebral metabolic rate. The ictal fluorodeoxyglucose patterns obtained from patients do not resemble autoradiographic patterns obtained from common experimental animal models of epilepsy.

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

    PubMed

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

    2012-05-01

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

  8. Increased expression of angiogenic factors in cultured human brain arteriovenous malformation endothelial cells.

    PubMed

    Xu, Ming; Xu, Hongzhi; Qin, Zhiyong; Zhang, Jie; Yang, Xiaoyu; Xu, Feng

    2014-09-01

    To compare the mRNA level of angiogenic factor vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-2, and MMP-9 in cultured human brain arteriovenous malformation (AVM) endothelial cells (ECs) and normal brain endothelial cells (BECs). Tissue explants both from deformed vessels of AVM and normal microvessel were put into culture for endothelial cells. After the monolayer adherent ECs reached confluence, they were tested with endothelial specific marker CD34 and von Willebrand factor (vWF) by immunochemical assay. mRNA levels of VEGF-A, MMP-2, and MMP-9 in AVM endothelial cells (AVMECs) and BECs were measured by PCR. Immunostaining confirmed that more than 95 % of the cultured cells were CD34 (Fig. 1b) and/or vWF positive. Expression levels of VEGF-A and MMP-2 mRNAs were significantly higher in AVMECs than in BECs. The MMP-9 level was also increased in AVMECs, but the difference was not statistically significant. Vascular tissue explants adherent method is a better approach for isolation and culture of AVMECs. Cultured AVMECs expressed higher angiogenic factors (VEGF, MMP-2) than the controlled BECs, implicating angiogenesis plays an important role in the pathogenesis of AVM.

  9. The effect of donor variation and senescence on endothelial differentiation of human mesenchymal stromal cells.

    PubMed

    Portalska, Karolina Janeczek; Groen, Nathalie; Krenning, Guido; Georgi, Nicole; Mentink, Anouk; Harmsen, Martin C; van Blitterswijk, Clemens; de Boer, Jan

    2013-11-01

    Application of autologous cells is considered for a broad range of regenerative therapies because it is not surrounded by the immunological and ethical issues of allo- or xenogenic cells. However, isolation, expansion, and application of autologous cells do suffer from variability in therapeutic efficacy due to donor to donor differences and due to prolonged culture. One important source of autologous cells is mesenchymal stromal cells (MSCs), which can differentiate toward endothelial-like cells, thus making them an ideal candidate as cell source for tissue vascularization. Here we screened MSCs from 20 donors for their endothelial differentiation capacity and correlated it with the gene expression profile of the whole genome in the undifferentiated state. Cells of all donors were able to form tubes on Matrigel and induced the expression of endothelial genes, although with quantitative differences. In addition, we analyzed the effect of prolonged in vitro expansion on the multipotency of human MSCs and found that endothelial differentiation is only mildly sensitive to expansion-induced loss of differentiation as compared to osteogenic and adipogenic differentiation. Our results show the robustness of the endothelial differentiation protocol and the gene expression data give insight in the differences in endothelial differentiation between donors.

  10. Characterization of cationic amino acid transporters and expression of endothelial nitric oxide synthase in human placental microvascular endothelial cells.

    PubMed

    Dye, J F; Vause, S; Johnston, T; Clark, P; Firth, J A; D'Souza, S W; Sibley, C P; Glazier, J D

    2004-01-01

    We investigated the expression and activity of arginine transporters and endothelial nitric oxide synthase (eNOS) in human placental microvascular endothelial cells (HPMEC). Using RT-PCR amplification products for eNOS, CAT1, CAT2A, CAT2B, CAT4, 4F2hc (CD98), rBAT and the light chains y+LAT1, y+LAT2, and b0+T1 were detected in HPMEC, but not B0+. Immunohistochemistry and Western blotting confirmed the presence of 4F2hc and CAT1 protein in HPMEC. 4F2hc-light chain dimers were indicated by a shift in molecular mass detected under nonreducing conditions. L-Arginine transport into HPMEC was independent of Na+ or Cl- and was inhibited by the neutral amino acid glutamine, but not by cystine. The Ki for glutamine inhibition was greater in the absence of Na+. Kinetic analysis supported a two-transporter model attributed to system y+L and system y+. Expression of eNOS in HPMEC was detectable by immunohistochemistry and ELISA but not by Western blotting. Activity of eNOS in HPMEC, measured over 48 h, either as the basal production of nitric oxide (NO) or as the accumulation of intracellular cGMP was not detectable. We conclude that HPMEC transport cationic amino acids by systems y+ and y+L and that basal eNOS expression and activity in these cells is low. PMID:14597568

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

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

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

  12. Endothelial Differentiation of Human Adipose-Derived Stem Cells on Polyglycolic Acid/Polylactic Acid Mesh

    PubMed Central

    Deng, Meng; Gu, Yunpeng; Liu, Zhenjun; Qi, Yue; Ma, Gui E.; Kang, Ning

    2015-01-01

    Adipose-derived stem cell (ADSC) is considered as a cell source potentially useful for angiogenesis in tissue engineering and regenerative medicine. This study investigated the growth and endothelial differentiation of human ADSCs on polyglycolic acid/polylactic acid (PGA/PLA) mesh compared to 2D plastic. Cell adhesion, viability, and distribution of hADSCs on PGA/PLA mesh were observed by CM-Dil labeling, live/dead staining, and SEM examination while endothelial differentiation was evaluated by flow cytometry, Ac-LDL/UEA-1 uptake assay, immunofluorescence stainings, and gene expression analysis of endothelial related markers. Results showed hADSCs gained a mature endothelial phenotype with a positive ratio of 21.4 ± 3.7% for CD31+/CD34− when induced in 3D mesh after 21 days, which was further verified by the expressions of a comprehensive range of endothelial related markers, whereas hADSCs in 2D induced and 2D/3D noninduced groups all failed to differentiate into endothelial cells. Moreover, compared to 2D groups, the expression for α-SMA was markedly suppressed in 3D cultured hADSCs. This study first demonstrated the endothelial differentiation of hADSCs on the PGA/PLA mesh and pointed out the synergistic effect of PGA/PLA 3D culture and growth factors on the acquisition of mature characteristic endothelial phenotype. We believed this study would be the initial step towards the generation of prevascularized tissue engineered constructs. PMID:26106426

  13. Mapping striate and extrastriate visual areas in human cerebral cortex.

    PubMed Central

    DeYoe, E A; Carman, G J; Bandettini, P; Glickman, S; Wieser, J; Cox, R; Miller, D; Neitz, J

    1996-01-01

    Functional magnetic resonance imaging (fMRI) was used to identify and map the representation of the visual field in seven areas of human cerebral cortex and to identify at least two additional visually responsive regions. The cortical locations of neurons responding to stimulation along the vertical or horizontal visual field meridia were charted on three-dimensional models of the cortex and on unfolded maps of the cortical surface. These maps were used to identify the borders among areas that would be topographically homologous to areas V1, V2, V3, VP, and parts of V3A and V4 of the macaque monkey. Visually responsive areas homologous to the middle temporal/medial superior temporal area complex and unidentified parietal visual areas were also observed. The topography of the visual areas identified thus far is consistent with the organization in macaque monkeys. However, these and other findings suggest that human and simian cortical organization may begin to differ in extrastriate cortex at, or beyond, V3A and V4. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8637882

  14. Stimulation of the cerebral cortex in the intact human subject

    NASA Astrophysics Data System (ADS)

    Merton, P. A.; Morton, H. B.

    1980-05-01

    One of the most fertile methods of investigating the brain is to stimulate a part of it electrically and observe the results. So far, however, use of the method in man has been restricted by the necessity of opening the skull surgically to apply the electrodes. Much could be done, both with healthy subjects and with neurological patients, if it were feasible to stimulate through electrodes on the scalp, although the localization of the stimulus on the cortex will always be much less sharp than with electrodes on the brain surface. In an intact man, however, the brain is protected from electricity by the skull and by the scalp, both of which normally offer considerable resistance. Furthermore, the cerebral cortex does not have a particularly low electrical threshold. It is probably for these reasons (despite an occasional contrary claim1) that attempts to stimulate the brain by applying stimuli from conventional stimulators to the scalp have been stopped by pain or have otherwise failed. These obstacles have now begun to yield. Recently, it was found that, on stimulating muscles in the human hand2 without any special preparation of the skin, the effective resistance fell to low values if brief but very high voltage shocks were used. Applying the same technique to the head, it has now proved possible at the first attempt to stimulate two areas of the human cortex, without undue discomfort.

  15. Anti-CD31 delays platelet adhesion/aggregation at sites of endothelial injury in mouse cerebral arterioles.

    PubMed

    Rosenblum, W I; Murata, S; Nelson, G H; Werner, P K; Ranken, R; Harmon, R C

    1994-07-01

    The arterioles on the surface of the mouse brain (pial arterioles) were observed by in vivo microscopy. A focus of minor endothelial damage was produced in a single pial arteriole in each mouse by briefly exposing the site to a helium neon laser after an intravenous injection of Evans blue. Mice were injected 10 minutes before injury with a monoclonal antibody (MAb) to mouse CD31, also known as platelet endothelial cell adhesion molecule. This treatment doubled (P < .01) the time required for the laser to produce a recognizable platelet aggregate. In additional experiments, an MAb to mouse CD61 and an MAb to mouse intercellular adhesion molecule 1 had no effect. The data support previous observations indicating that platelet adhesion/aggregation in this model is induced by endothelial injury without exposure of basal lamina. The data are consistent with the hypothesis that the endothelial injury exposes or activates a platelet endothelial cell adhesion molecule on the endothelium which is blocked by the MAb directed against CD31. This may be the first demonstration of an effect of an anti-platelet endothelial cell adhesion molecule on platelet endothelial cell adhesion molecule on platelet adhesion/aggregation in vivo. PMID:8030753

  16. Induction of heme oxygenase-1 by whisky congeners in human endothelial cells.

    PubMed

    Suzuki, Keiko; Nemoto, Asuka; Tanaka, Izumi; Koshimizu, Seiichi; Suwa, Yoshihide; Ishihara, Hiroshi

    2010-08-01

    It is expected that the production of the cytoprotective heme oxygenase-1 (HO-1) protein in endothelial cells would reduce severity of vascular injuries, while phenolic compounds are known to induce HO-1 mRNA and protein in various cells. We investigated the activation of HO-1 by whisky, which contains various phenolic substances. The congeners of whisky stored from 4 to 18 y in oak barrels were shown to induce an increase of HO-1 protein in human umbilical vein endothelial cells, while those of freshly distilled whisky spirit exhibited no activity. To determine the compounds with potent HO-1-inducing activity among the whisky congeners, several chemicals that had been reported to exist in whisky or oak barrels were screened, and coniferyl aldehyde and sinapyl aldehyde showed the activity. Thus, compounds that emerged in whisky during barrel storage induced cytoprotective protein, HO-1, in human endothelial cells.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  19. Cerebral oxygen metabolism and blood flow in human cerebral ischemic infarction

    SciTech Connect

    Lenzi, G.L.; Frackowiak, R.S.; Jones, T.

    1982-09-01

    Fifteen patients with acute cerebral hemispheric infarcts have been studied with positron emission tomography and the /sup 15/O steady-state inhalation technique. Thirteen follow-up studies were also performed. The values of cerebral oxygen metabolism (CMRO/sub 2/), cerebral blood flow (CBF), and oxygen extraction ration (OER) have been calculated for the infarcted regions, their borders, the symmetrical regions in contralateral cerebral hemispheres, and the cerebellar hemispheres. This study demonstrates that in the completed stroke there are thresholds for regional CMRO/sub 2/ and regional CBF below which the general clinical outcome of the patients is usually poor. The ischaemic lesions invariably produce an uncoupling between the greatly decreased metabolic demand and the less affected blood supply, with very frequent instances of relative hyperperfusion. Remote effects of the hemispheric infarcts have been demonstrated, such as crossed cerebellar diaschisis and contralateral transhemispheric depression. The level of consciousness correlates with oxygen uptake and blood flow both in the posterior fossa and in the contralateral cerebral hemispheres. The follow-up studies of individual patients underline the high variability of metabolism-to-flow balance during the acute phase of the illness, and stress the need for more studies focused on repeated assessments of homogeneous patient populations.

  20. 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

  1. Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase.

    PubMed

    Shao, Beili; Bayraktutan, Ulvi

    2014-01-01

    Blood-brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2 (•-) generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2 (•-) by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2 (•-) production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase.

  2. Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase

    PubMed Central

    Shao, Beili; Bayraktutan, Ulvi

    2014-01-01

    Blood–brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2•- generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2•- by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2•- production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase. PMID:24936444

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

    SciTech Connect

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

    1991-03-11

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

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

    SciTech Connect

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

    1994-01-15

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

  5. Marinobufagenin and cyclic strain may activate endothelial NADPH oxidase, contributing to the adverse impact of salty diets on vascular and cerebral health.

    PubMed

    McCarty, Mark F

    2012-02-01

    Limited but provocative ecologic epidemiology suggests that dietary salt may play a central role in the genesis of not only of stroke, but also dementia, including Alzheimer's disease. Impairment of nitric oxide bioactivity in the cerebral microvasculature is a likely mediator of this effect. Salted diets evoke increased adrenal secretion of the natriuretic steroid marinobufagenin (MBG), which promotes natriuresis via inhibition of renal tubular Na+/K+-ATPase; this effect is notably robust in salt-sensitive rodent strains in which other compensatory natriuretic mechanisms are subnormally efficient. MBG-mediated inhibition of sodium pumps in vascular smooth muscle likely plays a role in the hypertension induced by salty diets in these rodents. However, salt sensitivity in humans is associated with increased vascular mortality and ventricular hypertrophy independent of blood pressure; this suggests that MBG may be pathogenic via mechanisms unrelated to blood pressure control. Indeed, recent evidence indicates that MBG, via interaction with alpha1 isoforms of the sodium pump, can activate various intracellular signaling pathways at physiological concentrations too low to notably inhibit pump activity. An overview of current evidence suggests the hypothesis that MBG - as well as the cyclic strain induced by hypertension per se - may induce endothelial oxidative stress by activating NADPH oxidase. If so, this could rationalize the increase in vascular and systemic oxidative stress observed in salt-sensitive rodents fed salty diets, or in rodents infused with MBG; moreover, if this effect is a particularly prominent determinant of oxidative stress in cerebrovascular endothelium, it might help to explain the virtual absence of stroke and dementia in low-salt societies. As a corollary of this hypothesis, it can be predicted that spirulina-derived phycobilins, which appear to mimic the physiological role of bilirubin as an inhibitor of NAPDH oxidase complexes, may have

  6. Micro- and nanotopography with extracellular matrix coating modulate human corneal endothelial cell behavior.

    PubMed

    Koo, Stephanie; Muhammad, Rizwan; Peh, Gary S L; Mehta, Jodhbir S; Yim, Evelyn K F

    2014-05-01

    The human corneal endothelium plays an important role in maintaining corneal transparency. Human corneal endothelial cells have limited regenerative capability in vivo. Consequently, endothelial dysfunction can occur following corneal endothelial trauma or inherited diseases. To restore endothelial function, corneal transplantation is needed. However, there is a worldwide shortage of donor corneas, motivating the development of a tissue-engineered graft alternative using cultivated endothelial cells. To induce in vitro cell proliferation, much effort has been made to improve culture conditions and to mimic the native extracellular microenvironment. We incorporated topographical and biochemical cues in our in vitro culture of human corneal endothelial cell line B4G12 (HCEC-B4G12) and hypothesized that manipulation of the extracellular environment can modulate cell proliferation, morphometry and phenotype. The topographies tested were nanopillars, microwells and micropillars on polydimethylsiloxane, while the biochemical factors were extracellular matrix protein coatings of fibronectin-collagen I (FC), FNC® coating mix (FNC) and laminin-chondroitin sulfate (LC). Cellular morphometry, Na(+)/K(+)-ATPase and zona occludens 1 (ZO-1) gene and protein expression were analyzed 3days after cells had formed a confluent monolayer. The cell circularity on all patterns and coatings was above 0.78. On all coatings, cell area was the lowest on micropillars. The coefficient of variation (CV) of the cell area was the lowest on nanopillars with an LC coating. With an FC coating, micropillars induced a better cellular outcome as the cells had the greatest circularity, smallest cell area and highest Na(+)/K(+)-ATPase and ZO-1 gene and protein expression. With the LC coating, HCECs grown on nanopillars resulted in the lowest CV of the cell area and the highest ZO-1 gene expression. Thus, HCEC-B4G12 morphometry and phenotype can be improved using different topographical and biochemical

  7. Functional involvement of cerebral cortex in human narcolepsy.

    PubMed

    Oliviero, A; Della Marca, G; Tonali, P A; Pilato, F; Saturno, E; Dileone, M; Versace, V; Mennuni, G; Di Lazzaro, V

    2005-01-01

    The pathophysiology of human narcolepsy is still poorly understood. The hypoactivity of some neurotransmitter systems has been hypothesised on the basis of the canine model. To determine whether narcolepsy is associated with changes in excitability of the cerebral cortex, we assessed the excitability of the motor cortex with transcranial magnetic stimulation (TMS) in 13 patients with narcolepsy and in 12 control subjects. We used several TMS paradigms that can provide information on the excitability of the motor cortex. Resting and active motor thresholds were higher in narcoleptic patients than in controls and intracortical inhibition was more pronounced in narcoleptic patients. No changes in the other evaluated measures were detected. These results are consistent with an impaired balance between excitatory and inhibitory intracortical circuits in narcolepsy that leads to cortical hypoexcitability. We hypothesise that the deficiency of the excitatory hypocretin/orexin-neurotransmitter-system in narcolepsy is reflected in changes of cortical excitability since circuits originating in the lateral hypothalamus and in the basal forebrain project widely to the neocortex, including motor cortex. This abnormal excitability of cortical networks could be the physiological correlate of excessive daytime sleepiness and it could be the substrate for allowing dissociated states of wakefulness and sleep to emerge suddenly while patients are awake, which constitute the symptoms of narcolepsy. PMID:15654554

  8. Attenuation of signaling and nitric oxide production following prolonged leptin exposure in human aortic endothelial cells.

    PubMed

    Blanquicett, Carmelo; Graves, Anitra; Kleinhenz, Dean J; Hart, C Michael

    2007-11-01

    Acute leptin exposure stimulates endothelial nitric oxide (NO) production in vitro. In contrast, chronic elevations in circulating leptin levels in patients with obesity are associated with endothelial dysfunction and impaired endothelial NO production. Therefore, the goal of the current study was to examine the direct effects of acute and more sustained leptin stimulation on endothelial nitric oxide synthase (eNOS) and NO production in human aortic endothelial cells (HAECs). HAECs were treated with vehicle or with leptin (5 or 60 ng/mL) acutely (30-60 minutes) or for 72 hours. HAEC NO release into culture media was measured with a chemiluminescence technique, and superoxide (O(2)(-.)) production was measured with electron spin resonance (ESR) spectroscopy. HAEC eNOS activity was measured as the conversion of (3)H-arginine to (3)H-citrulline, and protein levels of eNOS, phospho-eNOS (serine 1177), Erk, phospho-Erk, suppressor of cytokine signaling (SOCS3), xanthine oxidase (XO), and the reduced nicotinamide adenine dinucleotide (NADPH) oxidase components p22phox, p67phox, Nox-4, and gp91phox were examined by Western blotting or immunoprecipitation. Acute leptin exposure increased eNOS serine 1177 phosphorylation and caused Erk activation. In contrast, prolonged leptin stimulation was not cytotoxic and failed to alter eNOS expression, phosphorylation, or HAEC NO release. Furthermore, prolonged leptin stimulation did not alter O(2)(-.) production or NADPH oxidase or XO expression but increased SOCS3 expression. In contrast to acute stimulation, prolonged (72 hours) stimulation does not alter endothelial cell NO or O(2)(-.) production. We postulate that chronic leptin stimulation, through increased SOCS3 expression, may attenuate the effects of leptin on vascular endothelial function. PMID:18062898

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Chen, Chu-Huang; Pellis, Neal R.

    1997-01-01

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

  11. 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.

  12. Increased expression of extracellular proteins as a hallmark of human endothelial cell in vitro senescence.

    PubMed

    Hampel, B; Fortschegger, K; Ressler, S; Chang, M W; Unterluggauer, H; Breitwieser, A; Sommergruber, W; Fitzky, B; Lepperdinger, G; Jansen-Dürr, P; Voglauer, R; Grillari, J

    2006-05-01

    A convenient way to study processes of aging in distinct human tissues consists of a molecular analysis of cells from the tissue in question, that were explanted and grown in vitro until they reach senescence. Using human umbilical vein endothelial cells (HUVEC), we have established an in vitro senescence model for human endothelial cells. A major hallmark of HUVEC in vitro senescence is the increased frequency of apoptotic cell death, which occurs as a determining feature of HUVEC senescence. Senescent endothelial cells are also found in vivo in atherosclerotic lesions, suggesting that the presence of such cells may contribute to the development of vascular pathology. To elucidate mechanisms underlying endothelial cell senescence and age-associated apoptosis, gene expression analyses were carried out. In these experiments, we observed the up-regulation of genes coding for extracellular proteins in senescent HUVEC. In particular, a significant upregulation of interleukin-8, VEGI, and the IGF-binding proteins 3 and 5 was observed. Upregulation of these genes was confirmed by both RT-PCR and Western blot. In the case of interleukin-8, a roughly 50-fold upregulation of the protein was also found in cellular supernatants. The extracellular proteins encoded by these genes are well known for their ability to modulate the apoptotic response of human cells, and in the case of interleukin-8, clear links to the establishment of atherosclerotic lesions have been defined. The results described here support a new model, where changes in the secretome of human endothelial cells contribute to vascular aging and vascular pathology. PMID:16626901

  13. Argonaute‐2 Promotes miR‐18a Entry in Human Brain Endothelial Cells

    PubMed Central

    Ferreira, Raquel; Santos, Tiago; Amar, Arun; Gong, Alex; Chen, Thomas C.; Tahara, Stanley M.; Giannotta, Steven L.; Hofman, Florence M.

    2014-01-01

    Background Cerebral arteriovenous malformation (AVM) is a vascular disease exhibiting abnormal blood vessel morphology and function. miR‐18a ameliorates the abnormal characteristics of AVM‐derived brain endothelial cells (AVM‐BEC) without the use of transfection reagents. Hence, our aim was to identify the mechanisms by which miR‐18a is internalized by AVM‐BEC. Since AVM‐BEC overexpress RNA‐binding protein Argonaute‐2 (Ago‐2) we explored the clinical potential of Ago‐2 as a systemic miRNA carrier. Methods and Results Primary cultures of AVM‐BEC were isolated from surgical specimens and tested for endogenous miR‐18a levels using qPCR. Conditioned media (CM) was derived from AVM‐BEC cultures (AVM‐BEC‐CM). AVM‐BEC‐CM significantly enhanced miR‐18a internalization. Ago‐2 was detected using western blotting and immunostaining techniques. Ago‐2 was highly expressed in AVM‐BEC; and siAgo‐2 decreased miR‐18a entry into brain‐derived endothelial cells. Only brain‐derived endothelial cells were responsive to the Ago‐2/miR‐18a complex and not other cell types tested. Secreted products (eg, thrombospondin‐1 [TSP‐1]) were tested using ELISA. Brain endothelial cells treated with the Ago‐2/miR‐18a complex in vitro increased TSP‐1 secretion. In the in vivo angiogenesis glioma model, animals were treated with miR‐18a in combination with Ago‐2. Plasma was obtained and tested for TSP‐1 and vascular endothelial growth factor (VEGF)‐A. In this angiogenesis model, the Ago‐2/miR‐18a complex caused a significant increase in TSP‐1 and decrease in VEGF‐A secretion in the plasma. Conclusions Ago‐2 facilitates miR‐18a entry into brain endothelial cells in vitro and in vivo. This study highlights the clinical potential of Ago‐2 as a miRNA delivery platform for the treatment of brain vascular diseases. PMID:24837588

  14. Effect of salicylic acid on invasion of human vascular endothelial cells by Staphylococcus aureus.

    PubMed

    Park, Wan Beom; Kim, Sung-Han; Cho, Jae Hyun; Bang, Ji Hwan; Kim, Hong Bin; Kim, Nam Joong; Oh, Myoung-don; Choe, Kang Won

    2007-02-01

    Invasion of vascular endothelial cells by Staphylococcus aureus is associated with diverse complications and recurrent infection. Little is known about the effect of salicylic acid, the major metabolite of aspirin, on the interaction between S. aureus and vascular endothelial cells. We examined the adhesion of S. aureus strain 8325-4 cultured with or without salicylic acid to human umbilical vein endothelial cells (HUVECs), and the ability of the strain to invade these cells. Strain 8325-4 cells grown in salicylic acid were significantly less adherent to and invasive in HUVECs. Production of cytokine interleukin (IL)-6 was lower from the HUVECs infected with clinical isolates of S. aureus cultured in salicylic acid compared with those unexposed to salicylic acid. This study raises the possibility of using salicylic acid as an adjuvant therapeutic agent in the treatment of S. aureus bacteremia to prevent its complications or recurrence.

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

    SciTech Connect

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

    2008-02-29

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

  16. Coenzyme Q10 prevents high glucose-induced oxidative stress in human umbilical vein endothelial cells.

    PubMed

    Tsuneki, Hiroshi; Sekizaki, Naoto; Suzuki, Takashi; Kobayashi, Shinjiro; Wada, Tsutomu; Okamoto, Tadashi; Kimura, Ikuko; Sasaoka, Toshiyasu

    2007-07-01

    Hyperglycemia-induced oxidative stress plays a crucial role in the pathogenesis of vascular complications in diabetes. Although some clinical evidences suggest the use of an antioxidant reagent coenzyme Q10 in diabetes with hypertension, the direct effect of coenzyme Q10 on the endothelial functions has not been examined. In the present study, we therefore investigated the protective effect of coenzyme Q10 against high glucose-induced oxidative stress in human umbilical vein endothelial cells (HUVEC). HUVEC exposed to high glucose (30 mM) exhibited abnormal properties, including the morphological and biochemical features of apoptosis, overproduction of reactive oxygen species, activation of protein kinase Cbeta2, and increase in endothelial nitric oxide synthase expression. Treatment with coenzyme Q10 strongly inhibited these changes in HUVEC under high glucose condition. In addition, coenzyme Q10 inhibited high glucose-induced cleavage of poly(ADP-ribose) polymerase, an endogenous caspase-3 substrate. These results suggest that coenzyme Q10 prevents reactive oxygen species-induced apoptosis through inhibition of the mitochondria-dependent caspase-3 pathway. Moreover, consistent with previous reports, high glucose caused upregulation of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in HUVEC, and promoted the adhesion of U937 monocytic cells. Coenzyme Q10 displayed potent inhibitory effects against these endothelial abnormalities. Thus, we provide the first evidence that coenzyme Q10 has a beneficial effect in protecting against the endothelial dysfunction by high glucose-induced oxidative stress in vitro.

  17. 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.

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

    PubMed Central

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

    1995-01-01

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

  19. 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

  20. Oxidative stress and apoptosis induced by iron oxide nanoparticles in cultured human umbilical endothelial cells.

    PubMed

    Zhu, Mo-Tao; Wang, Yun; Feng, Wei-Yue; Wang, Bing; Wang, Meng; Ouyang, Hong; Chai, Zhi-Fang

    2010-12-01

    Recent epidemiologic researches indicate that exposure to ultrafine particles (nanoparticles) is an independent risk factor for several cardiovascular diseases. The induction of endothelial injuries is hypothesized to be an attractive mechanism involved in these cardiovascular diseases. To investigate this hypothesis, the widely used iron nanomaterials, ferric oxide (Fe2O3) and ferriferrous oxide (Fe3O4) nanoparticles were incubated with human umbilical endothelial cells (ECV304 cells) at different concentrations of 2, 20, 100 microg/mL. The cell viability, the rate of apoptosis, the apoptotic nuclear morphology and the mitochondria membrane potential were measured to estimate the cell necrosis and apoptosis caused by the nanoparticle exposure. The stimulation of superoxide anion (O2*-) and nitric oxide (NO) were examined to evaluate the stress responses of endothelial cells. Our results indicated that both the Fe2O3 and Fe3O4 nanoparticles could generate oxidative stress as well as the significant increase of nitric oxide in ECV304 cells. The loss of mitochondria membrane potential and the apoptotic chromatin condensation in the nucleus were observed as the early signs of apoptosis. It is inferred the stress response might be an important mechanism involving in endothelial cells apoptosis and death, and these injuries in endothelial cells might play a key role in downstream cardiovascular diseases such as atheroscelerosis, hypertension and myocardial infarction (MI).

  1. Noninvasive method of estimating human newborn regional cerebral blood flow

    SciTech Connect

    Younkin, D.P.; Reivich, M.; Jaggi, J.; Obrist, W.; Delivoria-Papadopoulos, M.

    1982-12-01

    A noninvasive method of estimating regional cerebral blood flow (rCBF) in premature and full-term babies has been developed. Based on a modification of the /sup 133/Xe inhalation rCBF technique, this method uses eight extracranial NaI scintillation detectors and an i.v. bolus injection of /sup 133/Xe (approximately 0.5 mCi/kg). Arterial xenon concentration was estimated with an external chest detector. Cerebral blood flow was measured in 15 healthy, neurologically normal premature infants. Using Obrist's method of two-compartment analysis, normal values were calculated for flow in both compartments, relative weight and fractional flow in the first compartment (gray matter), initial slope of gray matter blood flow, mean cerebral blood flow, and initial slope index of mean cerebral blood flow. The application of this technique to newborns, its relative advantages, and its potential uses are discussed.

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

    PubMed

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

    2012-08-01

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

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

    PubMed Central

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

    1988-01-01

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

  4. Novel Nox inhibitor of oxLDL-induced reactive oxygen species formation in human endothelial cells.

    PubMed

    Stielow, Claudia; Catar, Rusan A; Muller, Gregor; Wingler, Kirstin; Scheurer, Peter; Schmidt, Harald H H W; Morawietz, Henning

    2006-05-26

    In this study, we investigated effects of a novel NAD(P)H oxidase (Nox)-inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870) on oxidized low-density lipoprotein (oxLDL)-mediated reactive oxygen species (ROS) formation in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were cultured to confluence and ROS formation was induced with 50microg/ml oxLDL for 2h. ROS formation was detected by chemiluminescence (CL) using the Diogenes reagent. OxLDL induced ROS formation in human endothelial cells (171+/-12%; n=10, P<0.05 vs. control). This augmented ROS formation in response to oxLDL was completely inhibited by the Nox inhibitor VAS2870 (101+/-9%; n=7, P<0.05 vs. oxLDL). Similar results were obtained with superoxide dismutase (91+/-7%; n=7, P<0.05 vs. oxLDL). However, the Nox4 mRNA expression level was neither changed by oxLDL nor VAS2870. We conclude that VAS2870 could provide a novel strategy to inhibit the augmented endothelial superoxide anion formation in response to cardiovascular risk factors. PMID:16603125

  5. Anaphylatoxin C5a fails to promote prostacyclin release from cultured human endothelial cells

    SciTech Connect

    Lunberg, C.; Marceau, F.; Huey, R.; Hugli, T.E.

    1986-03-01

    A predominantly relaxing effect of C5a on isolated blood vessels has been reported, which is associated with prostacyclin release from the vessel wall. Further, the well known hypothensive effect of C5a, also associated with increased prostacyclin output and preventable by indomethacin, indicates an involvement of endothelial cells in this reaction. In this study the authors characterized the response to C5a of cultured human endothelial cells from umbilical vein as measured by prostacyclin release. Prostacyclin was quantitated by radioimmunoassay as 6-keto-PGF/sub 1..cap alpha../. Subcultured cells respond to histamine and mellitin with increased prostacyclin production, but do not respond to leukotriene C4 (LTC/sub 4/). Primary cultures, on the other hand, respond to LTC/sub 4/ and the histamine response is 7-fold greater for these cells than for subcultured cells. However, neither primary nor subcultured cells release prostacyclin following application of either human C5a (100 nM) or C3a (1 ..mu..M). Also, these cells fail to show specific binding sites for /sup 125/I-C5a. In contrast, endothelial cells in the presence of human PMNs challenged with C5a release prostacyclin. These data suggest that C5a has no direct effect on the endothelial cell, but rather activates this cell indirectly via mediators from other cells known to respond to C5a.

  6. Transcriptional and functional adaptations of human endothelial cells to physiological chronic low oxygen.

    PubMed

    Jiang, Yi-Zhou; Wang, Kai; Li, Yan; Dai, Cai-Feng; Wang, Ping; Kendziorski, Christina; Chen, Dong-Bao; Zheng, Jing

    2013-05-01

    Endothelial cells chronically reside in low-O2 environments in vivo (2%-13% O2), which are believed to be critical for cell homeostasis. To elucidate the roles of this physiological chronic normoxia in human endothelial cells, we examined transcriptomes of human umbilical vein endothelial cells (HUVECs), proliferation and migration of HUVECs in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA), and underlying signaling mechanisms under physiological chronic normoxia. Immediately after isolation, HUVECs were cultured steadily under standard cell culture normoxia (SCN; 21% O2) or physiological chronic normoxia (PCN; 3% O2) up to 25 days. We found that PCN up-regulated 41 genes and down-regulated 21 genes, 90% of which differed from those previously reported from HUVECs cultured under SCN and exposed to acute low O2. Gene ontology analysis indicated that PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from benchtop assays that showed that PCN significantly enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. Interestingly, preexposing the PCN cells to 21% O2 up to 5 days did not completely diminish PCN-enhanced cell proliferation and migration. These PCN-enhanced cell proliferations and migrations were mediated via augmented activation of MEK1/MEK2/ERK1/ERK2 and/or PI3K/AKT1. Importantly, these PCN-enhanced cellular responses were associated with an increase in activation of VEGFR2 but not FGFR1, without altering their expression. Thus, PCN programs endothelial cells to undergo dramatic changes in transcriptomes and sensitizes cellular proliferative and migratory responses to FGF2 and VEGFA. These PCN cells may offer a unique endothelial model, more closely mimicking the in vivo states.

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2016-02-01

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

  9. Microwave irradiation of rats at 2. 45 GHz activates pinocytotic-like uptake of tracer by capillary endothelial cells of cerebral cortex

    SciTech Connect

    Neubauer, C.; Phelan, A.M.; Kues, H.; Lange, D.G. )

    1990-01-01

    Far-field exposures of male albino rats to 2.45-GHz microwaves (10-microseconds pulses, 100 pps) at a low average power density (10 mW/cm2; SAR approximately 2 W/kg) and short durations (30-120 min) resulted in increased uptakes of tracer through the blood-brain barrier (BBB). The uptake of systemically administered rhodamine-ferritin complex by capillary endothelial cells (CECs) of the cerebral cortex was dependent on power density and on duration of exposure. At 5 mW/cm2, for example, a 15-min exposure had no effect. Near-complete blockade of uptake resulted when rats were treated before exposure to microwaves with a single dose of colchicine, which inhibits microtubular function. A pinocytotic-like mechanism is presumed responsible for the microwave-induced increase in BBB permeability.

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

    PubMed

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

    2015-09-01

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

  11. Biocompatibility of common polyimides with human endothelial cells for a cardiovascular microsensor.

    PubMed

    Starr, Peter; Agrawal, C Mauli; Bailey, Steven

    2016-02-01

    The cardiovasculature is an emerging niche for polyimide microdevices, yet the biocompatibility of polyimides with human endothelial cells has not been reported in the literature. In this study, we have evaluated an experimental polyimide-based pressure sensor for biological safety to determine its suitability for intravascular operation by using an in vitro model of human endothelium, following ISO 10993-5 protocols for extract tests and direct contact tests. First, SV-HCEC cells were incubated with extracts derived from common microfabrication polyimides utilized in the transducer (PMDA-ODA, BPDA-PPD, and a proprietary thermoplastic adhesive), and then labeled with selective probes to evaluate the effect of the polyimides on mitochondria and cell viability. Flow cytometry analysis showed that incubation of SV-HCECs with polyimide extracts resulted in no significant change in mitochondrial membrane potential (detected by JC-1) or apoptotic (annexin V) and necrotic (propidium iodide) cell death, when compared to incubation with extracts of high-density polyethylene (HDPE) and untreated cells used as negative controls. Second, primary human endothelial cells were incubated in direct contact with the completed sensor and then labeled with selective probes for live-dead analysis (calcein-AM, ethidium homodimer-1). Endothelial cells showed no loss of viability when compared to negative controls. Combined, the studies show no significant change in early markers of stress or more strict markers of viability in endothelial cells treated with the polyimides tested. We conclude that these common microfabrication polyimides and the derived sensor are not cytotoxic to human endothelial cells, the primary cell type that cardiovascular sensors will contact in vivo. PMID:26418753

  12. Proangiogenic effects of environmentally relevant levels of bisphenol A in human primary endothelial cells.

    PubMed

    Andersson, Helén; Brittebo, Eva

    2012-03-01

    Bisphenol A (BPA) is widely used in the manufacturing of consumer products such as plastic food containers and food cans. Experimental studies suggest a relationship between exposure to BPA and changes in metabolic processes and reproductive organs. Also, epidemiological studies report an association between elevated exposure to BPA and cardiovascular disease and diabetes. Although alterations in the vascular endothelium are implicated in pathological conditions associated with BPA, little is known about the effects of BPA in the human endothelium. This study aimed to investigate the effects of 0.1 nM-1 μM of BPA on selected biomarkers of endothelial dysfunction, inflammation, and angiogenesis in human umbilical vein endothelial cells (HUVEC). The mRNA expression of biomarkers was assayed using qRT-PCR, and the production of nitric oxide and reactive oxygen species was measured using the H(2)DCFDA and the DAF-FM assays. The effect of BPA on phosphorylated eNOS was examined using Western blot and immunofluorescence, and the endothelial tube formation assay was used to investigate in vitro angiogenesis. BPA (≤1 μM) increased the mRNA expression of the proangiogenic genes VEGFR-2, VEGF-A, eNOS, and Cx43 and increased the production of nitric oxide in HUVEC. Furthermore, BPA increased the expression of phosphorylated eNOS and endothelial tube formation in HUVEC. These studies demonstrate that environmentally relevant levels of BPA have direct proangiogenic effects on human primary endothelial cells in vitro suggesting that the human endothelium may be an important target for BPA.

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

    PubMed Central

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

    2016-01-01

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

  14. Direct Antiangiogenic Actions of Cadmium on Human Vascular Endothelial Cells*

    PubMed Central

    Woods, J. M.; Leone, M.; Klosowska, K.; Lamar, P. C.; Shaknovsky, T. J.; Prozialeck, W. C.

    2008-01-01

    The vascular endothelium is a primary target of cadmium (Cd) toxicity, but little is known regarding a potential mechanism whereby Cd may inhibit angiogenesis. Recent findings showing that Cd can disrupt cadherin-mediated cell-cell adhesion suggested that Cd might inhibit angiogenesis by altering the function of VE-cadherin, a molecule that is essential for angiogenesis. To address this issue, endothelial cells (ECs) were exposed to Cd in the presence of serum and subjected to angiogenesis-related cell migration and tube formation assays. Initial examination of cytotoxicity showed that ECs are rather resistant to the acute cytotoxic effects of Cd even at concentrations up to 1mM. However, 10μM Cd decreased migration of ECs. Cd concentrations of 500nM and greater significantly reduced organization of microvascular ECs into tubes. These antiangiogenic effects were evident even when ECs were preincubated with Cd and then washed to remove free Cd, indicating that Cd acted directly on the cells rather than on the extracellular matrix. Immunolocalization studies showed that Cd caused the redistribution of VE-cadherin from cell-cell contacts. These findings indicate that Cd acts in an angiostatic manner on ECs, and that this effect may involve alterations in the localization and function of VE cadherin. PMID:18243643

  15. Bortezomib induces autophagic death in proliferating human endothelial cells

    SciTech Connect

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

    2010-04-01

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

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

    SciTech Connect

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

    2008-01-11

    Cell to cell interaction is one of the key processes effecting angiogenesis and endothelial cell function. Wnt signalling is mediated through cell-cell interaction and is involved in many developmental processes and cellular functions. In this study, we investigated the possible function of Wnt5a and the non-canonical Wnt pathway in human endothelial cells. We found that Wnt5a-mediated non-canonical Wnt signalling regulated endothelial cell proliferation. Blocking this pathway using antibody, siRNA or a down-stream inhibitor led to suppression of endothelial cell proliferation, migration, and monolayer wound closure. We also found that the mRNA level of Wnt5a is up-regulated when endothelial cells are treated with a cocktail of inflammatory cytokines. Our findings suggest non-canonical Wnt signalling plays a role in regulating endothelial cell growth and possibly in angiogenesis.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  19. Influence of the oxygen microenvironment on the proangiogenic potential of human endothelial colony forming cells.

    PubMed

    Decaris, Martin L; Lee, Chang I; Yoder, Mervin C; Tarantal, Alice F; Leach, J Kent

    2009-01-01

    Therapeutic angiogenesis is a promising strategy to promote the formation of new or collateral vessels for tissue regeneration and repair. Since changes in tissue oxygen concentrations are known to stimulate numerous cell functions, these studies have focused on the oxygen microenvironment and its role on the angiogenic potential of endothelial cells. We analyzed the proangiogenic potential of human endothelial colony-forming cells (hECFCs), a highly proliferative population of circulating endothelial progenitor cells, and compared outcomes to human dermal microvascular cells (HMVECs) under oxygen tensions ranging from 1% to 21% O2, representative of ischemic or healthy tissues and standard culture conditions. Compared to HMVECs, hECFCs (1) exhibited significantly greater proliferation in both ischemic conditions and ambient air; (2) demonstrated increased migration compared to HMVECs when exposed to chemotactic gradients in reduced oxygen; and (3) exhibited comparable or superior proangiogenic potential in reduced oxygen conditions when assessed using a vessel-forming assay. These data demonstrate that the angiogenic potential of both endothelial populations is influenced by the local oxygen microenvironment. However, hECFCs exhibit a robust angiogenic potential in oxygen conditions representative of physiologic, ischemic, or ambient air conditions, and these findings suggest that hECFCs may be a superior cell source for use in cell-based approaches for the neovascularization of ischemic or engineered tissues.

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

    SciTech Connect

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

    1989-03-01

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

  1. Human brain microvascular endothelial cells resist elongation due to shear stress.

    PubMed

    Reinitz, Adam; DeStefano, Jackson; Ye, Mao; Wong, Andrew D; Searson, Peter C

    2015-05-01

    Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm(-2). From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood-brain barrier.

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

    PubMed

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

    2015-04-01

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

  3. 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.

  4. 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

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

    NASA Astrophysics Data System (ADS)

    Sharma, Preety; Guida, Peter; Grabham, Peter

    2014-07-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2012-01-01

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

  8. Cerebral blood flow decreases with time whereas cerebral oxygen consumption remains stable during hypothermic cardiopulmonary bypass in humans

    SciTech Connect

    Prough, D.S.; Rogers, A.T.; Stump, D.A.; Roy, R.C.; Cordell, A.R.; Phipps, J.; Taylor, C.L. )

    1991-02-01

    Recent investigations demonstrate that cerebral blood flow (CBF) progressively declines during hypothermic, nonpulsatile cardiopulmonary bypass (CPB). If CBF declines because of brain cooling, the cerebral metabolic rate for oxygen (CMRO2) should decline in parallel with the reduction in CBF. Therefore we studied the response of CBF, the cerebral arteriovenous oxygen content difference (A-VDcereO2) and CMRO2 as a function of the duration of CPB in humans. To do this, we compared the cerebrovascular response to changes in the PaCO2. Because sequential CBF measurements using xenon 133 (133Xe) clearance must be separated by 15-25 min, we hypothesized that a time-dependent decline in CBF would accentuate the CBF reduction caused by a decrease in PaCO2, but would blunt the CBF increase associated with a rise in PaCO2. We measured CBF in 25 patients and calculated the cerebral arteriovenous oxygen content difference using radial arterial and jugular venous bulb blood samples. Patients were randomly assigned to management within either a lower (32-48 mm Hg) or higher (50-71 mm Hg) range of PaCO2 uncorrected for temperature. Each patient underwent two randomly ordered sets of measurements, one at a lower PaCO2 and the other at a higher PaCO2 within the respective ranges. Cerebrovascular responsiveness to changes in PaCO2 was calculated as specific reactivity (SR), the change in CBF divided by the change in PaCO2, expressed in mL.100 g-1.min-1.mm Hg-1.

  9. 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

  10. Experimental Models of Microvascular Immunopathology: The Example of Cerebral Malaria

    PubMed Central

    El-Assaad, Fatima; Combes, Valery; Grau, Georges ER

    2015-01-01

    Human cerebral malaria is a severe and often lethal complication of Plasmodium falciparum infection. Complex host and parasite interactions should the precise mechanisms involved in the onset of this neuropathology. Adhesion of parasitised red blood cells and host cells to endothelial cells lead to profound endothelial alterations that trigger immunopathological changes, varying degrees of brain oedema and can compromise cerebral blood flow, cause cranial nerve dysfunction and hypoxia. Study of the cerebral pathology in human patients is limited to clinical and genetic field studies in endemic areas, thus cerebral malaria (CM) research relies heavily on experimental models. The availability of malaria models allows study from the inoculation of Plasmodium to the onset of disease and permit invasive experiments. Here, we discuss some aspects of our current understanding of CM, the experimental models available and some important recent findings extrapolated from these models. PMID:26430675

  11. Endothelial ischemia-reperfusion injury in humans: association with age and habitual exercise

    PubMed Central

    Umpierre, Daniel; Harrison, Michelle L.; Lin, Hsin-Fu; Tarumi, Takashi; Renzi, Christopher P.; Dhindsa, Mandeep; Hunter, Stacy D.; Tanaka, Hirofumi

    2011-01-01

    Advancing age is a major risk factor for coronary artery disease. Endothelial dysfunction accompanied by increased oxidative stress and inflammation with aging may predispose older arteries to greater ischemia-reperfusion (I/R) injury. Because coronary artery ischemia cannot be induced safely, the effects of age and habitual endurance exercise on endothelial I/R injury have not been determined in humans. Using the brachial artery as a surrogate model of the coronary arteries, endothelial function, assessed by brachial artery flow-mediated dilation (FMD), was measured before and after 20 min of continuous forearm occlusion in young sedentary (n = 10, 24 ± 2 yr) and middle-aged (n = 9, 48 ± 2 yr) sedentary adults to gain insight into the effects of primary aging on endothelial I/R injury. Young (n = 9, 25 ± 1 yr) and middle-aged endurance-trained (n = 9, 50 ± 2 yr) adults were also studied to determine whether habitual exercise provides protection from I/R injury. Fifteen minutes after ischemic injury, FMD decreased significantly by 37% in young sedentary, 35% in young endurance-trained, 68% in middle-aged sedentary, and 50% in middle-aged endurance-trained subjects. FMD returned to baseline levels within 30 min in young sedentary and endurance-trained subjects but remained depressed in middle-aged sedentary and endurance-trained subjects. Circulating markers of antioxidant capacity and inflammation were not related to FMD. In conclusion, advancing age is associated with a greater magnitude and delayed recovery from endothelial I/R injury in humans. Habitual endurance exercise may provide partial protection to the endothelium against this form of I/R injury with advancing age. PMID:21239631

  12. The protective role of isorhamnetin on human brain microvascular endothelial cells from cytotoxicity induced by methylglyoxal and oxygen-glucose deprivation.

    PubMed

    Li, Wenlu; Chen, Zhigang; Yan, Min; He, Ping; Chen, Zhong; Dai, Haibin

    2016-02-01

    As the first target of stroke, cerebral endothelial cells play a key role in brain vascular repair and maintenance, and their function is impeded in diabetes. Methylglyoxal (MGO), a reactive dicarbonyl produced during glucose metabolism, accumulates in diabetic patients. MGO and MGO-induced advanced glycation end-products (AGEs) could ameliorate stroke-induced brain vascular damage, closely related with ECs dysfunction. Using MGO plus oxygen-glucose deprivation (OGD) to mimic diabetic stroke, we reported the protective effect of isorhamnetin on OGD-induced cytotoxicity after MGO treatment on primary human brain microvascular endothelial cells (HBMEC) and explored the underlying mechanisms. Treatment of MGO for 24 h significantly enhanced 3-h OGD-induced HBMEC toxic effect, which was inhibited by pretreatment of isorhamnetin (100 μmol/L). Moreover, the protective effect of isorhamnetin is multiple function dependent, which includes anti-inflammation, anti-oxidative stress and anti-apoptosis effects. Besides its well-known inhibition on the mitochondria-dependent or intrinsic apoptotic pathway, isorhamnetin also reduced activation of the extrinsic apoptotic pathway, as characterized by the decreased expression and activity of caspase 3 and caspase 8. Furthermore, pretreatment with isorhamnetin specifically inhibited FAS/FASL expression and suppressed nuclear factor-kappa B nuclear translocation. Taken together, our results indicated that isorhamnetin protected against OGD-induced cytotoxicity after MGO treatment in cultured HBMEC due to its multiple protective effects and could inhibit Fas-mediated extrinsic apoptosis. Therefore, isorhamnetin is a promising reagent for the treatment of hyperglycemia and ischemia-induced cerebral vascular degeneration. A proposed model of the potential protective mechanism of isorhamnetin, a metabolite of quercetin, on methylglyoxal (MGO) treatment plus oxygen-glucose deprivation (OGD) exposure-induced cytotoxicity in cultured human

  13. The protective role of isorhamnetin on human brain microvascular endothelial cells from cytotoxicity induced by methylglyoxal and oxygen-glucose deprivation.

    PubMed

    Li, Wenlu; Chen, Zhigang; Yan, Min; He, Ping; Chen, Zhong; Dai, Haibin

    2016-02-01

    As the first target of stroke, cerebral endothelial cells play a key role in brain vascular repair and maintenance, and their function is impeded in diabetes. Methylglyoxal (MGO), a reactive dicarbonyl produced during glucose metabolism, accumulates in diabetic patients. MGO and MGO-induced advanced glycation end-products (AGEs) could ameliorate stroke-induced brain vascular damage, closely related with ECs dysfunction. Using MGO plus oxygen-glucose deprivation (OGD) to mimic diabetic stroke, we reported the protective effect of isorhamnetin on OGD-induced cytotoxicity after MGO treatment on primary human brain microvascular endothelial cells (HBMEC) and explored the underlying mechanisms. Treatment of MGO for 24 h significantly enhanced 3-h OGD-induced HBMEC toxic effect, which was inhibited by pretreatment of isorhamnetin (100 μmol/L). Moreover, the protective effect of isorhamnetin is multiple function dependent, which includes anti-inflammation, anti-oxidative stress and anti-apoptosis effects. Besides its well-known inhibition on the mitochondria-dependent or intrinsic apoptotic pathway, isorhamnetin also reduced activation of the extrinsic apoptotic pathway, as characterized by the decreased expression and activity of caspase 3 and caspase 8. Furthermore, pretreatment with isorhamnetin specifically inhibited FAS/FASL expression and suppressed nuclear factor-kappa B nuclear translocation. Taken together, our results indicated that isorhamnetin protected against OGD-induced cytotoxicity after MGO treatment in cultured HBMEC due to its multiple protective effects and could inhibit Fas-mediated extrinsic apoptosis. Therefore, isorhamnetin is a promising reagent for the treatment of hyperglycemia and ischemia-induced cerebral vascular degeneration. A proposed model of the potential protective mechanism of isorhamnetin, a metabolite of quercetin, on methylglyoxal (MGO) treatment plus oxygen-glucose deprivation (OGD) exposure-induced cytotoxicity in cultured human

  14. Organization and chromosomal localization of the human platelet-derived endothelial cell growth factor gene.

    PubMed Central

    Hagiwara, K; Stenman, G; Honda, H; Sahlin, P; Andersson, A; Miyazono, K; Heldin, C H; Ishikawa, F; Takaku, F

    1991-01-01

    Human platelet-derived endothelial cell growth factor (hPD-ECGF) is a novel angiogenic factor which stimulates endothelial cell growth in vitro and promotes angiogenesis in vivo. We report here the cloning and sequencing of the gene for hPD-ECGF and its flanking regions. This gene is composed of 10 exons dispersed over a 4.3-kb region. Its promoter lacks a TATA box and a CCAAT box, structures characteristic of eukaryotic promoters. Instead, six copies of potential Sp1-binding sites (GGGCGG or CCGCCC) were clustered just upstream of the transcription start sites. Southern blot analysis using genomic DNAs from several vertebrates suggested that the gene for PD-ECGF is conserved phylogenetically among vertebrates. The gene for hPD-ECGF was localized to chromosome 22 by analysis of a panel of human-rodent somatic cell hybrid lines. Images PMID:2005900

  15. Comparative immunochemical analysis of membrane proteins from human platelets and endothelial cells.

    PubMed

    Sugiyama, T; Pidard, D; Wautier, M P; Wautier, J L

    1990-01-01

    We have studied the expression of immunochemically-related membrane proteins on human platelets and human umbilical vein endothelial cells (EC) by using cross-immunoprecipitation of 125I, surface-labeled cell extracts by cell-specific antisera, coupled to one- or two-dimensional SDS-PAGE. From 125I-labeled EC, an anti-platelet antiserum was found to immunoprecipitate major surface polypeptides comigrating with platelet GPIa, GPIIa and GPIIIa, and a protein with relative molecular mass Mr = 54,000. On the other hand, anti-EC antisera precipitated mostly GPIa, GPIc, GPIIa and GPIIIa from 125I-labeled platelets. These results supported previous reports demonstrating shared major membrane glycoproteins on platelets and EC, and suggested that the similar antigenicities in platelets and endothelial cells could play an important role in the pathogenesis of thrombosis and hemostasis in some immunologic disorders.

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  17. 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.

  18. 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.

  19. Toxic effects of iron oxide nanoparticles on human umbilical vein endothelial cells.

    PubMed

    Wu, Xinying; Tan, Yanbin; Mao, Hui; Zhang, Minming

    2010-01-01

    Iron oxide nanoparticles (IONPs) have been employed for hyperthermia treatments, stem cell therapies, cell labeling, and imaging modalities. The biocompatibility and cytotoxic effects of iron oxide nanoparticles when used in biomedical applications, however, are an ongoing concern. Endothelial cells have a critical role in this research dealing with tumors, cardiovascular disease and inflammation. However, there is little information dealing with the biologic effects of IONPs on the endothelial cell. This paper deals with the influence of dextran and citric acid coated IONPs on the behavior and function of human umbilical vein endothelial cells (HUVECs). After exposing endothelial cells to IONPs, dose-dependent effects on HUVECs viability, cytoskeleton and function were determined. Both citric acid and dextran coated particles appeared to be largely internalized by HUVECs through endocytosis and contribute to eventual cell death possibly by apoptosis. Cytoskeletal structures were greatly disrupted, as evidenced by diminished vinculin spots, and disorganized actin fiber and tubulin networks. The capacity of HUVECs to form a vascular network on Matrigel™ diminished after exposure to IONPs. Cell migration/invasion were inhibited significantly even at very low iron concentrations (0.1 mM). The results of this study indicate the great importance of thoroughly understanding nanoparticle-cell interactions, and the potential to exploit this understanding in tumor therapy applications involving IONPs as thermo/chemoembolization agents.

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

    SciTech Connect

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

    2010-02-12

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  3. Evidence for developmental programming of cerebral laterality in humans.

    PubMed

    Jones, Alexander; Osmond, Clive; Godfrey, Keith M; Phillips, David I W

    2011-01-01

    Adverse fetal environments are associated with depression, reduced cognitive ability and increased stress responsiveness in later life, but underlying mechanisms are unknown. Environmental pressures on the fetus, resulting from variations in placental function and maternal nutrition, health and stress might alter neurodevelopment, promoting the development of some brain regions over others. As asymmetry of cerebral activity, with greater right hemisphere activity, has been associated with psychopathology, we hypothesized that regional specialization during fetal life might be reflected persistently in the relative activity of the cerebral hemispheres. We tested this hypothesis in 140 healthy 8-9 year-old children, using tympanic membrane temperature to assess relative blood flow to the cerebral hemispheres at rest and following psychosocial stress (Trier Social Stress Test for Children). Their birth weight and placental weight had already been measured when their mothers took part in a previous study of pregnancy outcomes. We found that children who had a smaller weight at birth had evidence of greater blood flow to the right hemisphere than to the left hemisphere (r = -.09, P = .29 at rest; r = -.18, P = .04 following stress). This finding was strengthened if the children had a relatively low birth weight for their placental weight (r = -.17, P = .05 at rest; r = -.31, P = .0005 following stress). Our findings suggest that lateralization of cerebral activity is influenced persistently by early developmental experiences, with possible consequences for long-term neurocognitive function. PMID:21359174

  4. Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture.

    PubMed

    Trubetskaya, O V; Trubetskoy, V S; Domogatsky, S P; Rudin, A V; Popov, N V; Danilov, S M; Nikolayeva, M N; Klibanov, A L; Torchilin, V P

    1988-02-01

    A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells. Upon binding E25 is rapidly internalized and digested intracellularly. Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system. Up to 30% of the cell-adherent liposomal lipid is internalized.

  5. Paracrine crosstalk between human hair follicle dermal papilla cells and microvascular endothelial cells.

    PubMed

    Bassino, Eleonora; Gasparri, Franco; Giannini, Valentina; Munaron, Luca

    2015-05-01

    Human follicle dermal papilla cells (FDPC) are a specialized population of mesenchymal cells located in the skin. They regulate hair follicle (HF) development and growth, and represent a reservoir of multipotent stem cells. Growing evidence supports the hypothesis that HF cycling is associated with vascular remodeling. Follicular keratinocytes release vascular endothelial growth factor (VEGF) that sustains perifollicular angiogenesis leading to an increase of follicle and hair size. Furthermore, several human diseases characterized by hair loss, including Androgenetic Alopecia, exhibit alterations of skin vasculature. However, the molecular mechanisms underlying HF vascularization remain largely unknown. In vitro coculture approaches can be successfully employed to greatly improve our knowledge and shed more light on this issue. Here we used Transwell-based co-cultures to show that FDPC promote survival, proliferation and tubulogenesis of human microvascular endothelial cells (HMVEC) more efficiently than fibroblasts. Accordingly, FDPC enhance the endothelial release of VEGF and IGF-1, two well-known proangiogenic growth factors. Collectively, our data suggest a key role of papilla cells in vascular remodeling of the hair follicle.

  6. Human Haemato-Endothelial Precursors: Cord Blood CD34+ Cells Produce Haemogenic Endothelium

    PubMed Central

    Pelosi, Elvira; Castelli, Germana; Martin-Padura, Ines; Bordoni, Veronica; Santoro, Simona; Conigliaro, Alice; Cerio, Anna Maria; De Santis Puzzonia, Marco; Marighetti, Paola; Biffoni, Mauro; Alonzi, Tonino; Amicone, Laura; Alcalay, Myriam; Bertolini, Francesco; Testa, Ugo; Tripodi, Marco

    2012-01-01

    Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144−), triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45−) capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level. PMID:23226561

  7. Cyclic strain and motion control produce opposite oxidative responses in two human endothelial cell types.

    PubMed

    Sung, Hak-Joon; Yee, Andrew; Eskin, Suzanne G; McIntire, Larry V

    2007-07-01

    The phenotype of endothelial cells (ECs) is specific to the vascular bed from which they originate. To examine how mechanical forces alter the phenotype of different ECs, we compared the effects of cyclic strain and motion control on reactive oxygen species (ROS) production and metabolism and cell adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) vs. human aortic endothelial cells (HAEC). HUVEC and HAEC were subjected to cyclic strain (10% or 20%, 1 Hz), to a motion control that simulated fluid agitation over the cells without strain, or to static conditions for 24 h. We measured H(2)O(2) production with dichlorodihydrofluorescein acetate and superoxide with dihydroethidium fluorescence changes; superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities spectrophotometrically; and vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 protein expression with Western blot analyses. HUVEC under cyclic strain showed 1) higher intracellular H(2)O(2) levels, 2) increased SOD, catalase, and GPx activities, and 3) greater VCAM-1 and ICAM-1 protein expression, compared with motion control or static conditions. However, in HAEC, motion control induced higher levels of ROS, enzyme activities associated with ROS defense, and VCAM-1 and ICAM-1 expression than cyclic strain. The opposite responses obtained with these two human EC types may reflect their vessels of origin, in that HAEC are subjected to higher cyclic strain deformations in vivo than HUVEC.

  8. Human umbilical cord blood-derived mesenchymal stem cells improve functional recovery through thrombospondin1, pantraxin3, and vascular endothelial growth factor in the ischemic rat brain.

    PubMed

    Park, Hyung Woo; Moon, Hyo-Eun; Kim, Hye-Soo R; Paek, Seung Leal; Kim, Yona; Chang, Jong Wook; Yang, Yoon Sun; Kim, KwanWoo; Oh, Wonil; Hwang, Jae Ha; Kim, Jin Wook; Kim, Dong Gyu; Paek, Sun Ha

    2015-12-01

    Cell therapy is a potential therapeutic method for cerebral ischemia, which remains a serious problem. In the search for more effective therapeutic methods, many kinds of stem cells from various tissues have been developed and tested as candidate therapeutic agents. Among them, human umbilical cord blood (hUCB)-derived mesenchymal stem cells (MSCs) are widely used for cell therapy because of their genetic flexibility. To confirm that they are effective and understand how they affect ischemic neural cells, hUCB-MSCs were directly administered ipsilaterally into an ischemic zone induced by middle cerebral artery occlusion (MCAO). We found that the neurobehavioral performance of the hUCB-MSC group was significantly improved compared with that of the vehicle-injected control group. The infarct was also remarkably smaller in the hUCB-MSC group. Additionally, hUCB-MSC transplantation resulted in a greater number of newly generated cells and angiogenic and tissue repair factors and a lower number of inflammatory events in the penumbra zone. To determine why these events occurred, hUCB-MSCs were assayed under hypoxic and normoxic conditions in vitro. The results showed that hUCB-MSCs exhibit higher expression levels of thrombospondin1, pantraxin3, and vascular endothelial growth factor under hypoxic conditions than under normoxic conditions. These results were found to be correlated with our in vivo immunofluorescent staining results. On the basis of these findings, we suggest that hUCB-MSCs may have a beneficial effect on cerebral ischemia, especially through angiogenesis, neurogenesis, and anti-inflammatory effects, and thus could be used as a therapeutic agent to treat neurological disorders such as cerebral ischemia.

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

    PubMed

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

    2015-11-01

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

  10. Contradictory Effects of Superoxide and Hydrogen Peroxide on KCa3.1 in Human Endothelial Cells

    PubMed Central

    Choi, Shinkyu; Na, Hye-Young; Kim, Ji Aee; Cho, Sung-Eun

    2013-01-01

    Reactive oxygen species (ROS) are generated in various cells, including vascular smooth muscle and endothelial cells, and regulate ion channel functions. KCa3.1 plays an important role in endothelial functions. However, the effects of superoxide and hydrogen peroxide radicals on the expression of this ion channel in the endothelium remain unclear. In this study, we examined the effects of ROS donors on KCa3.1 expression and the K+ current in primary cultured human umbilical vein endothelial cells (HUVECs). The hydrogen peroxide donor, tert-butyl hydroperoxide (TBHP), upregulated KCa3.1 expression, while the superoxide donors, xanthine/xanthine oxidase mixture (X/XO) and lysopho-sphatidylcholine (LPC), downregulated its expression, in a concentration-dependent manner. These ROS donor effects were prevented by antioxidants or superoxide dismustase. Phosphorylated extracellular signal-regulated kinase (pERK) was upregulated by TBHP and downregulated by X/XO. In addition, repressor element-1-silencing transcription factor (REST) was downregulated by TBHP, and upregulated by X/XO. Furthermore, KCa3.1 current, which was activated by clamping cells with 1 µM Ca2+ and applying the KCa3.1 activator 1-ethyl-2-benzimidazolinone, was further augmented by TBHP, and inhibited by X/XO. These effects were prevented by antioxidants. The results suggest that hydrogen peroxide increases KCa3.1 expression by upregulating pERK and downregulating REST, and augments the K+ current. On the other hand, superoxide reduces KCa3.1 expression by downregulating pERK and upregulating REST, and inhibits the K+ current. ROS thereby play a key role in both physiological and pathological processes in endothelial cells by regulating KCa3.1 and endothelial function. PMID:23776393

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

    PubMed Central

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

    2013-01-01

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

  12. 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

  13. Induction of stearoyl-CoA desaturase protects human arterial endothelial cells against lipotoxicity.

    PubMed

    Peter, Andreas; Weigert, Cora; Staiger, Harald; Rittig, Kilian; Cegan, Alexander; Lutz, Philipp; Machicao, Fausto; Häring, Hans-Ulrich; Schleicher, Erwin

    2008-08-01

    Endothelial lipotoxicity has been implicated in the pathogenesis of multiple stages of cardiovascular disease from early endothelial dysfunction to manifest atherosclerosis and its complications. Saturated free fatty acids are the major inducers of endothelial cell apoptosis and inflammatory cytokines. In humans, the enzyme human stearoyl-CoA desaturase-1 (hSCD-1) is the limiting step of the desaturation of saturated to monounsaturated fatty acids. Since we could demonstrate the expression of SCD-1 in primary human arterial endothelial cells (HAECs), we aimed to prove a beneficial role of upregulated hSCD-1 expression. In contrast to other cells that are less susceptible to lipotoxicity, hSCD-1 was not upregulated in HAECs upon palmitate treatment. Following that, we could show that upregulation of hSCD-1 using the LXR activator TO-901317 in HAECs protects the cells against palmitate-induced lipotoxicity, cell apoptosis, and expression of inflammatory cytokines IL-6 and IL-8. Increased hSCD-1 activity was determined as increased C16:1/16:0 ratio and enhanced triglyceride storage in palmitate treated cells. The beneficial effect was clearly attributed to enhanced hSCD-1 activity. Overexpression of hSCD-1 blocked palmitate-induced cytotoxicity, and knockdown of hSCD-1 using siRNA abolished the protective effect of TO-901317 in HEK-293 cells. Additionally, inhibition of hSCD-1 with 10/12 CLA blocked the effect of TO-901317 on palmitate-induced lipotoxicity, cell apoptosis, and inflammatory cytokine induction in HAECs. We conclude that upregulation of hSCD-1 leads to a desaturation of saturated fatty acids and facilitates their esterification and storage, thereby preventing downstream effects of lipotoxicity in HAECs. These findings add a novel aspect to the atheroprotective actions of LXR activators in cardiovascular disease.

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

    SciTech Connect

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

    2012-06-22

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

  15. Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells

    PubMed Central

    2011-01-01

    Background ATP binding is essential for the bioactivity of several growth factors including nerve growth factor, fibroblast growth factor-2 and brain-derived neurotrophic factor. Vascular endothelial growth factor isoform 165 (VEGF-A165) induces the proliferation of human umbilical vein endothelial cells, however a dependence on ATP-binding is currently unknown. The aim of the present study was to determine if ATP binding is essential for the bioactivity of VEGF-A165. Results We found evidence that ATP binding toVEGF-A165 induced a conformational change in the secondary structure of the growth factor. This binding appears to be significant at the biological level, as we found evidence that nanomolar levels of ATP (4-8 nm) are required for the VEGF-A165-induced proliferation of human umbilical vein endothelial cells. At these levels, purinergic signaling by ATP via P2 receptors can be excluded. Addition of alkaline phosphate to cell culture lowered the ATP concentration in the cell culture medium to 1.8 nM and inhibited cell proliferation. Conclusions We propose that proliferation of endothelial cells is induced by a VEGF-A165-ATP complex, rather than VEGF-A165 alone. PMID:21619628

  16. Quantitative comparison of cerebral artery development in metatherians and monotremes with non-human eutherians.

    PubMed

    Ashwell, Ken W S; Shulruf, Boaz

    2016-03-01

    A quantitative comparison of the internal diameters of cerebral feeder arteries (internal carotid and vertebral) and the aorta in developing non-human eutherians, metatherians and monotremes has been made, with the aim of determining if there are differences in cerebral arterial flow between the three infraclasses of mammals such as might reflect differences in metabolism of the developing brain. There were no significant differences between eutherians and metatherians in the internal radius of the aorta or the thickness of the aortic wall, but aortic internal radius was significantly smaller in developing monotremes than therians at the < 10 mm body length range. Aortic thickness in the developing monotremes also rose at a slower rate relative to body length than in metatherians or eutherians. The sums of the internal calibres of the internal carotid and vertebral arteries were significantly lower in metatherians as a group and monotremes compared with non-human eutherians at body lengths up to 20 mm and in metatherians at > 20 mm body length. The internal calibre of the internal carotids relative to the sum of all cerebral feeder arteries was also significantly lower in monotremes at < 10 mm body length compared with eutherians. It was noted that dasyurids differed from other metatherians in several measures of cerebral arterial calibre and aortic internal calibre. The findings suggest that: (i) both aortic outflow and cerebral arterial inflow may be lower in developing monotremes than in therians, particularly at small body size (< 20 mm); (ii) cerebral inflow may be lower in some developing metatherians than non-human eutherians; and (iii) dasyurids have unusual features of cerebral arteries possibly related to the extreme immaturity and small size at which they are born. The findings have implications for nutritional sourcing of the developing brain in the three infraclasses of mammals.

  17. Quantitative comparison of cerebral artery development in metatherians and monotremes with non-human eutherians.

    PubMed

    Ashwell, Ken W S; Shulruf, Boaz

    2016-03-01

    A quantitative comparison of the internal diameters of cerebral feeder arteries (internal carotid and vertebral) and the aorta in developing non-human eutherians, metatherians and monotremes has been made, with the aim of determining if there are differences in cerebral arterial flow between the three infraclasses of mammals such as might reflect differences in metabolism of the developing brain. There were no significant differences between eutherians and metatherians in the internal radius of the aorta or the thickness of the aortic wall, but aortic internal radius was significantly smaller in developing monotremes than therians at the < 10 mm body length range. Aortic thickness in the developing monotremes also rose at a slower rate relative to body length than in metatherians or eutherians. The sums of the internal calibres of the internal carotid and vertebral arteries were significantly lower in metatherians as a group and monotremes compared with non-human eutherians at body lengths up to 20 mm and in metatherians at > 20 mm body length. The internal calibre of the internal carotids relative to the sum of all cerebral feeder arteries was also significantly lower in monotremes at < 10 mm body length compared with eutherians. It was noted that dasyurids differed from other metatherians in several measures of cerebral arterial calibre and aortic internal calibre. The findings suggest that: (i) both aortic outflow and cerebral arterial inflow may be lower in developing monotremes than in therians, particularly at small body size (< 20 mm); (ii) cerebral inflow may be lower in some developing metatherians than non-human eutherians; and (iii) dasyurids have unusual features of cerebral arteries possibly related to the extreme immaturity and small size at which they are born. The findings have implications for nutritional sourcing of the developing brain in the three infraclasses of mammals. PMID:26644330

  18. Transfer function analysis of dynamic cerebral autoregulation in humans

    NASA Technical Reports Server (NTRS)

    Zhang, R.; Zuckerman, J. H.; Giller, C. A.; Levine, B. D.; Blomqvist, C. G. (Principal Investigator)

    1998-01-01

    To test the hypothesis that spontaneous changes in cerebral blood flow are primarily induced by changes in arterial pressure and that cerebral autoregulation is a frequency-dependent phenomenon, we measured mean arterial pressure in the finger and mean blood flow velocity in the middle cerebral artery (VMCA) during supine rest and acute hypotension induced by thigh cuff deflation in 10 healthy subjects. Transfer function gain, phase, and coherence function between changes in arterial pressure and VMCA were estimated using the Welch method. The impulse response function, calculated as the inverse Fourier transform of this transfer function, enabled the calculation of transient changes in VMCA during acute hypotension, which was compared with the directly measured change in VMCA during thigh cuff deflation. Beat-to-beat changes in VMCA occurred simultaneously with changes in arterial pressure, and the autospectrum of VMCA showed characteristics similar to arterial pressure. Transfer gain increased substantially with increasing frequency from 0.07 to 0.20 Hz in association with a gradual decrease in phase. The coherence function was > 0.5 in the frequency range of 0.07-0.30 Hz and < 0.5 at < 0.07 Hz. Furthermore, the predicted change in VMCA was similar to the measured VMCA during thigh cuff deflation. These data suggest that spontaneous changes in VMCA that occur at the frequency range of 0.07-0.30 Hz are related strongly to changes in arterial pressure and, furthermore, that short-term regulation of cerebral blood flow in response to changes in arterial pressure can be modeled by a transfer function with the quality of a high-pass filter in the frequency range of 0.07-0.30 Hz.

  19. Effects of midazolam on cerebral blood flow in human volunteers

    SciTech Connect

    Forster, A.; Juge, O.; Morel, D.

    1982-06-01

    The effects of intravenously administered midazolam on cerebral blood flow were evaluated in eight healthy volunteers using the /sup 133/Xe inhalation technique. Six minutes after an intravenous dose of 0.15 mg/kg midazolam, the cerebral blood flow decreased significantly (P less than 0.001) from a value of 40.6 +/- 3.3 to a value of 27.0 +/- 5.0 ml . 100 g-1 . min-1. Cerebrovascular resistance (CVR) increased from 2.8 +/- 0.2 to 3.9 to 0.6 mmHg/(ml . 100 g-1 . min-1)(P less than 0.001). Mean arterial blood pressure decreased significantly (P less than 0.05) from 117 +/- 8 to 109 +/- 9 mmHg and arterial carbon dioxide tension increased from 33.9 +/- 2.3 to 38.6 +/- 3.2 mmHg (P less than 0.05). Arterial oxygen tension remained stable throughout the study, 484 +/- 95 mmHg before the administration of midazolam and 453 +/- 76 mmHg after. All the subjects slept after the injection of the drug and had anterograde amnesia of 24.5 +/- 5 min. The decrease in mean arterial blood pressure was probably not important since it remained in the physiologic range for cerebral blood flow autoregulation. The increase in arterial carbon dioxide tension observed after the midazolam injection may have partially counteracted the effect of this new benzodiazepine on cerebral blood flow. Our data suggest that midazolam might be a safe agent to use for the induction of anethesia in neurosurgical patients with intracranial hypertension.

  20. Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context*

    PubMed Central

    Hang, Ta-Chun; Tedford, Nathan C.; Reddy, Raven J.; Rimchala, Tharathorn; Wells, Alan; White, Forest M.; Kamm, Roger D.; Lauffenburger, Douglas A.

    2013-01-01

    The process of angiogenesis is under complex regulation in adult organisms, particularly as it often occurs in an inflammatory post-wound environment. As such, there are many impacting factors that will regulate the generation of new blood vessels which include not only pro-angiogenic growth factors such as vascular endothelial growth factor, but also angiostatic factors. During initial postwound hemostasis, a large initial bolus of platelet factor 4 is released into localized areas of damage before progression of wound healing toward tissue homeostasis. Because of its early presence and high concentration, the angiostatic chemokine platelet factor 4, which can induce endothelial anoikis, can strongly affect angiogenesis. In our work, we explored signaling crosstalk interactions between vascular endothelial growth factor and platelet factor 4 using phosphotyrosine-enriched mass spectrometry methods on human dermal microvascular endothelial cells cultured under conditions facilitating migratory sprouting into collagen gel matrices. We developed new methods to enable mass spectrometry-based phosphorylation analysis of primary cells cultured on collagen gels, and quantified signaling pathways over the first 48 h of treatment with vascular endothelial growth factor in the presence or absence of platelet factor 4. By observing early and late signaling dynamics in tandem with correlation network modeling, we found that platelet factor 4 has significant crosstalk with vascular endothelial growth factor by modulating cell migration and polarization pathways, centered around P38α MAPK, Src family kinases Fyn and Lyn, along with FAK. Interestingly, we found EphA2 correlational topology to strongly involve key migration-related signaling nodes after introduction of platelet factor 4, indicating an influence of the angiostatic factor on this ambiguous but generally angiogenic signal in this complex environment. PMID:24023389

  1. C. pneumoniae disrupts eNOS trafficking and impairs NO production in human aortic endothelial cells.

    PubMed

    Mueller, Konrad E; Wolf, Katerina

    2015-01-01

    Endothelial nitric oxide synthase (eNOS) generated NO plays a crucial physiological role in the regulation of vascular tone. eNOS is a constitutively expressed synthase whose enzymatic function is regulated by dual acylation, phosphorylation, protein-protein interaction and subcellular localization. In endothelial cells, the enzyme is primarily localized to the Golgi apparatus (GA) and the plasma membrane where it binds to caveolin-1. Upon stimulation, the enzyme is translocated from the plasma membrane to the cytoplasm where it generates NO. When activation of eNOS ceases, the majority of the enzyme is recycled back to the membrane fraction. An inability of eNOS to cycle between the cytosol and the membrane leads to impaired NO production and vascular dysfunction. Chlamydia pneumoniae is a Gram-negative obligate intracellular bacterium that primarily infects epithelial cells of the human respiratory tract, but unlike any other chlamydial species, C. pneumoniae displays tropism toward atherosclerotic tissues. In this study, we demonstrate that C. pneumoniae inclusions colocalize with eNOS, and the microorganism interferes with trafficking of the enzyme from the GA to the plasma membrane in primary human aortic endothelial cells. This mislocation of eNOS results in significant inhibition of NO release by C. pneumoniae-infected cells. Furthermore, we show that the distribution of eNOS in C. pneumoniae-infected cells is altered due to an intimate association of the Golgi complex with chlamydial inclusions rather than by direct interaction of the enzyme with the chlamydial inclusion membrane.

  2. Slit2-Robo4 receptor responses inhibit ANDV directed permeability of human lung microvascular endothelial cells.

    PubMed

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

    2013-08-01

    Hantaviruses nonlytically infect human endothelial cells (ECs) and cause edematous and hemorrhagic diseases. Andes virus (ANDV) causes hantavirus pulmonary syndrome (HPS), and Hantaan virus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses enhance vascular endothelial growth factor directed EC permeability resulting in the disassembly of inter-endothelial cell adherens junctions (AJs). Recent studies demonstrate that Slit2 binding to Robo1/Robo4 receptors on ECs has opposing effects on AJ disassembly and vascular fluid barrier functions. Here we demonstrate that Slit2 inhibits ANDV and HTNV induced permeability and AJ disassembly of pulmonary microvascular ECs (PMECs) by interactions with Robo4. In contrast, Slit2 had no effect on the permeability of ANDV infected human umbilical vein ECs (HUVECs). Analysis of Robo1/Robo4 expression determined that PMECs express Robo4, but not Robo1, while HUVECs expressed both Robo4 and Robo1 receptors. SiRNA knockdown of Robo4 in PMECs prevented Slit2 inhibition of ANDV induced permeability demonstrating that Robo4 receptors determine PMEC responsiveness to Slit2. Collectively, this data demonstrates a selective role for Slit2/Robo4 responses within PMECs that inhibits ANDV induced permeability and AJ disassembly. These findings suggest Slit2s utility as a potential HPS therapeutic that stabilizes the pulmonary endothelium and antagonizes ANDV induced pulmonary edema.

  3. Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

    PubMed

    Hecke, Anneke; Brooks, Hilary; Meryet-Figuière, Matthieu; Minne, Stephanie; Konstantinides, Stavros; Hasenfuss, Gerd; Lebleu, Bernard; Schäfer, Katrin

    2006-05-01

    Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

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

    PubMed Central

    Yang, Hana; Lee, Seung Eun; Kim, Gun-Dong; Park, Hye Rim; Park, Yong Seek

    2013-01-01

    Spermidine (SPD) is a ubiquitous polycation that is commonly distributed in living organisms. Intracellular levels of SPD are tightly regulated, and SPD controls cell proliferation and death. However, SPD undergoes oxidation in the presence of serum, producing aldehydes, hydrogen peroxide, and ammonia, which exert cytotoxic effect on cells. Hemeoxygenase-1 (HO-1) is thought to have a protective effect against oxidative stress. Upregulation of HO-1 in endothelial cells is considered to be beneficial in the cardiovascular disease. In the present study, we demonstrate that the ubiquitous polyamine, SPD, induces HO-1 in human umbilical vein endothelial cells (HUVECs). SPD-induced HO-1 expression was examined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Involvement of reactive oxygen species, serum amine oxidase, PI3K/Akt signaling pathway, and transcription factor Nrf2 in the induction of HO-1 by SPD was also investigated. Furthermore, small interfering RNA knockdown of Nrf2 or HO-1 and treatment with the specific HO-1 inhibitor ZnPP exhibited a noteworthy increase of death of SPD-stimulated HUVECs. In conclusion, these results suggest that SPD induces PI3K/Akt-Nrf2-mediated HO-1 expression in human endothelial cells, which may have a role in cytoprotection of the cells against oxidative stress-induced death. PMID:23983896

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

    PubMed

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

    1998-07-01

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

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

    PubMed

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

    2015-07-01

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

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

    PubMed Central

    Wong, Wing Tak; Cooke, John P

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed

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

    2016-06-01

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

  11. Evaluation of the protective potential of brain microvascular endothelial cell autophagy on blood-brain barrier integrity during experimental cerebral ischemia-reperfusion injury.

    PubMed

    Li, Haiying; Gao, Anju; Feng, Dongxia; Wang, Yang; Zhang, Li; Cui, Yonghua; Li, Bo; Wang, Zhong; Chen, Gang

    2014-10-01

    Brain microvascular endothelial cell (BMVEC) injury induced by ischemia-reperfusion (I/R) is the initial phase of blood-brain barrier (BBB) disruption, which results in a poor prognosis for ischemic stroke patients. Autophagy occurs in ischemic brain and has been shown to exhibit protective effects on endothelial cell against stress. However, the potential effects of BMVEC autophagy on BBB permeability during I/R and the mechanisms underlying these effects have yet to be elucidated. In the current study, we answered these questions by using chemical modulators of autophagy, including rapamycin and lithium carbonate acting, respectively, as mammalian target of rapamycin (mTOR)-dependent and mTOR-independent autophagy inducers and 3-methyladenine (3-MA) as an autophagy inhibitor. To mimic I/R injury, BMVECs were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R), and a rat transient middle cerebral artery occlusion/reperfusion (MCAO/R) model was performed. All the drugs were given at 0.5 h before OGD/R or MCAO/R. First, enhancement of autophagy by rapamycin and lithium carbonate attenuated, whereas suppression of autophagy by 3-MA intensified BMVEC apoptosis and the high level of ROS induced by OGD/R. In addition, rapamycin and lithium carbonate pretreatments significantly reversed the decreased level of tight junction protein zonula occludens-1 (ZO-1) induced by OGD/R and promoted the distribution of ZO-1 on cell membranes. Finally, pretreatments with rapamycin and lithium carbonate reduced evans blue extravasation and brain water content in the ischemic hemisphere of the rat. In contrast, 3-MA pretreatment exerted opposite effects both in vitro and in vivo. These results may indicate a beneficial effect of BMVEC autophagy on BBB integrity during I/R injury. PMID:25070048

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

    PubMed

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

    2011-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-11-01

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

  14. Role of asymmetrical dimethylarginine in inflammation-induced endothelial dysfunction in human atherosclerosis.

    PubMed

    Antoniades, Charalambos; Demosthenous, Michael; Tousoulis, Dimitris; Antonopoulos, Alexios S; Vlachopoulos, Charalambos; Toutouza, Marina; Marinou, Kyriakoula; Bakogiannis, Constantinos; Mavragani, Kleio; Lazaros, George; Koumallos, Nikolaos; Triantafyllou, Costas; Lymperiadis, Dimitris; Koutsilieris, Michael; Stefanadis, Christodoulos

    2011-07-01

    We explored the role of asymmetrical dimethylarginine (ADMA) as a cause of endothelial dysfunction induced by systemic inflammation. In vitro data suggest that ADMA bioavailability is regulated by proinflammatory stimuli, but it is unclear whether ADMA is a link between inflammation and endothelial dysfunction in humans. In study 1 we recruited 351 patients with coronary artery disease (CAD) and 87 healthy controls. In study 2 we recruited 69 CAD, 69 healthy, and 10 patients with rheumatoid arthritis, whereas in study 3, 22 healthy and 70 CAD subjects were randomly assigned to Salmonella typhii vaccination (n=11 healthy and n=60 CAD) or placebo (n=11 healthy and n=10 CAD). Circulating interleukin 6/ADMA and flow-mediated dilation (FMD) were measured at 0 and 8 hours. In study 1, ADMA was inversely correlated with FMD in healthy individuals and CAD patients (P<0.0001 for both). However, interleukin 6 was inversely correlated with FMD (P<0.0001) in healthy subjects but not in CAD patients. The positive correlation between ADMA and interleukin 6 was stronger in healthy (r=0.515; P<0.0001) compared with CAD (r=0.289; P=0.0001) subjects. In study 2, both patients with rheumatoid arthritis and CAD had higher interleukin 6 (P<0.0001) and ADMA (P=0.004) but lower FMD (P=0.001) versus healthy subjects. In study 3, vaccination increased interleukin 6 in healthy (P<0.001) and CAD (P<0.001) subjects. FMD was reduced in healthy subjects (P<0.05), but its reduction in CAD was borderline. Vaccination increased ADMA only in healthy subjects (P<0.001). Systemic, low-grade inflammation leads to increased ADMA that may induce endothelial dysfunction. This study demonstrated that ADMA may be a link between inflammation and endothelial dysfunction in humans.

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

    PubMed

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

    2016-09-01

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

  16. Quantitative comparison of cerebral artery development in human embryos with other eutherians.

    PubMed

    Ashwell, Ken W S; Shulruf, Boaz

    2015-09-01

    The embryonic and early fetal human brain is known to undergo extraordinary expansion of its cellular population during embryonic and early fetal life, and is critically dependant on a steady supply of nutrients and oxygen for proper brain development. Quantitative analysis of the internal radius of the aorta and cerebral arteries in a range of eutherian mammals has been used to compare arterial flow to the developing human brain with that to the brains of non-human eutherians. Human embryos showed a much steeper rise of internal radius of the aorta with increasing body size than the embryos of non-human eutherians, but the thickness of the aorta rose at the same pace relative to body size in both humans and non-humans, suggesting that aortic pressure is similar in all eutherian embryos of a similar size. The sums of internal radii of both the internal carotids and vertebral arteries of human embryos raised to the fourth power were much lower at embryonic stages (less than 22 mm body length) than in non-human eutherians, were similar between humans and non-humans at 22-30 mm body length, and exceeded the non-humans at body lengths of more than 30 mm. The relative size of the internal calibre of the cerebral feeder arteries (internal carotid and vertebral) to the aorta did not change between embryonic and fetal sizes in either humans or non-humans. The findings suggest that the developing human brain may actually receive less blood flow at embryonic sizes (less than 22 mm body length) than do other mammalian embryos of a similar body size, but that internal carotid and vertebral flow is higher in human fetuses (body length greater than 30 mm) than in developing non-humans of the same body size. Increased flow to the developing human brain relative to non-humans is achieved by simultaneous increases in both aortic and cerebral feeder artery internal calibre.

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

    PubMed

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

    2001-10-01

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

  18. Alternative polyadenylation sites of human endothelial nitric oxide synthase mRNA

    PubMed Central

    Ihara, Hideshi; Tsutsuki, Hiroyasu; Ida, Tomoaki; Kozaki, Shunji; Tsuyama, Shingo; Moss, Joel

    2010-01-01

    The mRNA 3′-untranslated region (3′-UTR) has been shown to have important roles in the regulation of mRNA function. In this study, we investigated the human endothelial nitric oxide synthase (eNOS) 3′-UTR to evaluate its potential regulatory role. 3′-RACE analysis revealed that the human eNOS mRNA has multiple alternative polyadenylation sites. Apart from the proximal site (418 bp downstream of the stop codon), we identified two additional distal sites approximately 770 and 1478 bp downstream of the stop codon. In addition, Northern analysis showed that the usage of these sites differed among human tissues. Further, amounts of these eNOS mRNAs were changed during growth of cultured human aortic endothelial cells; mRNAs with long 3′-UTRs decreased more rapidly than total mRNA, as cells approached confluency. Thus, the 3′-UTRs of human eNOS results from alternative polyadenylation sites and differ across tissues and during cell growth. PMID:17825792

  19. Endothelial-mesenchymal transition in normal human esophageal endothelial cells cocultured with esophageal adenocarcinoma cells: role of IL-1β and TGF-β2.

    PubMed

    Nie, Linghui; Lyros, Orestis; Medda, Rituparna; Jovanovic, Nebojsa; Schmidt, Jamie L; Otterson, Mary F; Johnson, Christopher P; Behmaram, Behnaz; Shaker, Reza; Rafiee, Parvaneh

    2014-11-01

    Endothelial-mesenchymal transition (EndoMT) has been recognized as a key determinant of tumor microenvironment in cancer progression and metastasis. Endothelial cells undergoing EndoMT lose their endothelial markers, acquire the mesenchymal phenotype, and become more invasive with increased migratory abilities. Early stages of esophageal adenocarcinoma (EAC) are characterized by strong microvasculature whose impact in tumor progression remains undefined. Our aim was to determine the role of EndoMT in EAC by investigating the impact of tumor cells on normal primary human esophageal microvascular endothelial cells (HEMEC). HEMEC were either cocultured with OE33 adenocarcinoma cells or treated with IL-1β and transforming growth factor-β2 (TGF-β2) for indicated periods and analyzed for EndoMT-associated changes by real-time PCR, Western blotting, immunofluorescence staining, and functional assays. Additionally, human EAC tissues were investigated for detection of EndoMT-like cells. Our results demonstrate an increased expression of mesenchymal markers [fibroblast-specific protein 1 (FSP1), collagen1α2, vimentin, α-smooth muscle actin (α-SMA), and Snail], decreased expression of endothelial markers [CD31, von Willebrand factor VIII (vWF), and VE-cadherin], and elevated migration ability in HEMEC following coculture with OE33 cells. The EndoMT-related changes were inhibited by IL-1β and TGF-β2 gene silencing in OE33 cells. Recombinant IL-1β and TGF-β2 induced EndoMT in HEMEC. Although the level of VEGF expression was elevated in EndoMT cells, the angiogenic property of these cells was diminished. In vivo, by immunostaining EndoMT-like cells were detected at the invasive front of EAC. Our findings underscore a significant role for EndoMT in EAC and provide new insights into the mechanisms and significance of EndoMT in the context of tumor progression.

  20. Transcriptional regulation of the human iNOS gene by IL-1beta in endothelial cells.

    PubMed Central

    Kolyada, A. Y.; Madias, N. E.

    2001-01-01

    BACKGROUND: Vascular endothelium participates in the control of vascular tone and function via the release of nitric oxide (NO) by the endothelial-type NO synthase (eNOS). Inducible NO synthase (iNOS) expression in endothelial cells occurs in many clinical conditions following induction by lipopolysaccharide or cytokines and generates large quantities of NO that result in endothelial cell activation and dysfunction. No information exists on the transcriptional regulation of the human iNOS gene (or that of other species) in endothelial cells. MATERIALS AND METHODS: We examined the transcriptional regulation of the human iNOS gene by interleukin-1beta (IL-1beta) in rat pulmonary microvascular endothelial cells (PVEC) by transient cotransfections of different iNOS-promoter constructs and cDNA of different transcription factors and regulatory proteins. RESULTS: The -1034/+88 bp iNOS promoter was strongly induced by IL-1beta, the regulatory elements for such induction being localized downstream of -205 bp. Cotransfection experiments with NF-kappaB isoforms, IkappaB isoforms, and IKK mutants suggested that the NF-kappaB site at -115/-106 bp is important, but not sufficient, for induction of iNOS promoter and that the role of NF-kappaB is partially independent of its binding site. C/EBP sites within the -205/+88 bp region were shown to be responsible, along with NF-kappaB site, for induction of iNOS promoter by IL-1beta. Overexpression of C/EBPalpha, C/EBPdelta, and liver-enriched activator protein (LAP) activated the promoter, whereas overexpression of liver-enriched inhibitory protein (LIP) strongly suppressed it. C/EBPbeta (LAP and LIP isoforms) was constitutively present in PVEC and was induced (approximately 2-fold) by IL-1beta, whereas C/EBPdelta was not constitutively expressed but was strongly induced by IL-1beta. Both C/EBPbeta and C/EBPdelta participated in DNA-protein complex formation. CONCLUSION: Both NF-kappaB and C/EBP pathways are important for the

  1. Birth weight and characteristics of endothelial and smooth muscle cell cultures from human umbilical cord vessels

    PubMed Central

    Martín de Llano, José Javier; Fuertes, Graciela; Torró, Isabel; García Vicent, Consuelo; Fayos, José Luis; Lurbe, Empar

    2009-01-01

    Background Low birth weight has been related to an increased risk for developing high blood pressure in adult life. The molecular and cellular analysis of umbilical cord artery and vein may provide information about the early vascular characteristics of an individual. We have assessed several phenotype characteristics of the four vascular cell types derived from human umbilical cords of newborns with different birth weight. Further follow-up studies could show the association of those vascular properties with infancy and adulthood blood pressure. Methods Endothelial and smooth muscle cell cultures were obtained from umbilical cords from two groups of newborns of birth weight less than 2.8 kg or higher than 3.5 kg. The expression of specific endothelial cell markers (von Willebrand factor, CD31, and the binding and internalization of acetylated low-density lipoprotein) and the smooth muscle cell specific α-actin have been evaluated. Cell culture viability, proliferation kinetic, growth fraction (expression of Ki67) and percentage of senescent cells (detection of β-galactosidase activity at pH 6.0) have been determined. Endothelial cell projection area was determined by morphometric analysis of cell cultures after CD31 immunodetection. Results The highest variation was found in cell density at the confluence of endothelial cell cultures derived from umbilical cord arteries (66,789 ± 5,093 cells/cm2 vs. 45,630 ± 11,927 cells/cm2, p < 0.05). Morphometric analysis indicated that the projection area of the artery endothelial cells (1,161 ± 198 and 1,544 ± 472 μm2, p < 0.05), but not those derived from the vein from individuals with a birth weight lower than 2.8 kg was lower than that of cells from individuals with a birth weight higher than 3.5 kg. Conclusion The analysis of umbilical cord artery endothelial cells, which demonstrated differences in cell size related to birth weight, can provide hints about the cellular and molecular links between lower birth

  2. Aldehyde dehydrogenase 2 protects human umbilical vein endothelial cells against oxidative damage and increases endothelial nitric oxide production to reverse nitroglycerin tolerance.

    PubMed

    Hu, X Y; Fang, Q; Ma, D; Jiang, L; Yang, Y; Sun, J; Yang, C; Wang, J S

    2016-06-10

    Medical nitroglycerin (glyceryl trinitrate, GTN) use is limited principally by tolerance typified by a decrease in nitric oxide (NO) produced by biotransformation. Such tolerance may lead to endothelial dysfunction by inducing oxidative stress. In vivo studies have demonstrated that aldehyde dehydrogenase 2 (ALDH2) plays important roles in GTN biotransformation and tolerance. Thus, modification of ALDH2 expression represents a potentially effective strategy to prevent and reverse GTN tolerance and endothelial dysfunction. In this study, a eukaryotic expression vector containing the ALDH2 gene was introduced into human umbilical vein endothelial cells (HUVECs) by liposome-mediated transfection. An indirect immunofluorescence assay showed that ALDH2 expression increased 24 h after transfection. Moreover, real-time polymerase chain reaction and western blotting revealed significantly higher ALDH2 mRNA and protein expression in the gene-transfected group than in the two control groups. GTN tolerance was induced by treating HUVECs with 10 mM GTN for 16 h + 10 min, which significantly decreased NO levels in control cells, but not in those transfected with ALDH2. Overexpression of ALDH2 increased cell survival against GTN-induced cytotoxicity and conferred protection from oxidative damage resulting from nitrate tolerance, accompanied by decreased production of intracellular reactive oxygen species and reduced expression of heme oxygenase 1. Furthermore, ALDH2 overexpression promoted Akt phosphorylation under GTN tolerance conditions. ALDH2 gene transfection can reverse and prevent tolerance to GTN through its bioactivation and protect against oxidative damage, preventing the development of endothelial dysfunction.

  3. Transfusion of CXCR4-primed endothelial progenitor cells reduces cerebral ischemic damage and promotes repair in db/db diabetic mice.

    PubMed

    Chen, Ji; Chen, Jianying; Chen, Shuzhen; Zhang, Cheng; Zhang, Liangqing; Xiao, Xiang; Das, Avik; Zhao, Yuhui; Yuan, Bin; Morris, Mariana; Zhao, Bin; Chen, Yanfang

    2012-01-01

    This study investigated the role of stromal cell-derived factor-1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) axis in brain and endothelial progenitor cells (EPCs), and explored the efficacy of CXCR4 primed EPCs in treating ischemic stroke in diabetes. The db/db diabetic and db/+ mice were used in this study. Levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were measured. Brain SDF-1α and CXCR4 expression were quantified at basal and after middle cerebral artery occlusion (MCAO). In in vitro study, EPCs were transfected with adenovirus carrying null (Ad-null) or CXCR4 (Ad-CXCR4) followed with high glucose (HG) treatment for 4 days. For pathway block experiments, cells were pre-incubated with PI3K inhibitor or nitric oxide synthase (NOS) inhibitor for two hours. The CXCR4 expression, function and apoptosis of EPCs were determined. The p-Akt/Akt and p-eNOS/eNOS expression in EPCs were also measured. In in vivo study, EPCs transfected with Ad-null or Ad-CXCR4 were infused into mice via tail vein. On day 2 and 7, the cerebral blood flow, neurologic deficit score, infarct volume, cerebral microvascular density, angiogenesis and neurogenesis were determined. We found: 1) The levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were decreased in db/db mice; 2) The basal level of SDF-1α and MCAO-induced up-regulation of SDF-1α/CXCR4 axis were reduced in the brain of db/db mice; 3) Ad-CXCR4 transfection increased CXCR4 expression in EPCs and enhanced EPC colonic forming capacity; 4) Ad-CXCR4 transfection prevented EPCs from HG-induced dysfunction (migration and tube formation) and apoptosis via activation of PI3K/Akt/eNOS signal pathway; 4) Ad-CXCR4 transfection enhanced the efficacy of EPC infusion in attenuating infarct volume and promoting angiogenesis and neurogenesis. Our data suggest that Ad-CXCR4 primed EPCs have better therapeutic effects for ischemia stroke in diabetes than unmodified EPCs do. PMID:23185548

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

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

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

  5. Uptake and transport of superparamagnetic iron oxide nanoparticles through human brain capillary endothelial cells.

    PubMed

    Thomsen, L B; Linemann, T; Pondman, K M; Lichota, J; Kim, K S; Pieters, R J; Visser, G M; Moos, T

    2013-10-16

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

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

    SciTech Connect

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

    2008-04-18

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

  7. Hyperbaric oxygen induces a cytoprotective and angiogenic response in human microvascular endothelial cells.

    PubMed

    Godman, Cassandra A; Chheda, Kousanee P; Hightower, Lawrence E; Perdrizet, George; Shin, Dong-Guk; Giardina, Charles

    2010-07-01

    A genome-wide microarray analysis of gene expression was carried out on human microvascular endothelial cells (HMEC-1) exposed to hyperbaric oxygen treatment (HBOT) under conditions that approximated clinical settings. Highly up-regulated genes included immediate early transcription factors (FOS, FOSB, and JUNB) and metallothioneins. Six molecular chaperones were also up-regulated immediately following HBOT, and all of these have been implicated in protein damage control. Pathway analysis programs identified the Nrf-2-mediated oxidative stress response as one of the primary responders to HBOT. Several of the microarray changes in the Nrf2 pathway and a molecular chaperone were validated using quantitative PCR. For all of the genes tested (Nrf2, HMOX1, HSPA1A, M1A, ACTC1, and FOS), HBOT elicited large responses, whereas changes were minimal following treatment with 100% O(2) in the absence of elevated pressure. The increased expression of immediate early and cytoprotective genes corresponded with an HBOT-induced increase in cell proliferation and oxidative stress resistance. In addition, HBOT treatment enhanced endothelial tube formation on Matrigel plates, with particularly dramatic effects observed following two daily HBO treatments. Understanding how HBOT influences gene expression changes in endothelial cells may be beneficial for improving current HBOT-based wound-healing protocols. These data also point to other potential HBOT applications where stimulating protection and repair of the endothelium would be beneficial, such as patient preconditioning prior to major surgery.

  8. Implanted neonatal human dermal fibroblasts influence the recruitment of endothelial cells in mice

    PubMed Central

    Guerreiro, Susana G.; Brochhausen, Christoph; Negrão, Rita; Barbosa, Mário A.; Unger, Ronald E.; Kirkpatrick, C. James; Soares, Raquel; Granja, Pedro L.

    2012-01-01

    The vascularization of new tissue within a reasonable time is a crucial prerequisite for the success of different cell- and material-based strategies. Considering that angiogenesis is a multi-step process involving humoral and cellular regulatory components, only in vivo assays provide the adequate information about vessel formation and the recruitment of endothelial cells. The present study aimed to investigate if neonatal human dermal fibroblasts could influence in vivo neovascularization. Results obtained showed that fibroblasts were able to recruit endothelial cells to vascularize the implanted matrix, which was further colonized by murine functional blood vessels after one week. The vessels exhibited higher levels of hemoglobin, compared with the control matrix, implanted without fibroblasts, in which no vessel formation could be observed. No significant differences were detected in systemic inflammation. The presence of vessels originated from the host vasculature suggested that host vascular response was involved, which constitutes a fundamental aspect in the process of neovascularization. Fibroblasts implanted within matrigel increased the presence of endothelial cells with positive staining for CD31 and for CD34 and the production of collagen influencing the angiogenic process and promoting the formation of microvessels. New strategies in tissue engineering could be delineated with improved angiogenesis using neonatal fibroblasts. PMID:23507785

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

    SciTech Connect

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

    2009-05-01

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

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

    PubMed Central

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

    1993-01-01

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

  11. Chloroquine stimulates nitric oxide synthesis in murine, porcine, and human endothelial cells.

    PubMed Central

    Ghigo, D; Aldieri, E; Todde, R; Costamagna, C; Garbarino, G; Pescarmona, G; Bosia, A

    1998-01-01

    Nitric oxide (NO) is a free radical involved in the regulation of many cell functions and in the expression of several diseases. We have found that the antimalarial and antiinflammatory drug, chloroquine, is able to stimulate NO synthase (NOS) activity in murine, porcine, and human endothelial cells in vitro: the increase of enzyme activity is dependent on a de novo synthesis of some regulatory protein, as it is inhibited by cycloheximide but is not accompanied by an increased expression of inducible or constitutive NOS isoforms. Increased NO synthesis is, at least partly, responsible for chloroquine-induced inhibition of cell proliferation: indeed, NOS inhibitors revert the drug-evoked blockage of mitogenesis and ornithine decarboxylase activity in murine and porcine endothelial cells. The NOS-activating effect of chloroquine is dependent on its weak base properties, as it is exerted also by ammonium chloride, another lysosomotropic agent. Both compounds activate NOS by limiting the availability of iron: their stimulating effects on NO synthesis and inhibiting action on cell proliferation are reverted by iron supplementation with ferric nitrilotriacetate, and are mimicked by incubation with desferrioxamine. Our results suggest that NO synthesis can be stimulated in endothelial cells by chloroquine via an impairment of iron metabolism. PMID:9691096

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

    PubMed Central

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

    2016-01-01

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

  13. Permeability characteristics of human endothelial monolayers seeded on different extracellular matrix proteins.

    PubMed Central

    Nooteboom, A; Hendriks, T; Ottehöller, I; van der Linden, C J

    2000-01-01

    OBJECTIVE: To investigate whether endothelial monolayer permeability changes induced by inflammatory mediators are affected by the extracellular matrix protein used for cell seeding. METHODS: Human umbilical venular endothelial cells (HUVEC) were grown to confluent monolayers on membranes coated with either collagen, fibronectin or gelatin. The permeability to albumin and dextran was then assessed, both under normal conditions and after treatment with tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide (LPS). RESULTS: With any of the three protein coatings, tight junctions were formed all over the monolayers. The permeability of the coated membranes to albumin and dextran was reduced strongly by confluent monolayers; the relative reduction was similar for the three matrix proteins used. Pre-incubation of the monolayers with either TNF-alpha or LPS increased permeability dose dependently. However, the relative increase due to either treatment was independent of the protein used for membrane coating. CONCLUSION: The extracellular matrix protein used for initial seeding of endothelial cultures plays a minor role in determining the permeability changes induced in HUVEC monolayers by inflammatory mediators. PMID:11200364

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

    PubMed

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

    2013-08-01

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

  15. Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells

    PubMed Central

    Zaske, Ana-María; Danila, Delia; Queen, Michael C.; Golunski, Eva; Conyers, Jodie L.

    2013-01-01

    Although atomic force microscopy (AFM) has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs) in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15–30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses. PMID:26555999

  16. 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

  17. 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

  18. Aspirin inhibits human coronary artery endothelial cell proliferation by upregulation of p53.

    PubMed

    Ranganathan, Subramanian; Joseph, Jacob; Mehta, Jawahar L

    2003-01-31

    Aspirin (acetylsalicylic acid, ASA) is effective in the primary and secondary prevention of vascular events. This effect is mediated in large part by platelet inhibition; however, non-platelet-mediated effects may also be relevant in the overall efficacy of ASA. We determined the effect of ASA on the synthesis of DNA and total proteins in cultured human coronary endothelial cells (HCAECs). Fourth generation HCAECs were cultured and treated with ASA and rate of synthesis of DNA and total proteins was determined by incorporation of [3H]thymidine and [3H]proline, respectively. ASA inhibited DNA synthesis by 50% at a concentration of 1mM and protein synthesis by 50% at a concentration of 2mM. The inhibitory effect of ASA was observed as early as 2h after treatment of HCAECs. The inhibition of DNA and protein synthesis could be reversed within 24h after removal of the drug from the culture medium. Indomethacin also inhibited DNA and protein synthesis. Western blot analysis revealed that the expression of p53 protein was increased after treatment of the cells with ASA. These observations indicate that ASA decreases endothelial cell proliferation through cell cycle arrest mediated by enhanced p53 expression. Arrest of endothelial proliferation and activation may be an important mechanism of the beneficial effect of ASA in acute coronary syndromes.

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

    SciTech Connect

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

    2007-10-10

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

  20. Dissecting human cerebral organoids and fetal neocortex using single-cell RNAseq

    NASA Astrophysics Data System (ADS)

    Treutlein, Barbara

    Cerebral organoids - three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells - have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

  1. A New Presentation and Exploration of Human Cerebral Vasculature Correlated with Surface and Sectional Neuroanatomy

    ERIC Educational Resources Information Center

    Nowinski, Wieslaw L.; Thirunavuukarasuu, Arumugam; Volkau, Ihar; Marchenko, Yevgen; Aminah, Bivi; Gelas, Arnaud; Huang, Su; Lee, Looi Chow; Liu, Jimin; Ng, Ting Ting; Nowinska, Natalia G.; Qian, Guoyu Yu; Puspitasari, Fiftarina; Runge, Val M.

    2009-01-01

    The increasing complexity of human body models enabled by advances in diagnostic imaging, computing, and growing knowledge calls for the development of a new generation of systems for intelligent exploration of these models. Here, we introduce a novel paradigm for the exploration of digital body models illustrating cerebral vasculature. It enables…

  2. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development

    PubMed Central

    Camp, J. Gray; Badsha, Farhath; Florio, Marta; Kanton, Sabina; Gerber, Tobias; Wilsch-Bräuninger, Michaela; Lewitus, Eric; Sykes, Alex; Hevers, Wulf; Lancaster, Madeline; Knoblich, Juergen A.; Lachmann, Robert; Pääbo, Svante; Huttner, Wieland B.; Treutlein, Barbara

    2015-01-01

    Cerebral organoids—3D cultures of human cerebral tissue derived from pluripotent stem cells—have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures. PMID:26644564

  3. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.

    PubMed

    Camp, J Gray; Badsha, Farhath; Florio, Marta; Kanton, Sabina; Gerber, Tobias; Wilsch-Bräuninger, Michaela; Lewitus, Eric; Sykes, Alex; Hevers, Wulf; Lancaster, Madeline; Knoblich, Juergen A; Lachmann, Robert; Pääbo, Svante; Huttner, Wieland B; Treutlein, Barbara

    2015-12-22

    Cerebral organoids-3D cultures of human cerebral tissue derived from pluripotent stem cells-have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures. PMID:26644564

  4. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development

    PubMed Central

    Camp, J. Gray; Badsha, Farhath; Florio, Marta; Kanton, Sabina; Gerber, Tobias; Wilsch-Bräuninger, Michaela; Lewitus, Eric; Sykes, Alex; Hevers, Wulf; Lancaster, Madeline; Knoblich, Juergen A.; Lachmann, Robert; Pääbo, Svante; Huttner, Wieland B.; Treutlein, Barbara

    2015-01-01

    Cerebral organoids—3D cultures of human cerebral tissue derived from pluripotent stem cells—have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures. PMID:26644564

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

    SciTech Connect

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

    2011-03-01

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

  6. Plasmodium falciparum Adhesion on Human Brain Microvascular Endothelial Cells Involves Transmigration-Like Cup Formation and Induces Opening of Intercellular Junctions

    PubMed Central

    Jambou, Ronan; Combes, Valery; Jambou, Marie-Jose; Weksler, Babeth B.; Couraud, Pierre-Olivier; Grau, Georges E.

    2010-01-01

    Cerebral malaria, a major cause of death during malaria infection, is characterised by the sequestration of infected red blood cells (IRBC) in brain microvessels. Most of the molecules implicated in the adhesion of IRBC on endothelial cells (EC) are already described; however, the structure of the IRBC/EC junction and the impact of this adhesion on the EC are poorly understood. We analysed this interaction using human brain microvascular EC monolayers co-cultured with IRBC. Our study demonstrates the transfer of material from the IRBC to the brain EC plasma membrane in a trogocytosis-like process, followed by a TNF-enhanced IRBC engulfing process. Upon IRBC/EC binding, parasite antigens are transferred to early endosomes in the EC, in a cytoskeleton-dependent process. This is associated with the opening of the intercellular junctions. The transfer of IRBC antigens can thus transform EC into a target for the immune response and contribute to the profound EC alterations, including peri-vascular oedema, associated with cerebral malaria. PMID:20686652

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

    NASA Astrophysics Data System (ADS)

    Critser, Paul J.; Yoder, Mervin C.

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

  8. 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

  9. Functional Characterization and Expression Profiling of Human Induced Pluripotent Stem Cell- and Embryonic Stem Cell-Derived Endothelial Cells

    PubMed Central

    Li, Zongjin; Hu, Shijun; Ghosh, Zhumur; Han, Zhongchao

    2011-01-01

    With regard to human induced pluripotent stem cells (hiPSCs), in which adult cells are reprogrammed into embryonic-like cells using defined factors, their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study, hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method, and CD31+ cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the “distances” among hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and HUVECs. We further conclude that hiPSCs can differentiate into functional endothelial cells, but with limited expansion potential compared with hESC-ECs; thus, extensive studies should be performed to explore the cause and extent of such differences before clinical application of hiPSC-ECs can begin. PMID:21235328

  10. Factor VIIa binding to endothelial cell protein C receptor: Differences between mouse and human systems

    PubMed Central

    Sen, Prosenjit; Clark, Curtis A.; Gopalakrishnan, Ramakrishnan; Hedner, Ulla; Esmon, Charles T.; Pendurthi, Usha R.; Rao, L. Vijaya Mohan

    2013-01-01

    Summary Recent in vitro studies have shown that the zymogen and activated form of FVII bind to endothelial cell protein C receptor (EPCR). At present, there is no evidence that FVIIa binds to EPCR on vascular endothelium in vivo in the presence of circulating protein C, a primary ligand for EPCR. The present study was carried out to investigate the interaction of murine and human ligands with murine EPCR both in vivo and in vitro. Measurement of endogenous plasma levels of FVII in wild-type, EPCR-deficient and EPCR-over expressing mice showed slightly lower levels of FVII in EPCR-over expressing mice. However, infusion of high concentrations of competing ligands, either human APCi or FVIIai, to EPCR-over expressing mice failed to increase plasma levels of mouse FVII whereas they increased the plasma levels of protein C by 2 to 3-fold. Examining the association of exogenously administered mouse FVIIa or human FVIIa by immunohistochemistry revealed that human, but not murine FVIIa, binds to the murine endothelium in an EPCR-dependent manner. In vitro binding studies performed using surface plasmon resonance and endothelial cells revealed that murine FVIIa binds murine EPCR negligibly. Human FVIIa binding to EPCR, particularly to mouse EPCR, is markedly enhanced by availability of Mg2+ ions. In summary, our data show that murine FVIIa binds poorly to murine EPCR, whereas human FVIIa binds efficiently to both murine and human EPCR. Our data suggest that one should consider the use of human FVIIa in mouse models to investigate the significance of FVIIa and EPCR interaction. PMID:22370814

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2015-11-01

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

  13. Generation of novel monoclonal antibodies for the enrichment and characterization of human corneal endothelial cells (hCENC) necessary for the treatment of corneal endothelial blindness

    PubMed Central

    Ding, Vanessa; Chin, Angela; Peh, Gary; Mehta, Jodhbir S; Choo, Andre

    2014-01-01

    Corneal transplantation is the primary treatment option to restore vision for patients with corneal endothelial blindness. Although the success rate of treatment is high, limited availability of transplant grade corneas is a major obstacle. Tissue-engineered corneal endothelial grafts constructed using cultivated human corneal endothelial cells (hCENC) isolated from cadaveric corneas may serve as a potential graft source. Currently, tools for the characterization of cultured hCENC and enrichment of hCENC from potential contaminating cells such as stromal fibroblasts are lacking. In this study, we describe the generation and characterization of novel cell surface monoclonal antibodies (mAbs) specific for hCENC. These mAbs could be used for enrichment and characterization of hCENC. Out of a total of 389 hybridomas, TAG-1A3 and TAG-2A12 were found to be specific to the corneal endothelial monolayer by immunostaining of frozen tissue sections. Both mAbs were able to clearly identify hCENC with good ‘cobblestone-like’ morphology from multiple donors. The antigen targets for TAG-1A3 and TAG-2A12 were found to be CD166/ALCAM and Peroxiredoxin-6 (Prdx-6), respectively, both of which have not been previously described as markers of hCENC. Additionally, unlike other Prdx-6 mAbs, TAG-2A12 was found to specifically bind cell surface Prdx-6, which was only expressed on hCENC and not on other cell types screened such as human corneal stromal fibroblasts (hCSF) and human pluripotent stem cells (hPSC). From our studies, we conclude that TAG-1A3 and TAG-2A12 are promising tools to quantitatively assess hCENC quality. It is also noteworthy that the binding specificity of TAG-2A12 could be used for the enrichment of hCENC from cell mixtures of hCSF and hPSC. PMID:25484056

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-03-01

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

  16. A biomimetic physiological model for human adipose tissue by adipocytes and endothelial cell cocultures with spatially controlled distribution.

    PubMed

    Yao, Rui; Du, Yanan; Zhang, Renji; Lin, Feng; Luan, Jie

    2013-08-01

    An in vitro model that recapitulates the characteristics of native human adipose tissue would largely benefit pathology studies and therapy development. In this paper, we fabricated a physiological model composed of both human adipocytes and endothelial cells with spatially controlled distribution that biomimics the structure and composition of human adipose tissue. Detailed studies into the cell-cell interactions between the adipocytes and endothelial cells revealed a mutual-enhanced effect which resembles the in vivo routine. Furthermore, comparisons between planar coculture and model coculture demonstrated improved adipocyte function as well as endothelial cell proliferation under the same conditions. This research provided a reliable model for human adipose tissue development studies and potential obesity-related therapy development.

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    2015-09-01

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

  19. Endothelial lipase is highly expressed in macrophages in advanced human atherosclerotic lesions.

    PubMed

    Bartels, Emil D; Nielsen, John E; Lindegaard, Marie L S; Hulten, Lillemor M; Schroeder, Torben V; Nielsen, Lars B

    2007-12-01

    Endothelial lipase (EL) is expressed in endothelial cells, and affects plasma lipoprotein metabolism by hydrolyzing phospholipids in HDL. To determine the cellular expression of EL mRNA and protein in human atherosclerotic lesions, we performed in situ hybridization and immunohistochemical studies on sections of carotid endarterectomy specimens from patients with symptomatic cerebrovascular disease. In each of eight patients, EL mRNA and/or protein were seen in areas between the necrotic core and the fibrotic cap where they colocalized with LPL and macrophage-specific CD68. Moreover, there was a positive association between the expression of EL mRNA and CD68 mRNA in plaques from 26 patients. The impact of differentiation from monocytes into macrophages, and subsequently foam cells (by incubation with acetylated LDL) on expression was studied using THP-1 monocytes and primary human monocytes. EL mRNA expression increased markedly when either type of monocytes was differentiated into macrophages. Upon further differentiation into foam cells EL mRNA decreased whereas protein levels remained high compared to monocytes. In conclusion, macrophages in advanced human atherosclerotic lesions display high levels of EL expression, and the level of EL expression varies greatly during transformation of blood monocytes into foam cells.

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

    PubMed

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

    2011-12-01

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

  1. Human dermal microvascular endothelial cells in vitro: effect of cyclic AMP on cellular morphology and proliferation rate.

    PubMed

    Davison, P M; Karasek, M A

    1981-02-01

    Macrovascular endothelial cells isolated from the human umbilical vein and microvessel endothelium from the newborn foreskin dermis differ in their requirements for optimal growth in vitro. In the presence of 5 X 10(-4) M dibutyryl cyclic AMP (Bt2cAMP), human dermal microvessel endothelial cell proliferation rate increased to give a cell number of 203% of controls values by day 10 in culture. The cells retained their characteristic endothelial cell morphology, reached confluence, and could be serially passaged. Cells grown in the absence of Bt2cAMP did not proliferate readily and grew in a disorganized pattern. The effect of Bt2cAMP on microvascular endothelial cell proliferation rate and morphology could be duplicated by cholera toxin (CT) used together with isobutyl methylxanthine (IMX). These agents were found to elevate intracellular levels of cyclic AMP in microvascular endothelium over 40-fold. Human umbilical vein cells in culture failed to respond to either Bt2cAMP or CT together with IMX. The growth-promoting effect of dibutyryl cyclic AMP (Bt2cAMP) on human foreskin dermal microvascular endothelium in vitro is in marked contrast to the lack of response of human umbilical vein cells. These results provide further evidence of differences in the mechanisms that regulate macro and microvessel endothelial cell proliferation in vitro.

  2. Reproducibility of cerebral glucose metabolic measurements in resting human subjects.

    PubMed

    Bartlett, E J; Brodie, J D; Wolf, A P; Christman, D R; Laska, E; Meissner, M

    1988-08-01

    Positron emission tomography with 11C-2-deoxyglucose was used to determine the test-retest variability of regional cerebral glucose metabolism in 22 young normal right-handed men scanned twice in a 24-h period under baseline (resting) conditions. To assess the effects of scan order and time of day on variability, 12 subjects were scanned in the morning and afternoon of the same day (a.m.-p.m.) and 10 in the reverse order (p.m.-a.m.) with a night in between. The effect of anxiety on metabolism was also assessed. Seventy-three percent of the total subject group showed changes in whole brain metabolism from the first to the second measurement of 10% or less, with comparable changes in various cortical and subcortical regions. When a scaling factor was used to equate the whole brain metabolism in the two scans for each individual, the resulting average regional changes for each group were no more than 1%. This suggests that the proportion of the whole brain metabolism utilized regionally is stable in a group of subjects over time. Both groups of subjects had lower morning than afternoon metabolism, but the differences were slight in the p.m.-a.m. group. One measure of anxiety (pulse at run 1) was correlated with run 1 metabolism and with the percentage of change from run 1 to run 2. No significant run 2 correlations were observed. This is the first study to measure test-retest variability in cerebral glucose metabolism in a large sample of young normal subjects. It demonstrates that the deoxyglucose method yields low intrasubject variability and high stability over a 24-h period. PMID:3260593

  3. Cerebral blood volume in humans by NIRS and PET

    NASA Astrophysics Data System (ADS)

    Pott, Frank; Knudsen, Gitte M.; Rostrup, Egill; Ide, Kojiro; Secher, Niels H.; Paulson, Olaf B.

    1998-01-01

    Near infrared spectroscopy (NIRS) determined changes in the cerebral blood volume (CBV) were compared to those obtained by positron emission tomography (PET) in five healthy volunteers (2 females). Two NIRS optodes were placed on the left forehead and NIRS-CBV was derived from the sum of oxyhemoglobin and deoxyhemoglobin. CBV changes were induced by hyperventilation and inhalation of 6% CO2. After 2 min inhalation of labeled carbon monoxide, data were sampled during 8 min for both PET- and NIRS-CBV as well as for the arterial carbon dioxide tension (PaCO2). The region of interest for PET-CBV was `banana-shaped' with boundaries corresponding to the position of the NIRS optodes on the transmission scan and to a depth of approximately 2 cm. During hyperventilation, PaCO2 decreased from 5.2 (4.6 - 5.8) to 4.6 (4.2 - 4.9) kPa and equally PET-CBV (from 3.9 (2.5 - 5.2) to 3.6 (3.0 - 4.8) ml (DOT) 100 g-1) and NIRS-CBV were reduced (by -0.14 [-0.38 - 0.50] ml (DOT) 100 g-1). During hypercapnia PaCO2 increased to 6.0 (5.9 - 7.0) kPa accompanied by parallel changes in PET- (to 4.5 (3.9 - 4.9) ml (DOT) 100 g-1) and NIRS-CBV (by 0.04 [-0.02 - 0.30] ml (DOT) 100 g-1) and the two variables were correlated (r equals 0.78, p < 0.05). In conclusion, with a moderate change in the arterial carbon dioxide tension, the cerebral blood volumes determined by near infrared spectroscopy and by positron emission tomography change in parallel but the change in NIRS-CBV is small compared to that obtained by PET.

  4. Cerebral blood volume in humans by NIRS and PET

    NASA Astrophysics Data System (ADS)

    Pott, Frank; Knudsen, Gitte M.; Rostrup, Egill; Ide, Kojiro; Secher, Niels H.; Paulson, Olaf B.

    1997-12-01

    Near infrared spectroscopy (NIRS) determined changes in the cerebral blood volume (CBV) were compared to those obtained by positron emission tomography (PET) in five healthy volunteers (2 females). Two NIRS optodes were placed on the left forehead and NIRS-CBV was derived from the sum of oxyhemoglobin and deoxyhemoglobin. CBV changes were induced by hyperventilation and inhalation of 6% CO2. After 2 min inhalation of labeled carbon monoxide, data were sampled during 8 min for both PET- and NIRS-CBV as well as for the arterial carbon dioxide tension (PaCO2). The region of interest for PET-CBV was `banana-shaped' with boundaries corresponding to the position of the NIRS optodes on the transmission scan and to a depth of approximately 2 cm. During hyperventilation, PaCO2 decreased from 5.2 (4.6 - 5.8) to 4.6 (4.2 - 4.9) kPa and equally PET-CBV (from 3.9 (2.5 - 5.2) to 3.6 (3.0 - 4.8) ml (DOT) 100 g-1) and NIRS-CBV were reduced (by -0.14 [-0.38 - 0.50] ml (DOT) 100 g-1). During hypercapnia PaCO2 increased to 6.0 (5.9 - 7.0) kPa accompanied by parallel changes in PET- (to 4.5 (3.9 - 4.9) ml (DOT) 100 g-1) and NIRS-CBV (by 0.04 [-0.02 - 0.30] ml (DOT) 100 g-1) and the two variables were correlated (r equals 0.78, p < 0.05). In conclusion, with a moderate change in the arterial carbon dioxide tension, the cerebral blood volumes determined by near infrared spectroscopy and by positron emission tomography change in parallel but the change in NIRS-CBV is small compared to that obtained by PET.

  5. Lectins as markers of endothelial cells: comparative study between human and animal cells.

    PubMed

    Roussel, F; Dalion, J

    1988-04-01

    Vascular endothelial cells were labelled with 10 vegetal lectins and 3 more monoclonal antibodies antiblood group ABO substances, in major organs of 14 common laboratory animals. After fixation in PLPa and paraffin embedding, cells were examined to determine their likeness to human cells. The most interesting reactive used was EEA, whose positivity defines upper mammalians. Blood B substance positivity and CSA negativity defines primates among which man is unique and defined by UEA I positivity and variability in ABO substance. CSA positivity defines non-primate upper mammalians. Rodents and birds were negative with all reactives tested. From the histochemical point of view, the animals closest to humans are monkeys, followed by swine and oxen, then by cat and dog and lastly by sheep. Rodents appear unrelated to humans in this system.

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

    PubMed

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

    2009-08-01

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

  7. Human adipose tissue-resident monocytes exhibit an endothelial-like phenotype and display angiogenic properties

    PubMed Central

    2014-01-01

    Introduction Adipose tissue has the unique property of expanding throughout adult life, and angiogenesis is required for its growth. However, endothelial progenitor cells contribute minimally to neovascularization. Because myeloid cells have proven to be angiogenic, and monocytes accumulate in expanding adipose tissue, they might contribute to vascularization. Methods The stromal vascular fraction (SVF) cells from human adipose tissue were magnetically separated according to CD45 or CD14 expression. Adipose-derived mesenchymal stromal cells (MSCs) were obtained from SVF CD45- cells. CD14+ monocytes were isolated from peripheral blood (PB) mononuclear cells and then cultured with SVF-derived MSCs. Freshly isolated or cultured cells were characterized with flow cytometry; the conditioned media were analyzed for the angiogenic growth factors, angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) with Luminex Technology; their angiogenic capacity was determined in an in vivo gelatinous protein mixture (Matrigel) plug angiogenesis assay. Results CD45+ hematopoietic cells within the SVF contain CD14+ cells that co-express the CD34 progenitor marker and the endothelial cell antigens VEGF receptor 2 (VEGFR2/KDR), VEGFR1/Flt1, and Tie2. Co-culture experiments showed that SVF-derived MSCs promoted the acquisition of KDR and Tie-2 in PB monocytes. MSCs secreted significant amounts of Ang-2 and HGF, but minimal amounts of bFGF, G-CSF, or GM-CSF, whereas the opposite was observed for SVF CD14+ cells. Additionally, SVF CD14+ cells secreted significantly higher levels of VEGF and bFGF than did MSCs. Culture supernatants of PB monocytes cultured with MSCs contained significantly higher concentrations of VEGF, HGF, G-CSF, and GM-CSF than did the supernatants from cultures without MSCs

  8. Dual mechanism of vascular endothelial growth factor upregulation by hypoxia in human hepatocellular carcinoma

    PubMed Central

    von Marschall, Z; Cramer, T; Hocker, M; Finkenzeller, G; Wiedenmann, B; Rosewicz, S

    2001-01-01

    BACKGROUND/AIMS—Vascular endothelial growth factor (VEGF) plays a key role in regulation of tumour associated angiogenesis. In the current study we analysed expression of VEGF and its receptors in human hepatocellular carcinoma (HCC) and investigated the molecular mechanisms of VEGF regulation by hypoxia.
METHODS—VEGF, kinase domain region (KDR)/fetal liver kinase 1 (flk-1), and flt-1 expression were examined by immunohistochemistry and in situ hybridisation in 15 human HCC tissues. Expression of VEGF and regulation by hypoxia were assessed in three human HCC cell lines using a quantitative competitive reverse transcription-polymerase chain reaction, ELISA, and a series of 5' deletion reporter gene constructs of the human VEGF promoter in transient transfection assays.
RESULTS—We observed over expression of VEGF mRNA and protein in HCC compared with cirrhosis or normal liver. Expression of VEGF in tumour cells was strongly increased in areas directly adjacent to necrotic/hypoxic regions. Both VEGF receptors were detected in vascular endothelia of HCC while only KDR/flk-1 receptors were detected in endothelial cells of cirrhotic livers. Expression of VEGF was observed in all human HCC cell lines examined. Hypoxia (1% oxygen) resulted in profound upregulation of VEGF mRNA and protein levels. Furthermore, hypoxia treatment resulted in a doubling of VEGF mRNA stability. Deletion analysis of the human VEGF 5' flanking region −2018 and +50 demonstrated induction of VEGF promoter activity under hypoxic conditions which was significantly decreased following deletion of the region −1286 and −789 suggesting a substantial contribution of the −975 putative hypoxia inducible factor 1 binding site to hypoxia mediated transcriptional activation of the VEGF gene.
CONCLUSION—These data suggest hypoxia as a central stimulus of angiogenesis in human HCC through upregulation of VEGF gene expression by at least two distinct molecular mechanisms: activation of

  9. Stromal cell-derived factor-1α prevents endothelial progenitor cells senescence and enhances re-endothelialization of injured arteries via human telomerase reverse transcriptase.

    PubMed

    Shen, Xiaohua; Zhou, Yucheng; Bi, Xukun; Zhang, Jiefang; Fu, Guosheng; Zheng, Hao

    2015-08-01

    Recent studies have suggested that endothelial progenitor subpopulation (EPCs) number and activity were associated with EPCs senescence. Our previous study had shown that stromal cell-derived factor-1alpha (SDF-1α) could prevent EPCs senescence, which may be via telomerase. In this study, we further investigated the role of human telomerase reverse transcriptase (h-TERT) on the protective effect of SDF-1α against senescence. Knockdown h-TERT abrogated the protective effect of SDF-1α and abolished the effects of SDF-1α on migration and proliferation. Moreover, it inhibited EPCs recruitment. In conclusion, h-TERT served a critical role in the progress that SDF-1α prevented EPCs senescence and enhanced re-endothelialization of the injured arteries.

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

    PubMed

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

    2008-08-01

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

  11. Selective induction of cell adhesion molecules by proinflammatory mediators in human cardiac microvascular endothelial cells in culture

    PubMed Central

    Yan, Jun; Nunn, Adrian D; Thomas, Regi

    2010-01-01

    Pro-inflammatory mediators can dramatically alter many responses of cultured endothelial cells in vitro, which are relevant to understanding the role played by the endothelium in inflammation in vivo. The aim of this study was to determine the ability of a comprehensive array of pro-inflammatory stimuli to modulate Cell Adhesion Molecule (CAM) expression in cultures of human microvascular cardiac endothelial cells (HMVEC.c). Cell ELISA, immunocy-tochemistry and flow cytometry were used to measure the CAM expressions in HMVEC.c in response to interleukins, TNF-α and LPS. Passage matched HMVEC.c from different donors showed different CAM expression profiles, confirming inherent variability in endothelial cells. Endothelial cells from different parts of the vasculature are exposed to different cytokines and thus different protein expression profiles. A thorough understanding of these innate differences in expression pattern of the microvasculatures of cardiac tissues might allow us the opportunity to target these tissues selectively. PMID:21072266

  12. 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.

  13. Adherence of Staphylococcus epidermidis to human endothelial cells is associated with a polysaccharidic component of its extracellular mucous layer.

    PubMed

    Krevvata, Maria I; Spiliopoulou, Anastasia; Anastassiou, Evangelos D; Karamanos, Nikos; Kolonitsiou, Fevronia

    2011-06-01

    Bacterial adherence to eukaryotic cells is highly contributing to microbial pathogenesis. Bacterial adhesins, macromolecules, and glycosaminoglycan chains of the endothelial cell surface have been implicated in staphylococcal attachment. Our research group has isolated an antigenic polysaccharidic component of Staphylococcus epidermidis extracellular layer, known as 20-kDa PS (PS), and showed that antibodies against this polysaccharide protect from infections. Therefore, the role of PS in S. epidermidis adherence to endothelial cells was studied. For this purpose we examined the impact of PS on the ability of two S. epidermidis strains (a PS-producing and a non-PS-producing strain) to adhere to human endothelial cells in the presence or absence of specific antibodies to this polysaccharide. Hence, it is established that exogenous chondroitin sulfate (CS) decreases, in part, the S. epidermidis' attachment to endothelial cells and the antagonistic binding effect of CS and PS was also studied. The results obtained demonstrate that PS facilitates the adherence of S. epidermidis to both strains. CS abolished the PS-induced adherence in PS-producing strain and partially in the non-PS-producing one. Conclusively, the adherence of S. epidermidis to human endothelial cells is associated with its extracellular PS component and it is suggested that the bacterial binding via glycosaminoglycan chains is an important mechanism underlining the PS-induced binding to endothelial cells.

  14. Effect of extracellular human immunodeficiency virus type 1 glycoprotein 120 on primary human vascular endothelial cell cultures.

    PubMed

    Huang, M B; Hunter, M; Bond, V C

    1999-09-20

    During the course of an HIV-1 infection, free infectious and noninfectious virus particles, and free HIV-1 proteins, circulate within the host, exposing the host endothelium to these viral factors, even if the endothelium is not infected. This suggests that extracellular HIV-1 proteins could influence endothelial cell function, leading to pathogenesis. In light of this, we have used primary cultured human vascular endothelial cells (HUVECs) to screen for effects of the HIV-1 protein gp120 on endothelial cell function. The results of this study show that short exposure of HUVEC cultures to this protein causes significant levels of cytotoxicity. Further, using several different assays, we have shown that this cytotoxic effect on HUVECs appears to be due to induction of an apoptotic program. The biphasic nature of gp120 titration curves suggests that multiple cellular factors are mediating these gp120-induced effects. Competition studies appear to confirm this by showing that the apoptotic effect is mediated through two cell surface receptors on HUVECs, CCR5 and CXCR4. Alternatively, competition studies examining CD4 receptors suggests that CD4 played no role in gp12O-induced effects on HUVECs.

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

    SciTech Connect

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

    2010-09-10

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-05-01

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

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

    PubMed Central

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

    2016-01-01

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

  18. Identification and characterization of angiogenesis targets through proteomic profiling of endothelial cells in human cancer tissues.

    PubMed

    Mesri, Mehdi; Birse, Charlie; Heidbrink, Jenny; McKinnon, Kathy; Brand, Erin; Bermingham, Candy Lee; Feild, Brian; Fitzhugh, William; He, Tao; Ruben, Steve; Moore, Paul A

    2013-01-01

    Genomic and proteomic analysis of normal and cancer tissues has yielded abundant molecular information for potential biomarker and therapeutic targets. Considering potential advantages in accessibility to pharmacological intervention, identification of targets resident on the vascular endothelium within tumors is particularly attractive. By employing mass spectrometry (MS) as a tool to identify proteins that are over-expressed in tumor-associated endothelium relative to normal cells, we aimed to discover targets that could be utilized in tumor angiogenesis cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. First, we isolated endothelial cells (ECs) from human normal and kidney cancer tissues by FACS using CD146 as a marker. Additionally, dispersed human colon and lung cancer tissues and their corresponding normal tissues were cultured ex-vivo and their endothelial content were preferentially expanded, isolated and passaged. Cell surface proteins were then preferentially captured, digested with trypsin and subjected to MS-based proteomic analysis. Peptides were first quantified, and then the sequences of differentially expressed peptides were resolved by MS analysis. A total of 127 unique non-overlapped (157 total) tumor endothelial cell over-expressed proteins identified from directly isolated kidney-associated ECs and those identified from ex-vivo cultured lung and colon tissues including known EC markers such as CD146, CD31, and VWF. The expression analyses of a panel of the identified targets were confirmed by immunohistochemistry (IHC) including CD146, B7H3, Thy-1 and ATP1B3. To determine if the proteins identified mediate any functional role, we performed siRNA studies which led to previously unidentified functional dependency for B7H3 and ATP1B3.

  19. Identification and Characterization of Angiogenesis Targets through Proteomic Profiling of Endothelial Cells in Human Cancer Tissues

    PubMed Central

    Mesri, Mehdi; Birse, Charlie; Heidbrink, Jenny; McKinnon, Kathy; Brand, Erin; Bermingham, Candy Lee; Feild, Brian; FitzHugh, William; He, Tao; Ruben, Steve; Moore, Paul A.

    2013-01-01

    Genomic and proteomic analysis of normal and cancer tissues has yielded abundant molecular information for potential biomarker and therapeutic targets. Considering potential advantages in accessibility to pharmacological intervention, identification of targets resident on the vascular endothelium within tumors is particularly attractive. By employing mass spectrometry (MS) as a tool to identify proteins that are over-expressed in tumor-associated endothelium relative to normal cells, we aimed to discover targets that could be utilized in tumor angiogenesis cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. First, we isolated endothelial cells (ECs) from human normal and kidney cancer tissues by FACS using CD146 as a marker. Additionally, dispersed human colon and lung cancer tissues and their corresponding normal tissues were cultured ex-vivo and their endothelial content were preferentially expanded, isolated and passaged. Cell surface proteins were then preferentially captured, digested with trypsin and subjected to MS-based proteomic analysis. Peptides were first quantified, and then the sequences of differentially expressed peptides were resolved by MS analysis. A total of 127 unique non-overlapped (157 total) tumor endothelial cell over-expressed proteins identified from directly isolated kidney-associated ECs and those identified from ex-vivo cultured lung and colon tissues including known EC markers such as CD146, CD31, and VWF. The expression analyses of a panel of the identified targets were confirmed by immunohistochemistry (IHC) including CD146, B7H3, Thy-1 and ATP1B3. To determine if the proteins identified mediate any functional role, we performed siRNA studies which led to previously unidentified functional dependency for B7H3 and ATP1B3. PMID:24236063

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

    PubMed

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

    2013-01-01

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

  1. Provocation of pulmonary vascular endothelial injury in rabbits by human recombinant interleukin-1 beta.

    PubMed Central

    Goldblum, S E; Yoneda, K; Cohen, D A; McClain, C J

    1988-01-01

    Interleukin-1 (IL-1) mediates components of the acute-phase response, stimulates granulocyte metabolism, and induces endothelial cell surface changes. We studied the effects of human recombinant IL-1 beta (rIL-1 beta) or rIL-1 alpha on circulating granulocytes, their sequestration within the pulmonary microvasculature, pulmonary edema formation, and changes in pulmonary vascular permeability to 125I-labeled albumin. rIL-1 beta administration induced significant (P less than 0.03) but transient granulocytopenia followed by significant (P less than 0.04) neutrophilia and significant (P less than 0.04) pulmonary leukostasis compared with saline-infused rabbits. Rabbits preinfused with 125I-labeled rabbit serum albumin and administered saline, rIL-1 beta, or rIL-1 alpha were sacrificed, and lung wet/dry weight ratios and bronchoalveolar lavage fluid and plasma 125I activities determined. Both rIL-1 beta and rIL-1 alpha increased lung wet/dry weight ratios (P less than 0.025 and P less than 0.01, respectively) compared with saline controls. rIL-1 beta increased bronchoalveolar lavage fluid/plasma 125I radioactivity ratios (P less than 0.025). Electron microscopic analysis of lung sections obtained from rIL-1 beta-infused animals demonstrated endothelial injury, perivascular edema, and extravasation of an ultrastructural permeability tracer. The observation that human rIL-1 can evoke acute pulmonary vascular endothelial injury and lung edema in rabbits supports the hypothesis that IL-1 may play a role in the pathogenesis of the adult respiratory distress syndrome. Images PMID:3261716

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

    PubMed Central

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

    2003-01-01

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

  3. Human liver sinusoidal endothelial cells respond to interaction with Entamoeba histolytica by changes in morphology, integrin signalling and cell death.

    PubMed

    Faust, Daniela M; Marquay Markiewicz, Jacques; Danckaert, Anne; Soubigou, Guillaume; Guillen, Nancy

    2011-07-01

    Invasive infection with Entamoeba histolytica causes intestinal and hepatic amoebiasis. In liver, parasites cross the endothelial barrier before abscess formation in the parenchyma. We focussed on amoebae interactions with human hepatic endothelial cells, the latter potentially playing a dual role in the infection process: as a barrier and as modulators of host defence responses. We characterized early responses of a human liver sinusoidal endothelial cell line to virulent and virulence-attenuated E. histolytica. Within the first minutes human cells start to retract, enter into apoptosis and die. In the presence of virulent amoebae, expression of genes related to cell cycle, cell death and integrin-mediated adhesion signalling was modulated, and actin fibre, focal adhesion kinase and paxillin localizations changed. Effects of inhibitors and amoeba strains not expressing pathogenic factors amoebapore A and cysteine protease A5 indicated that cell death and cytoskeleton disorganization depend upon parasite adhesion and amoebic cysteine proteinase activities. The data establish a relation between cytotoxic effects of E. histolytica and altered human target cell adhesion and suggest that interference with adhesion signalling triggers endothelial cell retraction and death. Understanding the roles of integrin signalling in endothelial cells will provide clues to unravel host-pathogen interactions during amoebic liver infection. PMID:21624031

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

    PubMed

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

    2015-10-01

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

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

    PubMed

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

    2012-01-01

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

  6. A comprehensive characterization of membrane vesicles released by autophagic human endothelial cells.

    PubMed

    Pallet, Nicolas; Sirois, Isabelle; Bell, Christina; Hanafi, Laïla-Aïcha; Hamelin, Katia; Dieudé, Mélanie; Rondeau, Christiane; Thibault, Pierre; Desjardins, Michel; Hebert, Marie-Josée

    2013-04-01

    The stress status of the apoptotic cell can promote phenotypic changes that have important consequences on the immunogenicity of the dying cell. Autophagy is one of the biological processes activated in response to a stressful condition. It is an important mediator of intercellular communications, both by regulating the unconventional secretion of molecules, including interleukin 1β, and by regulating the extracellular release of ATP from early stage apoptotic cells. Additionally, autophagic components can be released in a caspase-dependent manner by serum-starved human endothelial cells that have engaged apoptotic and autophagic processes. The nature and the components of the extracellular vesicles released by dying autophagic cells are not known. In this study, we have identified extracellular membrane vesicles that are released by human endothelial cells undergoing apoptosis and autophagy, and characterized their biochemical, ultrastructural, morphological properties as well as their proteome. These extracellular vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho-associated, coiled-coil containing protein kinase 1 activation. Instead, they are enriched with autophagosomes and mitochondria and convey various danger signals, including ATP, suggesting that they could be involved in the modulation of innate immunity. PMID:23436686

  7. Prunella vulgaris L. Upregulates eNOS expression in human endothelial cells.

    PubMed

    Xia, Ning; Bollinger, Larissa; Steinkamp-Fenske, Katja; Förstermann, Ulrich; Li, Huige

    2010-01-01

    The purported effects of "circulation-improving" herbs used in traditional Chinese medicine (TCM) show striking similarities with the vascular actions of nitric oxide (NO) produced by the endothelial NO synthase (eNOS). We have previously reported that Salviae miltiorrhizae radix and Zizyphi spinosae semen upregulate eNOS expression. In the present study, we studied the effect on eNOS gene expression of 15 Chinese herbs with potential effects on the vasculature, and identified Prunella vulgaris L. (PVL) (flowering spike) as a potent eNOS-upregulating agent. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVEC), an aqueous extract of PVL increased eNOS promoter activity, eNOS mRNA and protein expressions, as well as NO production in concentration- and time-dependent manners. We have previously shown that ursolic acid (a constituent of Salviae miltiorrhizae radix), betulinic acid (a compound present in Zizyphi spinosae semen), luteolin and cynaroside (ingredients of artichoke, Cynara scolymus L.) are capable of enhancing eNOS gene expression. These compounds are also present in significant quantities in PVL. Thus, PVL contains active principles that stimulate human eNOS gene expression, and such compounds may have therapeutic potential against cardiovascular diseases. PMID:20503475

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

    PubMed Central

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

    1999-01-01

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

  9. Experimental Arrest of Cerebral Blood Flow in Human Subjects

    PubMed Central

    Smith, Brian A.; Clayton, Ellen Wright; Robertson, David

    2013-01-01

    Loss of consciousness in pilots during rapid ascent after bombing missions was a major problem in World War II, and experiments were undertaken to study the cause of this phenomenon. Postulating impaired cerebral blood flow as a likely mechanism, the investigators developed a neck device, the KRA Cuff, which when inflated could shut off blood supply to the brain. With cessation of blood flow for up to 100 seconds, the investigators observed a sequence of responses, including unconsciousness, followed by dilated pupils, tonic/clonic movements, loss of bladder and eventually bowel control, and appearance of pathological reflexes. This study, carried out in prisoners and patients with schizophrenia in 1941–42, largely disappeared from public discourse for a number of years. It has received occasional attention subsequently and been considered controversial. Recently discovered records, including extensive written and photographic data from the studies, shed new light on the methods and motives of the research team. We describe here this new information and its implications for the scientific and ethical assessment of the study. PMID:21532128

  10. Capillary morphogenesis during human endothelial cell invasion of three-dimensional collagen matrices.

    PubMed

    Davis, G E; Black, S M; Bayless, K J

    2000-09-01

    Here, we describe assay systems that utilize serum-free defined media to evaluate capillary morphogenesis during human endothelial cell (EC) invasion of three-dimensional collagen matrices. ECs invade these matrices over a 1-3-d period to form capillary tubes. Blocking antibodies to the alpha2beta1 integrin interfere with invasion and morphogenesis while other integrin blocking antibodies do not. Interestingly, we observed increased invasion of ECs toward a population of underlying ECs undergoing morphogenesis. In addition, we have developed assays on microscope slides that display the invasion process horizontally, thereby enhancing our ability to image these events. Thus far, we have observed intracellular vacuoles that appear to regulate the formation of capillary lumens, and extensive cell processes that facilitate the interconnection of ECs during morphogenic events. These assays should enable further investigation of the morphologic steps and molecular events controlling human capillary tube formation in three-dimensional extracellular matrices.

  11. 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.

  12. Phthalimide neovascular factor 1 (PNF1) modulates MT1-MMP activity in human microvascular endothelial cells.

    PubMed

    Wieghaus, Kristen A; Gianchandani, Erwin P; Neal, Rebekah A; Paige, Mikell A; Brown, Milton L; Papin, Jason A; Botchwey, Edward A

    2009-07-01

    We are creating synthetic pharmaceuticals with angiogenic activity and potential to promote vascular invasion. We previously demonstrated that one of these molecules, phthalimide neovascular factor 1 (PNF1), significantly expands microvascular networks in vivo following sustained release from poly(lactic-co-glycolic acid) (PLAGA) films. In addition, to probe PNF1 mode of action, we recently applied a novel pathway-based compendium analysis to a multi-timepoint, controlled microarray data set of PNF1-treated (vs. control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-alpha) and, subsequently, transforming growth factor-beta (TGF-beta) signaling networks by PNF1. Here we validate this microarray data set with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Subsequently, we probe this data set and identify three specific TGF-beta-induced genes with regulation by PNF1 conserved over multiple timepoints-amyloid beta (A4) precursor protein (APP), early growth response 1 (EGR-1), and matrix metalloproteinase 14 (MMP14 or MT1-MMP)-that are also implicated in angiogenesis. We further focus on MMP14 given its unique role in angiogenesis, and we validate MT1-MMP modulation by PNF1 with an in vitro fluorescence assay that demonstrates the direct effects that PNF1 exerts on functional metalloproteinase activity. We also utilize endothelial cord formation in collagen gels to show that PNF1-induced stimulation of endothelial cord network formation in vitro is in some way MT1-MMP-dependent. Ultimately, this new network analysis of our transcriptional footprint characterizing PNF1 activity 1-48 h post-supplementation in HMVECs coupled with corresponding validating experiments suggests a key set of a few specific targets that are involved in PNF1 mode of action and important for successful promotion of the neovascularization that we have observed by the drug in vivo. PMID:19326468

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

    PubMed Central

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

    2013-01-01

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

  14. The inhibitory effect of simvastatin and aspirin on histamine responsiveness in human vascular endothelial cells.

    PubMed

    Absi, Mais; Bruce, Jason I; Ward, Donald T

    2014-04-01

    Statins and aspirin deliver well-established cardiovascular benefits resulting in their increased use as combined polypills to decrease risk of stroke and heart disease. However, the direct endothelial effect of combined statin/aspirin cotreatment remains unclear. Histamine is an inflammatory mediator that increases vascular permeability, and so we examined the effect of treating human umbilical vein endothelial cells (HUVECs) for 24 h with 1 μM simvastatin and 100 μM aspirin on histamine responsiveness. Subsequent histamine (1 μM) challenge increased intracellular calcium (Ca(2+)i) concentration, an effect that was significantly inhibited by combined simvastatin/aspirin pretreatment but not when then the compounds were given separately, even at 10-fold higher concentrations. In contrast, the Ca(2+)i mobilization response to ATP challenge (10 μM) was not inhibited by combined simvastatin/aspirin pretreatment. The H1 receptor antagonist pyrilamine significantly inhibited both histamine-induced Ca(2+)i mobilization and extracellular signal-regulated kinase (ERK) activation, whereas ranitidine (H2 receptor antagonist) was without effect. However, combined simvastatin/aspirin pretreatment failed to decrease H1 receptor protein expression ruling out receptor downregulation as the mechanism of action. Histamine-induced ERK activation was also inhibited by atorvastatin pretreatment, while simvastatin further inhibited histamine-induced vascular endothelial cadherin phosphorylation as well as altered HUVEC morphology and inhibited actin polymerization. Therefore, in addition to the known therapeutic benefits of statins and aspirin, here we provide initial cellular evidence that combined statin/aspirin treatment inhibits histamine responsiveness in HUVECs.

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

    PubMed Central

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

    2013-01-01

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

  16. α-Tocopherol suppresses antiangiogenic effect of δ-tocotrienol in human umbilical vein endothelial cells.

    PubMed

    Shibata, Akira; Nakagawa, Kiyotaka; Tsuduki, Tsuyoshi; Miyazawa, Teruo

    2015-04-01

    Recently, tocotrienol (T3), a less well-known form of vitamin E, has gained attention as a potent hypocholesterolemic, anticancer and antiangiogenic agent. However, tocopherol (Toc), a commonly consumed form of vitamin E, has been reported to inhibit T3's effects (hypocholesterolemic and anticancer activity). There has been no report on Toc's effect on the antiangiogenic action of T3 during cotreatment. The aim of this study is to determine if and to what extent Toc affects the antiangiogenic effects of δ-T3 (the most potent isomer). This was achieved through cotreatment of human umbilical vein endothelial cells (HUVECs) with δ-T3 and Toc (α-, β-, γ- and δ-isomers). Toc, especially α-Toc, attenuated δ-T3-induced cytotoxicity and tube degradation in cotreated HUVECs, while α-Toc treatments did not exhibit any effects. A rat aortic ring assay also showed inhibition of δ-T3's antiangiogenic effects by α-Toc. Further, in HUVEC study, cell cycle arrest and proapoptotic gene expression (p21, p27, caspase-3 and caspase-9) which were induced by δ-T3 were decreased by α-Toc treatment. α-Toc also suppressed δ-T3-induced dephosphorylation of vascular endothelial growth factor receptor 2 and Akt pathway proteins. Additionally, uptake of δ-T3 into HUVECs was decreased by α-Toc. Here we demonstrate that α-Toc not only has little antiangiogenic effect on endothelial cells but also reduces the antiangiogenic effects of δ-T3 through modulation of its cellular uptake and of relevant signal transduction pathways. Understanding T3's antiangiogenic effects and interaction with Toc is important for developing medical applications.

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

    PubMed Central

    2013-01-01

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

  18. Monoclonal Antibody against Angiotensin-Converting Enzyme: Its Use as a Marker for Murine, Bovine, and Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Auerbach, R.; Alby, L.; Grieves, J.; Joseph, J.; Lindgren, C.; Morrissey, L. W.; Sidky, Y. A.; Tu, M.; Watt, S. L.

    1982-12-01

    A monoclonal antibody has been prepared against rat angiotensin-converting enzyme (ACE). By selection for antibody binding to endothelial cells of bovine rather than rat origin we have obtained a reagent that has broad cross-species binding properties and that can at the same time serve as a useful marker for the surface of endothelial cells. The IgM-producing clone that we have established, α -ACE 3.1.1, has been grown in ascites form to yield ascites fluid that binds selectively to immobilized ACE at a >1:10,000 dilution. By use of enzyme-linked immunosorbent assays, immunofluorescence histology, and flow cytometry, we have demonstrated the presence of ACE on endothelial cells of murine, bovine, and human origin. By means of a fluorescence-activated cell sorter (FACS-IV) we have been able to selectively isolate viable endothelial cells from a mixture of endothelial cells and fibroblasts. We believe the antibody will be useful not only for the selection and in vitro cultivation of endothelial cells but also as a tool for the identification and pharmacological study of ACE.

  19. Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis

    SciTech Connect

    Sun, Hui-Yan; Wei, Shu-Ping; Xu, Rui-Cheng; Xu, Peng-Xiao; Zhang, Wen-Cheng

    2010-05-07

    Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.

  20. Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

    PubMed

    Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

    2002-11-01

    Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

  1. Microarray Analysis of Cell Cycle Gene Expression in Adult Human Corneal Endothelial Cells

    PubMed Central

    Ha Thi, Binh Minh; Campolmi, Nelly; He, Zhiguo; Pipparelli, Aurélien; Manissolle, Chloé; Thuret, Jean-Yves; Piselli, Simone; Forest, Fabien; Peoc'h, Michel; Garraud, Olivier; Gain, Philippe; Thuret, Gilles

    2014-01-01

    Corneal endothelial cells (ECs) form a monolayer that controls the hydration of the cornea and thus its transparency. Their almost nil proliferative status in humans is responsible, in several frequent diseases, for cell pool attrition that leads to irreversible corneal clouding. To screen for candidate genes involved in cell cycle arrest, we studied human ECs subjected to various environments thought to induce different proliferative profiles compared to ECs in vivo. Donor corneas (a few hours after death), organ-cultured (OC) corneas, in vitro confluent and non-confluent primary cultures, and an immortalized EC line were compared to healthy ECs retrieved in the first minutes of corneal grafts. Transcriptional profiles were compared using a cDNA array of 112 key genes of the cell cycle and analysed using Gene Ontology classification; cluster analysis and gene map presentation of the cell cycle regulation pathway were performed by GenMAPP. Results were validated using qRT-PCR on 11 selected genes. We found several transcripts of proteins implicated in cell cycle arrest and not previously reported in human ECs. Early G1-phase arrest effectors and multiple DNA damage-induced cell cycle arrest-associated transcripts were found in vivo and over-represented in OC and in vitro ECs. Though highly proliferative, immortalized ECs also exhibited overexpression of transcripts implicated in cell cycle arrest. These new effectors likely explain the stress-induced premature senescence that characterizes human adult ECs. They are potential targets for triggering and controlling EC proliferation with a view to increasing the cell pool of stored corneas or facilitating mass EC culture for bioengineered endothelial grafts. PMID:24747418

  2. Regulation of Thrombomodulin Expression and Release in Human Aortic Endothelial Cells by Cyclic Strain

    PubMed Central

    Davenport, Colin; Murphy, Ronan P.; Cummins, Philip M.

    2014-01-01

    Background and Objectives Thrombomodulin (TM), an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain) intrinsic to endothelial-mediated vascular homeostasis. Methods This study employed human aortic endothelial cells (HAECs) to investigate the effects of equibiaxial cyclic strain (7.5%, 60 cycles/min, 24 hrs), and to a lesser extent, laminar shear stress (10 dynes/cm2, 24 hrs), on TM expression and release. Time-, dose- and frequency-dependency studies were performed. Results Our initial studies demonstrated that cyclic strain strongly downregulated TM expression in a p38- and receptor tyrosine kinase-dependent manner. This was in contrast to the upregulatory effect of shear stress. Moreover, both forces significantly upregulated TM release over a 48 hr period. With continuing focus on the cyclic strain-induced TM release, we noted both dose (0–7.5%) and frequency (0.5–2.0 Hz) dependency, with no attenuation of strain-induced TM release observed following inhibition of MAP kinases (p38, ERK-1/2), receptor tyrosine kinase, or eNOS. The concerted impact of cyclic strain and inflammatory mediators on TM release from HAECs was also investigated. In this respect, both TNFα (100 ng/ml) and ox-LDL (10–50 µg/ml) appeared to potentiate strain-induced TM release. Finally, inhibition of neither MMPs (GM6001) nor rhomboids (3,4-dichloroisocoumarin) had any effect on strain-induced TM release. However, significantly elevated levels (2.1 fold) of TM were observed in isolated microparticle fractions following 7.5% strain for 24 hrs. Conclusions A preliminary in vitro investigation into the effects of cyclic strain on TM in HAECs is presented

  3. Proinflammatory and prothrombotic effects on human vascular endothelial cells of Immune-cell-derived LIGHT

    PubMed Central

    2009-01-01

    Objective LIGHT (TNFSF 14) belongs to the tumor necrosis factor superfamily and is expressed by activated T cells as well as various types of antigen presenting cells. LIGHT binds to its cellular receptors TR2 and LTßR and has a co-stimulatory role in T cell activation. Here, we compared the relative expression of LIGHT in different immune cells and the biological activity of immune cell-derived LIGHT on endothelial cells. Methods and Results Surface expression of LIGHT and mRNA production by PBMC and isolated T cells (CD4+ or CD8+) significantly increased after stimulation with PMA (Phorbolester-12-Myristat-13-Acetat) + ionomycin. No LIGHT expression on PMA stimulated monocytes or monocytic-like THP-1 cells could be detected; differentiation of monocytes and THP-1 cells into macrophages, however, resulted in up-regulation of LIGHT. Supernatants of stimulated T cells contained higher concentrations of soluble LIGHT than macrophage supernatants normalized to cell numbers; release of soluble LIGHT was found to be dependent on metalloproteinase activity. Size determination of released soluble LIGHT by size exclusion chromatography revealed a molecular mass of ~60 kDa, suggesting a trimeric form. Released soluble LIGHT induced expression of proinflammatory antigens ICAM-1, tissue factor and IL-8 in human endothelial cells and caused apoptosis of IFN-γ pretreated endothelial cells. Soluble LIGHT was detected at low levels in sera of healthy controls and was significantly enhanced in sera of patients with chronic hepatitis C and rheumatoid arthritis (24.93 ± 9.41 vs.129.53 ± 49.14 and 172.13 ± 77.64; p < 0.0005). Conclusion These findings suggest that among immune cells activated T lymphocytes are the main source of soluble LIGHT with released amounts of soluble LIGHT markedly higher compared to platelets. Immune cell-derived membrane-bound and soluble trimeric LIGHT is biologically active, inducing proinflammatory changes in endothelial cells. Enhanced plasma

  4. Defining minimum essential factors to derive highly pure human endothelial cells from iPS/ES cells in an animal substance-free system.

    PubMed

    Wu, Yu-Ting; I-Shing Yu; Tsai, Kuen-Jer; Shih, Chien-Yu; Hwang, Shiaw-Min; Su, Ih-Jen; Chiang, Po-Min

    2015-04-13

    It is desirable to obtain unlimited supplies of endothelial cells for research and therapeutics. However, current methods of deriving endothelial cells from humans suffer from issues, such as limited supplies, contamination from animal substances, and lengthy/complicated procedures. In this article we developed a way to differentiate human iPS and ES cells to highly pure endothelial cells in 5 days. The chemically defined system is robust, easy to perform, and free of animal substances. Using the system, we verified that combined TGFβ and canonical Wnt agonists are essential and sufficient for iPS/ES cell-to-mesoderm transition. Besides, VEGF-KDR signaling alone is required for endothelial formation at high density while supplementation with FGF allows for colonial endothelial differentiation. Finally, anti-adsorptive agents could enrich the endothelial output by allowing selective attachment of the endothelial precursors. The system was validated to work on multiple iPS/ES cells lines to produce endothelial cells capable of forming capillary-like structures in vitro and integrating into host vasculature in vivo. In sum, the simple yet robust differentiation system permits the unlimited supply of human endothelial cells. The defined and animal substance-free nature of the system is compatible with clinical applications and characterization of endothelial differentiation in an unbiased manner.

  5. Defining Minimum Essential Factors to Derive Highly Pure Human Endothelial Cells from iPS/ES Cells in an Animal Substance-Free System

    PubMed Central

    Wu, Yu-Ting; I.-Shing Yu; Tsai, Kuen-Jer; Shih, Chien-Yu; Hwang, Shiaw-Min; Su, Ih-Jen; Chiang, Po-Min

    2015-01-01

    It is desirable to obtain unlimited supplies of endothelial cells for research and therapeutics. However, current methods of deriving endothelial cells from humans suffer from issues, such as limited supplies, contamination from animal substances, and lengthy/complicated procedures. In this article we developed a way to differentiate human iPS and ES cells to highly pure endothelial cells in 5 days. The chemically defined system is robust, easy to perform, and free of animal substances. Using the system, we verified that combined TGFβ and canonical Wnt agonists are essential and sufficient for iPS/ES cell-to-mesoderm transition. Besides, VEGF-KDR signaling alone is required for endothelial formation at high density while supplementation with FGF allows for colonial endothelial differentiation. Finally, anti-adsorptive agents could enrich the endothelial output by allowing selective attachment of the endothelial precursors. The system was validated to work on multiple iPS/ES cells lines to produce endothelial cells capable of forming capillary-like structures in vitro and integrating into host vasculature in vivo. In sum, the simple yet robust differentiation system permits the unlimited supply of human endothelial cells. The defined and animal substance-free nature of the system is compatible with clinical applications and characterization of endothelial differentiation in an unbiased manner. PMID:25864432

  6. High glucose and palmitate increases bone morphogenic protein 4 expression in human endothelial cells

    PubMed Central

    Hong, Oak-Kee; Yoo, Soon-Jib; Son, Jang-Won; Kim, Mee-Kyoung; Baek, Ki-Hyun; Song, Ki-Ho; Cha, Bong-Yun; Jo, Hanjoong

    2016-01-01

    Here, we investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) aff ect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this eff ect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a proinflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of infl ammatory adhesion molecules and ROS production. Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL. PMID:26937213

  7. In Vitro Effects of Hollow Gold Nanoshells on Human Aortic Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Gu, Chunrong; Wu, Hengfang; Ge, Gaoyuan; Li, Xiongzhi; Guo, Zhirui; Bian, Zhiping; Xu, Jindan; Lu, Hua; Chen, Xiangjian; Yang, Di

    2016-09-01

    Gold nanoparticles are emerging as promising biomedical tools due to their unique nanoscale characteristics. Our purpose was to synthesize a hollow-shaped gold nanoparticle and to investigate its effect on human aortic endothelial cells (HAECs) in vitro. Hollow gold nanoshells with average 35-nm diameters and 10-nm shell thickness were obtained by galvanic replacement using quasi-spherical nanosilver as sacrifice-template. Our results showed that hollow gold nanoshells in the culture medium could be internalized into the cytoplasm of HAECs. No cytotoxicity effect of hollow gold nanoshells on HAECs was observed within the test concentrations (0-0.8 μg/mL) and test exposure period (0-72 h) by tetrazolium dye assay. Meanwhile, the release of cell injury biomarker, lactate dehydrogenase, was not significantly higher than that from control cells (without hollow gold nanoshells). The concentrations of vasodilators, nitric oxide, and prostacyclin I-2 were not changed, but the vasoconstrictor endothelin-1 was decreased by hollow gold nanoshells treatment in HAECs. HAECs exposed to hollow gold nanoshells resulted in suppressing expressions of genes involved in apoptosis and activating expressions of genes of adhesion molecules. Moreover, we demonstrated by in vitro endothelial tube formation that hollow gold nanoshells (0.8 μg/mL) could not inhibit angiogenesis by the HAECs. Altogether, these results indicate that the structure and major function of HAECs would not be disrupted by hollow gold nanoshell treatment.

  8. Caveolae may enable albumin to enter human renal glomerular endothelial cells.

    PubMed

    Moriyama, Takahito; Takei, Takashi; Itabashi, Mitsuyo; Uchida, Keiko; Tsuchiya, Ken; Nitta, Kosaku

    2015-06-01

    Caveolae on human renal glomerular endothelial cells (HRGECs) are increased in glomerular disease and correlate with the degree of albuminuria. To assess the mechanism by which caveolae contribute to albuminuria, we investigated whether albumin enters into HRGECs through caveolae. HRGECs were incubated with Alexa Fluor 488 labeled BSA or transferrin, followed by immunofluorescence localization with antibody to caveolin-1 (Cav-1), the main structural protein of caveolae, or clathrin, the major structural protein of clathrin coated pits, to assess whether BSA colocalized with Cav-1. HRGECs were also incubated with albumin and caveolae disrupting agents, including methyl beta cyclodextrin (MBCD) and nystatin, to determine whether disrupting caveolae interfered with albumin endocytosis into HRGECs. HRGECs were also incubated with albumin after transfection with Cav-1 small interfering RNAs (siRNAs). Labeled BSA colocalized with Cav-1, but not with clathrin. In contrast, labeled transferrin colocalized with clathrin, but not with Cav-1. Incubation of HRGECs with MBCD or nystatin, or transfection with Cav-1 siRNA, significantly reduced the intracellular amounts of albumin and Cav-1, relative to normal HRGECs, as shown by western blotting and immunofluorescence. These findings indicate that albumin enters HRGECs through the caveolae, suggesting that caveolae play an important role in the pathogenesis of albuminuria by providing a pathway through which albumin can enter glomerular endothelial cells.

  9. Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells.

    PubMed

    Neri, Tommaso; Pergoli, Laura; Petrini, Silvia; Gravendonk, Lotte; Balia, Cristina; Scalise, Valentina; Amoruso, Angela; Pedrinelli, Roberto; Paggiaro, Pierluigi; Bollati, Valentina; Celi, Alessandro

    2016-04-01

    Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells. Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution. Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50-1000 nm). Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity. PMID:26876346

  10. Improved Cryopreservation of Human Umbilical Vein Endothelial Cells: A Systematic Approach

    PubMed Central

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

    2016-01-01

    Cryopreservation of human umbilical vein endothelial cells (HUVECs) facilitated their commercial availability for use in vascular biology, tissue engineering and drug delivery research; however, the key variables in HUVEC cryopreservation have not been comprehensively studied. HUVECs are typically cryopreserved by cooling at 1 °C/min in the presence of 10% dimethyl sulfoxide (DMSO). We applied interrupted slow cooling (graded freezing) and interrupted rapid cooling with a hold time (two-step freezing) to identify where in the cooling process cryoinjury to HUVECs occurs. We found that linear cooling at 1 °C/min resulted in higher membrane integrities than linear cooling at 0.2 °C/min or nonlinear two-step freezing. DMSO addition procedures and compositions were also investigated. By combining hydroxyethyl starch with DMSO, HUVEC viability after cryopreservation was improved compared to measured viabilities of commercially available cryopreserved HUVECs and viabilities for HUVEC cryopreservation studies reported in the literature. Furthermore, HUVECs cryopreserved using our improved procedure showed high tube forming capability in a post-thaw angiogenesis assay, a standard indicator of endothelial cell function. As well as presenting superior cryopreservation procedures for HUVECs, the methods developed here can serve as a model to optimize the cryopreservation of other cells. PMID:27708349

  11. Thioredoxin reductase 1 upregulates MCP-1 release in human endothelial cells

    SciTech Connect

    Liu, Zhen-Bo; Shen, Xun

    2009-09-04

    To know if thioredoxin reductase 1 (TrxR1) plays a role in antioxidant defense mechanisms against atherosclerosis, effect of TrxR1 on expression/release of monocyte chemoattractant protein (MCP-1) was investigated in activated human endothelial-like EAhy926 cells. The MCP-1 release and expression, cellular generation of reactive oxygen species (ROS), nuclear translocation and DNA-binding activity of NF-{kappa}B subunit p65 were assayed in cells either overexpressing recombinant TrxR1 or having their endogenous TrxR1 knocked down. It was found that overexpression of TrxR1 enhanced, while knockdown of TrxR1 reduced MCP-1 release and expression. Upregulation of MCP-1 by TrxR1 was associated with increasing generation of intracellular ROS generation, enhanced nuclear translocation and DNA-binding activity of NF-{kappa}B. Assay using NF-{kappa}B reporter revealed that TrxR1 upregulated transcriptional activity of NF-{kappa}B. This study suggests that TrxR1 enhances ROS generation, NF-{kappa}B activity and subsequent MCP-1 expression in endothelial cells, and may promote rather than prevent vascular endothelium from forming atherosclerotic plaque.

  12. In Vitro Effects of Hollow Gold Nanoshells on Human Aortic Endothelial Cells.

    PubMed

    Gu, Chunrong; Wu, Hengfang; Ge, Gaoyuan; Li, Xiongzhi; Guo, Zhirui; Bian, Zhiping; Xu, Jindan; Lu, Hua; Chen, Xiangjian; Yang, Di

    2016-12-01

    Gold nanoparticles are emerging as promising biomedical tools due to their unique nanoscale characteristics. Our purpose was to synthesize a hollow-shaped gold nanoparticle and to investigate its effect on human aortic endothelial cells (HAECs) in vitro. Hollow gold nanoshells with average 35-nm diameters and 10-nm shell thickness were obtained by galvanic replacement using quasi-spherical nanosilver as sacrifice-template. Our results showed that hollow gold nanoshells in the culture medium could be internalized into the cytoplasm of HAECs. No cytotoxicity effect of hollow gold nanoshells on HAECs was observed within the test concentrations (0-0.8 μg/mL) and test exposure period (0-72 h) by tetrazolium dye assay. Meanwhile, the release of cell injury biomarker, lactate dehydrogenase, was not significantly higher than that from control cells (without hollow gold nanoshells). The concentrations of vasodilators, nitric oxide, and prostacyclin I-2 were not changed, but the vasoconstrictor endothelin-1 was decreased by hollow gold nanoshells treatment in HAECs. HAECs exposed to hollow gold nanoshells resulted in suppressing expressions of genes involved in apoptosis and activating expressions of genes of adhesion molecules. Moreover, we demonstrated by in vitro endothelial tube formation that hollow gold nanoshells (0.8 μg/mL) could not inhibit angiogenesis by the HAECs. Altogether, these results indicate that the structure and major function of HAECs would not be disrupted by hollow gold nanoshell treatment. PMID:27624340

  13. Differential Cytokine Responses in Human and Mouse Lymphatic Endothelial Cells to Cytokines in Vitro

    PubMed Central

    Chaitanya, G.V.; Franks, S.E.; Cromer, W.; Wells, S.R.; Bienkowska, M.; Jennings, M.H.; Ruddell, A.; Ando, T.; Wang, Y.; Gu, Y.; Sapp, M.; Mathis, J.M.; Jordan, P.A.; Minagar, A.

    2010-01-01

    Abstract Background Inflammatory cytokines dysregulate microvascular function, yet how cytokines affect lymphatic endothelial cells (LEC) are unclear. Methods and Results We examined effects of TNF-α, IL-1β, and IFN-γ on LEC proliferation, endothelial cell adhesion molecule (ECAM) expression, capillary formation, and barrier changes in murine (SV-LEC) and human LECs (HMEC-1a). Results All cytokines induced ICAM-1, VCAM-1, MAdCAM-1, and E-selectin in SV-LECs; TNF-α, IL-1β and IFN-γ induced ECAMs (but not MAdCAM-1) in HMEC-1a. IL-1β increased, while IFN-γ and TNF-α reduced SV-LEC proliferation. While TNF-α induced, IFN-γ decreased, and IL-1β did not show any effect on HMEC-1a proliferation. TNF-α, IL-1β, and IFN-γ each reduced capillary formation in SV-LEC and in HMEC-1a. TNF-α and IL-1β reduced barrier in SV-LEC and HMEC-1a; IFN-γ did not affect SV-LEC barrier, but enhanced HMEC-1a barrier. Inflammatory cytokines alter LEC growth, activation and barrier function in vitro and may disturb lymphatic clearance increasing tissue edema in vivo. Conclusion Therapies that maintain or restore lymphatic function (including cytokines blockade), may represent important strategies for limiting inflammation. PMID:20863268

  14. Knockdown of ezrin suppresses the migration and angiogenesis of human umbilical vein endothelial cells in vitro.

    PubMed

    Zhao, Liang-ping; Huang, Lei; Tian, Xun; Liang, Feng-qi; Wei, Jun-cheng; Zhang, Xian; Li, Sha; Zhang, Qing-hua

    2016-04-01

    Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells (ECs) are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radixin moesin (ERM) family members are key regulators of cellular activities such as adhesion, morphogenetic change, and migration. We hypothesized that ezrin, one of the ERM family members, may play important roles in ECs organization during angiogenesis, and new vessels formation in preexisting tissues. To test this hypothesis, in this study, we investigated the effects of ezrin gene silencing on the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs were transfected with plasmids with ezrin-targeting short hairpin RNA by using the lipofectamine-2000 system. Wound assay in vitro and three-dimensional culture were used to detect the migration and angiogenesis capacity of HUVECs. The morphological changes of transfected cells were observed by confocal and phase contrast microscopy. Our results demonstrated that the decreased expression of ezrin in HUVECs significantly induced the morphogenetic changes and cytoskeletal reorganization of the transfected cells, and also reduced cell migration and angiogenesis capacity in vitro, suggesting that ezrin play an important role in the process of HUVECs migration and angiogenesis. PMID:27072970

  15. Improved Cryopreservation of Human Umbilical Vein Endothelial Cells: A Systematic Approach

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

    Cryopreservation of human umbilical vein endothelial cells (HUVECs) facilitated their commercial availability for use in vascular biology, tissue engineering and drug delivery research; however, the key variables in HUVEC cryopreservation have not been comprehensively studied. HUVECs are typically cryopreserved by cooling at 1 °C/min in the presence of 10% dimethyl sulfoxide (DMSO). We applied interrupted slow cooling (graded freezing) and interrupted rapid cooling with a hold time (two-step freezing) to identify where in the cooling process cryoinjury to HUVECs occurs. We found that linear cooling at 1 °C/min resulted in higher membrane integrities than linear cooling at 0.2 °C/min or nonlinear two-step freezing. DMSO addition procedures and compositions were also investigated. By combining hydroxyethyl starch with DMSO, HUVEC viability after cryopreservation was improved compared to measured viabilities of commercially available cryopreserved HUVECs and viabilities for HUVEC cryopreservation studies reported in the literature. Furthermore, HUVECs cryopreserved using our improved procedure showed high tube forming capability in a post-thaw angiogenesis assay, a standard indicator of endothelial cell function. As well as presenting superior cryopreservation procedures for HUVECs, the methods developed here can serve as a model to optimize the cryopreservation of other cells.

  16. Biocompatibility of pure titanium modified by human endothelial cell-derived extracellular matrix

    NASA Astrophysics Data System (ADS)

    Xue, Xiaoqing; Wang, Jin; Zhu, Ying; Tu, Qiufen; Huang, Nan

    2010-04-01

    Extracellular matrix (ECM) used to modify biomaterial surface is a promising method for improving cardiovascular material hemocompatibility. In the present work, human umbilical vein endothelial cells (HUVECs) are cultured and native ECM is obtained on pure titanium surface. Fourier infrared spectrum (FTIR) test proves the existence of amide I and amide II band on the modified titanium surface. X-ray photoelectron spectroscopy (XPS) further confirms the chemical composition and binding types of the ECM proteins on the titanium substrate. The results of light microscopy and atomic force microscopy (AFM) exhibit the morphology of HUVEC derived ECM. There are higher water contact angles on the ECM modified samples. Furthermore, some ECM components, including fibronectin (FN), laminin (LN) and type IV collagen (IV-COL) are presented on ECM-covered titanium surface by immunofluorescence staining. The biological behavior of cultured HUVECs and adherent platelets on different samples are investigated by in vitro HUVECs culture and platelet adhesion. Cells exhibit better morphology and their proliferation ability greatly improve on the ECM-covered titanium. At the same time, the platelet adhesion and spreading are inhibited on ECM-covered titanium surface. These investigations demonstrate that ECM produced by HUVECs cannot only improve adhesion and proliferation ability of endothelial cell but also inhibit adhesion and activation of platelets. Thus, the approach described here may provide a basis for preparation of modified surface in cardiovascular implants application.

  17. High glucose and palmitate increases bone morphogenic protein 4 expression in human endothelial cells.

    PubMed

    Hong, Oak-Kee; Yoo, Soon-Jib; Son, Jang-Won; Kim, Mee-Kyoung; Baek, Ki-Hyun; Song, Ki-Ho; Cha, Bong-Yun; Jo, Hanjoong; Kwon, Hyuk-Sang

    2016-03-01

    Here, we investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) aff ect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this eff ect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a proinflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of infl ammatory adhesion molecules and ROS production. Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL. PMID:26937213

  18. Concise Review: Tissue-Specific Microvascular Endothelial Cells Derived from Human Pluripotent Stem Cells

    PubMed Central

    Wilson, Hannah K.; Canfield, Scott G.; Shusta, Eric V.; Palecek, Sean P.

    2014-01-01

    Accumulating evidence suggests that endothelial cells (ECs) display significant heterogeneity across tissue types, playing an important role in tissue regeneration and homeostasis. Recent work demonstrating the derivation of tissue-specific microvascular endothelial cells (TS-MVECs) from human pluripotent stem cells (hPSCs) has ignited the potential to generate tissue-specific models which may be applied to regenerative medicine and in vitro modeling applications. Here we review techniques by which hPSC-derived TS-MVECs have been made to date and discuss how current hPSC-EC differentiation protocols may be directed towards tissue-specific fates. We begin by discussing the nature of EC tissue specificity in vivo and review general hPSC-EC differentiation protocols generated over the last decade. Finally, we describe how specificity can be integrated into hPSC-EC protocols to generate hPSC-derived TS-MVECs in vitro, including EC and parenchymal cell co-culture, directed differentiation, and direct reprogramming strategies. PMID:25070152

  19. Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells.

    PubMed

    Neri, Tommaso; Pergoli, Laura; Petrini, Silvia; Gravendonk, Lotte; Balia, Cristina; Scalise, Valentina; Amoruso, Angela; Pedrinelli, Roberto; Paggiaro, Pierluigi; Bollati, Valentina; Celi, Alessandro

    2016-04-01

    Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells. Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution. Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50-1000 nm). Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity.

  20. Calcitonin gene-related peptide stimulates proliferation of human endothelial cells

    SciTech Connect

    Haegerstrand, A.; Larsson, O. ); Dalsgaard, C.J. Karolinska Hospital, Stockholm ); Jonzon, B. ); Nilsson, J. )

    1990-05-01

    The effects of the vasoactive perivascular neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) on proliferation of cultured human umbilical vein endothelial cells (HUVECs) were investigated. CGRP was shown to increase both cell number and DNA synthesis, whereas NKA, NPY, and VIP were ineffective. {sup 125}I-labeled CGRP was shown to bind to HUVECs and this binding was displaced by addition of unlabeled CGRP, suggesting the existence of specific CGRP receptors. The effect of CGRP on formation of adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) and inositol phosphates (InsP), two intracellular messengers known to be involved in regulation of cell proliferation, was investigated. CGRP stimulated cAMP formation but was without effect on the formation of InsP. Proliferation, as well as cAMP formation, was also stimulated by cholera toxin. Basic fibroblast growth factor stimulated growth without affecting cAMP or InsP formation, whereas thrombin, which increased InsP formation, did not stimulate proliferation. The authors thus suggest that CGRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physicological and pathophysiological events such as ischemia, inflammation, and wound healing.

  1. Tissue factor: A potent stimulator of Von Willebrand factor synthesis by human umbilical vein endothelial cells

    PubMed Central

    Meiring, Muriel; Allers, W.; Le Roux, E.

    2016-01-01

    Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of Von Willebrand factor. The aim of this study was to examine the effects of the inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) and the coagulation factors, tissue factor and thrombin on the release and cleavage potential of ultra-large von Willebrand factor (ULVWF) and its cleavage protease by cultured human umbilical vein endothelial cells (HUVEC). HUVEC were treated with IL-6, IL-8, and TNF-α, tissue factor (TF) and thrombin, and combinations thereof for 24 hours under static conditions. The cells were then exposed to shear stress after which the VWF-propeptide levels and the VWF cleavage protease, ADAMTS13 content were measured. All treatments and their combinations, excluding IL-6, significantly stimulated the secretion of VWF from HUVEC. The VWF secretion from the HUVEC was stimulated most by the combination of TF with TNF-α. Slightly lower levels of ADAMTS13 secretion were found with all treatments. This may explain the thrombogenicity of patients with inflammation where extremely high VWF levels and slightly lower ADAMTS13 levels are present. PMID:27766025

  2. Hypericin-photodynamic therapy induces human umbilical vein endothelial cell apoptosis

    PubMed Central

    Zhang, Qian; Li, Zhuo-heng; Li, Yuan-yuan; Shi, San-jun; Zhou, Shi-wen; Fu, Yuan-yuan; Zhang, Qing; Yang, Xue; Fu, Ruo-qiu; Lu, Lai-chun

    2015-01-01

    The conventional photosensitizers used in photodynamic therapy (PDT), such as haematoporphyrin (HP), have not yet reached satisfactory therapeutic effects on port-wine stains (PWSs), due largely to the long-term dark toxicity. Previously we have showed that hypericin exhibited potent photocytotoxic effects on Roman chicken cockscomb model of PWSs. However, the molecular mechanism of hypericin-mediated photocytotoxicity remains unclear. In this study, we employed human umbilical vein endothelial cells (HUVECs) to investigate the hypericin-photolytic mechanism. Our study showed that hypericin-PDT induced reactive oxygen species (ROS), resulting in cell killings and an activation of the inflammatory response. Importantly, we have also discovered that photoactivated hypericin induced apoptosis by activating the mitochondrial caspase pathway and inhibiting the activation of the vascular endothelial growth factor-A (VEGF-A)-mediated PI3K/Akt pathway. Notably, we found that hypericin exhibited a more potent photocytotoxic effect than HP, and largely addressed the inconvenience issue associated with the use of HP. Thereby, hypericin may be a better alternative to HP in treating PWSs. PMID:26673286

  3. The Effect of Shiga Toxin on Weibel-Palade Bodies in Primary Human Endothelial Cells

    PubMed Central

    Geelen, Joyce; van den Biggelaar, Maartje; Linssen, Peter; van der Velden, Thea; Mertens, Koen; Monnens, Leo

    2014-01-01

    Background/Aims Diarrhea-associated hemolytic uremic syndrome is associated with the presence of Shiga toxin (Stx1, Stx2 and several variants) in the circulation. The aim of this study is to examine the possible triggering effect of Stx1 on the exocytosis of Weibel-Palade bodies (WPbs). Methods Cultured human umbilical venous endothelial cells (HUVECs) and glomerular microvascular endothelial cells (GMVECs) were stimulated by thrombin and Stx1 in both static and flowing conditions. The amount of secreted von Willebrand factor (VWF) in the supernatant as well as the remaining intracellular fraction was determined. Results In HUVECs and in 2 out of 4 GMVECs, the stimulation of Stx1 in flow at 1 dyne/cm2 resulted in a decrease of intracellular VWF. This is contrary to the results of Stx1 applied in static conditions. At a higher flow rate of 5 dyne/cm2, no effect in GMVECs was observed. Conclusion Stx1 can contribute, via an effect on WPbs, to the exocytosis of WPbs in flow conditions in HUVECs and probably in GMVECs. This results in the release of VWF, suggesting an initiating role of the coagulation system in the pathogenesis. PMID:25177338

  4. Cooperation between human fibrocytes and endothelial colony forming cells increases angiogenesis via CXCR4 pathway

    PubMed Central

    Smadja, David M.; Dorfmüller, Peter; Bieche, Ivan; Guerin, Coralie; Badoual, Cécile; Boscolo, Elisa; Kambouchner, Marianne; Cazes, Aurélie; Mercier, Olaf; Humbert, Marc; Gaussem, Pascale; Bischoff, Joyce; Israël-Biet, Dominique

    2016-01-01

    Background Fibrotic diseases of the lung are associated with a vascular remodeling process. Fibrocytes (Fy) are a distinct population of blood-borne cells that coexpress hematopoietic cell antigens and fibroblast markers, which have been shown to contribute to organ fibrosis. The purpose of this study was to test the hypothesis that Fy might cooperate with endothelial colony forming cells to induce angiogenesis. Methods/Results We successfully isolated Fy from blood of idiopathic pulmonary fibrosis (IPF) patients, which were further characterized by flow cytometry, Reverse Transcriptase quantitative-PCR (RTQ-PCR), and confocal analysis. We investigated the interaction between Fy and cord blood derived endothelial colony forming cells (ECFC) angiogenic potential in vitro and in vivo in a Matrigel implant model. Compared to fibroblast culture media, secreted media from Fy increase ECFC proliferation and their differentiation ability via SDF-1/CXCR4 pathway. IPF-Fy co-implanted with human ECFC in a matrigel plug in immunodeficient mice formed functional microvascular beds, whereas fibroblasts did not. Evaluation of implants after 2 weeks revealed an extensive network of blood vessels containing erythrocytes. CXCR4 blockade significantly inhibited blood vessel formation in the implants. The clinical relevance of these data was confirmed by the high expression level of CXCR4 in vessels close to fibrotic areas in biopsy specimens from patients with IPF, in contrast to control lungs. Conclusions Circulating Fy might be contribute to the intense remodeling of the pulmonary vasculature in patients with IPF. PMID:25103869

  5. In Vitro Effects of Hollow Gold Nanoshells on Human Aortic Endothelial Cells.

    PubMed

    Gu, Chunrong; Wu, Hengfang; Ge, Gaoyuan; Li, Xiongzhi; Guo, Zhirui; Bian, Zhiping; Xu, Jindan; Lu, Hua; Chen, Xiangjian; Yang, Di

    2016-12-01

    Gold nanoparticles are emerging as promising biomedical tools due to their unique nanoscale characteristics. Our purpose was to synthesize a hollow-shaped gold nanoparticle and to investigate its effect on human aortic endothelial cells (HAECs) in vitro. Hollow gold nanoshells with average 35-nm diameters and 10-nm shell thickness were obtained by galvanic replacement using quasi-spherical nanosilver as sacrifice-template. Our results showed that hollow gold nanoshells in the culture medium could be internalized into the cytoplasm of HAECs. No cytotoxicity effect of hollow gold nanoshells on HAECs was observed within the test concentrations (0-0.8 μg/mL) and test exposure period (0-72 h) by tetrazolium dye assay. Meanwhile, the release of cell injury biomarker, lactate dehydrogenase, was not significantly higher than that from control cells (without hollow gold nanoshells). The concentrations of vasodilators, nitric oxide, and prostacyclin I-2 were not changed, but the vasoconstrictor endothelin-1 was decreased by hollow gold nanoshells treatment in HAECs. HAECs exposed to hollow gold nanoshells resulted in suppressing expressions of genes involved in apoptosis and activating expressions of genes of adhesion molecules. Moreover, we demonstrated by in vitro endothelial tube formation that hollow gold nanoshells (0.8 μg/mL) could not inhibit angiogenesis by the HAECs. Altogether, these results indicate that the structure and major function of HAECs would not be disrupted by hollow gold nanoshell treatment.

  6. Hyperthermia stimulates plasminogen activator inhibitor type 1 expression in human umbilical vein endothelial cells in vitro.

    PubMed Central

    Wojta, J.; Holzer, M.; Hufnagl, P.; Christ, G.; Hoover, R. L.; Binder, B. R.

    1991-01-01

    The effect of exposure to hyperthermia on the fibrinolytic potential of human umbilical vein endothelial cells (HUVEC) in culture was studied. HUVEC responded to exposure to 42 degrees C with a time-dependent increase in plasminogen activator inhibitor type 1 (PAI-1) activity and antigen accompanied by a four- to fivefold increase in PAI-1 specific m-RNA and a decrease in tissue-type plasminogen activator (t-PA) antigen. The effect of 8 hours exposure to hyperthermia on PAI-1 activity and antigen could not be reversed by reexposure of the cells to 37 degrees C for 24 hours as evidenced by continuously increased amounts of PAI-1 released into the conditioned media. t-PA release, however, decreased during the 24-hour period at 37 degrees C after exposure to hyperthermia. No difference in PAI-1 antigen present in the extracellular matrix of heat treated HUVEC as compared to HUVEC kept at 37 degrees C could be found. Our data supports the idea that hyperthermia is one stress factor that influences the fibrinolytic potential of endothelial cells. Images Figure 6 PMID:1928306

  7. HIF-2α Expression Regulates Sprout Formation into 3D Fibrin Matrices in Prolonged Hypoxia in Human Microvascular Endothelial Cells

    PubMed Central

    Nauta, Tessa D.; Duyndam, Monique C. A.; Weijers, Ester M.; van Hinsbergh, Victor M. W.; Koolwijk, Pieter

    2016-01-01

    Background During short-term hypoxia, Hypoxia Inducible Factors (particular their subunits HIF-1α and HIF-2α) regulate the expression of many genes including the potent angiogenesis stimulator VEGF. However, in some pathological conditions chronic hypoxia occurs and is accompanied by reduced angiogenesis. Objectives We investigated the effect of prolonged hypoxia on the proliferation and sprouting ability of human microvascular endothelial cells and the involvement of the HIFs and Dll4/Notch signaling. Methods and Results Human microvascular endothelial cells (hMVECs), cultured at 20% oxygen for 14 days and seeded on top of 3D fibrin matrices, formed sprouts when stimulated with VEGF-A/TNFα. In contrast, hMVECs precultured at 1% oxygen for 14 days were viable and proliferative, but did not form sprouts into fibrin upon VEGF-A/TNFα stimulation at 1% oxygen. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting, whereas HIF-1α or HIF-3α by si-RNA had no effect. No involvement of Dll4/Notch pathway in the inhibitory effect on endothelial sprouting by prolonged hypoxia was found. In addition, hypoxia decreased the production of urokinase-type plasminogen activator (uPA), needed for migration and invasion, without a significant effect on its inhibitor PAI-1. This was independent of HIF-2α, as si-HIF-2α did not counteract uPA reduction. Conclusion Prolonged culturing of hMVECs at 1% oxygen inhibited endothelial sprouting into fibrin. Two independent mechanisms contribute. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting pointing to a HIF-2α-dependent mechanism. In addition, reduction of uPA contributed to reduced endothelial tube formation in a fibrin matrix during prolonged hypoxia. PMID:27490118

  8. A permanently growing human endothelial cell line supports productive infection with human cytomegalovirus under conditional cell growth arrest.

    PubMed

    Lieber, Diana; Hochdorfer, Daniel; Stoehr, Dagmar; Schubert, Axel; Lotfi, Ramin; May, Tobias; Wirth, Dagmar; Sinzger, Christian

    2015-09-01

    Infection of vascular endothelial cells (ECs) is assumed to contribute to dissemination of human cytomegalovirus (HCMV). Investigation of virus-host interactions in ECs such as human umbilical vein endothelial cells (HUVECs) is limited due to the low maximal passage numbers of these primary cells. We tested a conditionally immortalized EC line (HEC-LTT) and a permanent cell line (EA.hy926) for their susceptibility to HCMV infection. Both cell lines resembled HUVECs in that they allowed for entry and immediate early protein expression of highly endotheliotropic HCMV strains but not of poorly endotheliotropic strains, rendering them suitable for analysis of the viral entry mechanism in ECs. The late phase of viral replication and release, however, was supported by growth-controlled HEC-LTT cells but not by EA.hy926 cells. HEC-LTT cells support both the early and late phase of viral replication and release infectious progeny virus at titers comparable to primary HUVECs; thus, the HEC-LTT cell line is a cell culture model representing the full viral replicative cycle of HCMV in ECs. The implementation of permanent HEC-LTT and EA.hy926 cell lines in HCMV research will facilitate long-term approaches that are not feasible in primary HUVECs.

  9. Isolation and Culture of Human Endothelial Cells from Micro- and Macro-vessels.

    PubMed

    Hewett, Peter W

    2016-01-01

    The endothelium from different vascular beds exhibits a high degree of phenotypic heterogeneity. Endothelial cells (EC) can be harvested easily from large vessels by mechanical removal or collagenase digestion. In particular, the human umbilical vein has been used due to its wide availability, and the study of ECs derived from it has undoubtedly greatly advanced our knowledge of vascular biology. However, the majority of the body's endothelium (>95 %) forms the microvasculature, and it is these cells providing the interface between the blood and tissues that play a critical role in the development of new blood vessels. This has led to the establishment of techniques for the isolation of microvascular ECs (MEC) from different tissues to provide more physiologically relevant in vitro models of angiogenesis and EC function.In this chapter the use of superparamagnetic beads (Dynabeads) coated with anti-PECAM-1 (CD31) antibodies (PECA-beads) to culture MECs from human adipose tissue is described along with the standard methods used to characterize them. Adipose tissue is an ideal source of MECs as it is composed mainly of adipocytes with a very rich microvasculature and is easy to disaggregate. Furthermore, it can be obtained in large quantities during plastic surgery procedures. Adipose obtained at reduction mammoplasty or abdominoplasty is first dissected free of the connective tissue, minced finely, and subjected to collagenase type II digestion. The adipocytes are removed by centrifugation to obtain a microvessel rich pellet, which is further disaggregated with trypsin/EDTA solution. Following filtration to remove fragments of the connective tissue, the pellet is incubated with PECA-beads and microvessel fragments/ECs and washed and harvested using a magnet. In addition, the adaptation of this basic technique for the isolation of the human lung and stomach MECs is also described along with common methods for the preparation of large vessel endothelial cells. PMID

  10. Isolation and Culture of Human Endothelial Cells from Micro- and Macro-vessels.

    PubMed

    Hewett, Peter W

    2016-01-01

    The endothelium from different vascular beds exhibits a high degree of phenotypic heterogeneity. Endothelial cells (EC) can be harvested easily from large vessels by mechanical removal or collagenase digestion. In particular, the human umbilical vein has been used due to its wide availability, and the study of ECs derived from it has undoubtedly greatly advanced our knowledge of vascular biology. However, the majority of the body's endothelium (>95 %) forms the microvasculature, and it is these cells providing the interface between the blood and tissues that play a critical role in the development of new blood vessels. This has led to the establishment of techniques for the isolation of microvascular ECs (MEC) from different tissues to provide more physiologically relevant in vitro models of angiogenesis and EC function.In this chapter the use of superparamagnetic beads (Dynabeads) coated with anti-PECAM-1 (CD31) antibodies (PECA-beads) to culture MECs from human adipose tissue is described along with the standard methods used to characterize them. Adipose tissue is an ideal source of MECs as it is composed mainly of adipocytes with a very rich microvasculature and is easy to disaggregate. Furthermore, it can be obtained in large quantities during plastic surgery procedures. Adipose obtained at reduction mammoplasty or abdominoplasty is first dissected free of the connective tissue, minced finely, and subjected to collagenase type II digestion. The adipocytes are removed by centrifugation to obtain a microvessel rich pellet, which is further disaggregated with trypsin/EDTA solution. Following filtration to remove fragments of the connective tissue, the pellet is incubated with PECA-beads and microvessel fragments/ECs and washed and harvested using a magnet. In addition, the adaptation of this basic technique for the isolation of the human lung and stomach MECs is also described along with common methods for the preparation of large vessel endothelial cells.

  11. Visfatin as a Novel Mediator Released by Inflamed Human Endothelial Cells

    PubMed Central

    Romacho, Tania; Villalobos, Laura A.; Cercas, Elena; Carraro, Raffaele; Sánchez-Ferrer, Carlos F.; Peiró, Concepción

    2013-01-01

    Background Visfatin is a multifaceted adipokine whose circulating levels are enhanced in different metabolic diseases. Extracellular visfatin can exert various deleterious effects on vascular cells, including inflammation and proliferation. Limited evidence exists, however, on the capacity of human vascular cells to synthesize and release visfatin by themselves, under basal or pro-inflammatory conditions. Methods and Results Intracellular visfatin was detected by Western blot in non-stimulated human umbilical vein endothelial cells (HUVEC). However, exposing HUVEC for 18 h to a series of pro-inflammatory stimulus, such as interleukin (IL)-1β (1 to 10 ng/mL), tumor necrosis factor-α (1 to 10 ng/mL) or angiotensin II (10 pmol/L to 1 μmol/L) markedly enhanced intracellular visfatin content. Using IL-1β (10 ng/mL; 18 h), it was determined that the increase in intracellular visfatin, which was paralleled by enhanced visfatin mRNA levels, relied on a signalling mechanism involving both nuclear factor-κB and poly (ADP ribose) polymerase-1 activation. Moreover, IL-1β modified the sub-cellular localization of visfatin; while in non-stimulated HUVEC immunoreactive visfatin predominantly showed an intra-nuclear granular pattern, in IL-1β-inflamed cells an extra-nuclear filamentous staining, co-localising with F-actin fibers and suggesting a secretory pattern, was mainly found. Indeed, IL-1β promoted visfatin secretion, as determined by both ELISA and immunocytochemistry. Conclusions Human endothelial cells synthesize and release visfatin, particularly in response to inflammation. We suggest that the inflamed endothelium can be a source of visfatin, which arises as a local inflammatory mediator and a potential therapeutic target to interfere with vascular inflammation. PMID:24130902

  12. Spreading convulsions, spreading depolarization and epileptogenesis in human cerebral cortex

    PubMed Central

    Major, Sebastian; Pannek, Heinz-Wolfgang; Woitzik, Johannes; Scheel, Michael; Wiesenthal, Dirk; Martus, Peter; Winkler, Maren K.L.; Hartings, Jed A.; Fabricius, Martin; Speckmann, Erwin-Josef; Gorji, Ali

    2012-01-01

    Spreading depolarization of cells in cerebral grey matter is characterized by massive ion translocation, neuronal swelling and large changes in direct current-coupled voltage recording. The near-complete sustained depolarization above the inactivation threshold for action potential generating channels initiates spreading depression of brain activity. In contrast, epileptic seizures show modest ion translocation and sustained depolarization below the inactivation threshold for action potential generating channels. Such modest sustained depolarization allows synchronous, highly frequent neuronal firing; ictal epileptic field potentials being its electrocorticographic and epileptic seizure its clinical correlate. Nevertheless, Leão in 1944 and Van Harreveld and Stamm in 1953 described in animals that silencing of brain activity induced by spreading depolarization changed during minimal electrical stimulations. Eventually, epileptic field potentials were recorded during the period that had originally seen spreading depression of activity. Such spreading convulsions are characterized by epileptic field potentials on the final shoulder of the large slow potential change of spreading depolarization. We here report on such spreading convulsions in monopolar subdural recordings in 2 of 25 consecutive aneurismal subarachnoid haemorrhage patients in vivo and neocortical slices from 12 patients with intractable temporal lobe epilepsy in vitro. The in vitro results suggest that γ-aminobutyric acid-mediated inhibition protects from spreading convulsions. Moreover, we describe arterial pulse artefacts mimicking epileptic field potentials in three patients with subarachnoid haemorrhage that ride on the slow potential peak. Twenty-one of the 25 subarachnoid haemorrhage patients (84%) had 656 spreading depolarizations in contrast to only three patients (12%) with 55 ictal epileptic events isolated from spreading depolarizations. Spreading depolarization frequency and depression

  13. Spreading convulsions, spreading depolarization and epileptogenesis in human cerebral cortex.

    PubMed

    Dreier, Jens P; Major, Sebastian; Pannek, Heinz-Wolfgang; Woitzik, Johannes; Scheel, Michael; Wiesenthal, Dirk; Martus, Peter; Winkler, Maren K L; Hartings, Jed A; Fabricius, Martin; Speckmann, Erwin-Josef; Gorji, Ali

    2012-01-01

    Spreading depolarization of cells in cerebral grey matter is characterized by massive ion translocation, neuronal swelling and large changes in direct current-coupled voltage recording. The near-complete sustained depolarization above the inactivation threshold for action potential generating channels initiates spreading depression of brain activity. In contrast, epileptic seizures show modest ion translocation and sustained depolarization below the inactivation threshold for action potential generating channels. Such modest sustained depolarization allows synchronous, highly frequent neuronal firing; ictal epileptic field potentials being its electrocorticographic and epileptic seizure its clinical correlate. Nevertheless, Leão in 1944 and Van Harreveld and Stamm in 1953 described in animals that silencing of brain activity induced by spreading depolarization changed during minimal electrical stimulations. Eventually, epileptic field potentials were recorded during the period that had originally seen spreading depression of activity. Such spreading convulsions are characterized by epileptic field potentials on the final shoulder of the large slow potential change of spreading depolarization. We here report on such spreading convulsions in monopolar subdural recordings in 2 of 25 consecutive aneurismal subarachnoid haemorrhage patients in vivo and neocortical slices from 12 patients with intractable temporal lobe epilepsy in vitro. The in vitro results suggest that γ-aminobutyric acid-mediated inhibition protects from spreading convulsions. Moreover, we describe arterial pulse artefacts mimicking epileptic field potentials in three patients with subarachnoid haemorrhage that ride on the slow potential peak. Twenty-one of the 25 subarachnoid haemorrhage patients (84%) had 656 spreading depolarizations in contrast to only three patients (12%) with 55 ictal epileptic events isolated from spreading depolarizations. Spreading depolarization frequency and depression

  14. 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.

  15. Subacute methotrexate neurotoxicity and cerebral venous sinus thrombosis in a 12-year-old with acute lymphoblastic leukemia and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism: homocysteine-mediated methotrexate neurotoxicity via direct endothelial injury.

    PubMed

    Mahadeo, Kris M; Dhall, Girish; Panigrahy, Ashok; Lastra, Carlos; Ettinger, Lawrence J

    2010-02-01

    From as early as the 1970s methotrexate has been associated with disseminated necrotizing leukoencephalopathy and other neurotoxic sequelae. Yet, a clear mechanism for methotrexate-induced neurotoxicity has not been established. The authors describe the case of a 12-year-old male with acute lymphoblastic leukemia and a homozygous methylenetetrahydrofolate reductase C677T mutation, who developed subacute methotrexate-induced toxicity and cerebral venous thrombosis after receiving intrathecal methotrexate. The role of homocysteine as a possible mediator in methotrexate-induced neurotoxicity via direct endothelial injury is discussed. PMID:20121554

  16. Triple combination of irradiation, chemotherapy (pemetrexed), and VEGFR inhibition (SU5416) in human endothelial and tumor cells

    SciTech Connect

    Bischof, Marc; Abdollahi, Amir; Gong Ping; Stoffregen, Clemens; Lipson, Kenneth E.; Debus, Juergen; Weber, Klaus J.; Huber, Peter E. . E-mail: p.huber@dkfz.de

    2004-11-15

    Purpose: This is the first preclinical report evaluating a trimodal therapy consisting of irradiation, chemotherapy, and antiangiogenesis in the context of a multimodal anticancer strategy. The combination of the folate antimetabolite pemetrexed, SU5416, a receptor tyrosine kinase inhibitor of VEGFR2, and irradiation was investigated in human endothelial cells and tumor cell lines. Methods and materials: Primary isolated human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HDMEC), and human glioblastoma (U87) and prostate cancer cells (PC3) were exposed to pemetrexed (2 h) alone and in combination with SU5416 (2 h). When combined with irradiation up to 8 Gy, fixed concentrations of pemetrexed (1.06 {mu}M) and SU5416 (1.0 {mu}M) were used. Proliferation and clonogenic assays were conducted with endothelial and tumor cells. The migration/invasion ability of endothelial cells and the ability to produce tubular structures were tested in Matrigel and tube formation assays. Apoptosis was measured by sub-G1 DNA and caspase-3 flow cytometry. To investigate underlying cell signaling, immunocytochemistry was used to detect Akt survival signaling involvement. Results: Triple combination using only a low-toxicity drug exposure of pemetrexed and SU5416 results in greater response than each treatment alone or than each combination of two modalities in all tested endothelial and tumor cell models. Triple combination substantially inhibits proliferation, migration/invasion, tube formation, and clonogenic survival. Triple combination also induced the highest rate of apoptosis in HDMEC and HUVEC as indicated by sub-1 G1 and caspase-3 assessment. Interestingly, triple combination therapy also reduces proliferation and clonogenic survival significantly in U87 and PC3 tumor cell lines. SU5416 potently inhibited Akt phosphorylation which could be induced by radiation and radiochemotherapy in human endothelial cells. Conclusions: Our findings

  17. Lepidotol A from Mesua lepidota Inhibits Inflammatory and Immune Mediators in Human Endothelial Cells.

    PubMed

    Rouger, Caroline; Derbré, Séverine; Charreau, Béatrice; Pabois, Angélique; Cauchy, Thomas; Litaudon, Marc; Awang, Khalijah; Richomme, Pascal

    2015-09-25

    Phytochemical investigation on the fruits of Mesua lepidota (Calophyllaceae) led to the isolation of seven new phenylcoumarin derivatives named lepidotols A-E (1-5) and lepidotins A and B (6, 7). These structures were elucidated by spectroscopic and spectrometric methods including UV, NMR, and HRMS. Lepidotol A (1), the major compound, was evaluated for its inhibitory effect on inflammation and immunity using endothelial cell-based cellular assays. At 10 μM, 1 exhibited an anti-inflammatory activity, with a significant inhibition of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression induced by tumor necrosis factor-α. Lepidotol A also showed a mild immunosuppressive effect, with inhibition of the major histocompatibility complex molecules, namely, human leukocyte antigen (HLA)-DR and HLA-E.

  18. Antiangiogenic properties of cafestol, a coffee diterpene, in human umbilical vein endothelial cells

    SciTech Connect

    Wang, Shuaiyu; Yoon, Yeo Cho; Sung, Mi-Jeong; Hur, Haeng-Jeon; Park, Jae-Ho

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Cafestol inhibits tube formation and migration of VEGF-stimulated HUVEC. Black-Right-Pointing-Pointer Cafestol inhibits phosphorylation of FAK and Akt. Black-Right-Pointing-Pointer Cafestol decreases NO production. -- Abstract: As angiogenesis plays important roles in tumor growth and metastasis, searching for antiangiogenic compounds is a promising tactic for treating cancers. Cafestol, a diterpene found mainly in unfiltered coffee, provides benefit through varied biological activity, including antitumorigenic, antioxidative, and anti-inflammatory effects. This study aimed to investigate the effects of cafestol on angiogenesis and to uncover the associated mechanism. We show that cafestol inhibits angiogenesis of human umbilical vascular endothelial cells. This inhibition affects the following specific steps of the angiogenic process: proliferation, migration, and tube formation. The inhibitory effects of cafestol are accompanied by decreasing phosphorylation of FAK and Akt and by a decrease in nitric oxide production. Overall, cafestol inhibits angiogenesis by affecting the angiogenic signaling pathway.

  19. Aspirin Triggered-Lipoxin A4 Reduces the Adhesion of Human Polymorphonuclear Neutrophils to Endothelial Cells Initiated by Preeclamptic Plasma

    PubMed Central

    Gil-Villa, AM; Norling, LV; Serhan, CN; Cordero, D; Rojas, M; Cadavid, A

    2012-01-01

    Introduction Preeclampsia is a disorder of pregnancy, characterized by hypertension and proteinuria after 20 weeks of gestation. Here, we evaluated the role of aspirin triggered-lipoxin A4 (ATL, 15-epi-LXA4) on the modulation of the adhesion of human polymorphonuclear neutrophils (PMN) to endothelial cells initiated by preeclamptic plasma. Materials and methods Plasma from preeclamptic, normotensive pregnant, and non-pregnant women were analysed for factors involved in regulating angiogenesis, inflammation and lipid peroxidation. Plasma from preeclamptic women was added to human umbilical vein endothelial cells, and the adhesion of PMN (incubated with or without ATL) to cells was evaluated. Results Preeclampsia was associated with some augmented anti-angiogenic, oxidative and pro-inflammatory markers, as well as increasing human PMN-endothelial cell adhesion. This cell adhesion was reduced when human PMN were incubated with ATL prior to addition to endothelial monolayers. Discussions and Conclusions Our results are the starting point for further research on the efficacy and rational use of aspirin in preeclampsia. PMID:22974760

  20. RESIDUAL OIL FLY ASH (ROFA) AND VANADIUM-INDUCED GENE EXPRESSION PROFILES IN HUMAN VASCULAR ENDOTHELIAL CELLS

    EPA Science Inventory


    Residual oil fly ash (ROFA) and vanadium-induced gene expression profiles in human vascular endothelial cells.
    Srikanth S. Nadadur, Urmila P. Kodavanti, Mary Jane Selgrade and Daniel L. Costa, Pulmonary Toxicology Branch, ETD, NHEERL, ORD, US EPA, Research Triangle Park, N...

  1. GENE EXPRESSION PROFILES IN HUMAN AND RAT VASCULAR ENDOTHELIAL CELLS EXPOSED TO RESIDUAL OIL FLY ASH (ROFA) AND VANADIUM (V)

    EPA Science Inventory

    Gene expression profiles in human and rat vascular endothelial cells exposed to residual oil fly ash (ROFA) or vanadium (V).
    Srikanth S. Nadadur, Darrell W. Winsett and Daniel L. Costa, US EPA, ORD, NHEERL (ETD, Pulmonary Toxicology Branch), Research Triangle Park, NC 27711.

  2. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation

    PubMed Central

    Kirkby, Nicholas S.; Reed, Daniel M.; Edin, Matthew L.; Rauzi, Francesca; Mataragka, Stefania; Vojnovic, Ivana; Bishop-Bailey, David; Milne, Ginger L.; Longhurst, Hilary; Zeldin, Darryl C.; Mitchell, Jane A.; Warner, Timothy D.

    2016-01-01

    Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.—Kirkby, N. S., Reed, D. M., Edin, M. L., Rauzi, F., Mataragka, S., Vojnovic, I., Bishop-Bailey, D., Milne, G. L., Longhurst, H., Zeldin, D. C., Mitchell, J. A., Warner, T. D. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation. PMID:26183771

  3. Human chorionic villus mesenchymal stromal cells reveal strong endothelial conversion properties.

    PubMed

    Meraviglia, Viviana; Vecellio, Matteo; Grasselli, Annalisa; Baccarin, Marco; Farsetti, Antonella; Capogrossi, Maurizio C; Pompilio, Giulio; Coviello, Domenico A; Gaetano, Carlo; Di Segni, Marina; Rossini, Alessandra

    2012-06-01

    Chorion, amnion and villi are reservoirs of mesenchymal stromal cells (StC) and the hypothesis that StC from fetal tissues retain higher plasticity compared to adult StC has been suggested. Aimed at investigating this aspect, a series of in vitro experiments were performed with StC isolated from first trimester human chorionic villi (CVStC). CVStC were cultured in: (i) standard mesenchymal medium (MM) and (ii) AmniomaxII® (AM), specifically designed to grow amnion-derived cells in prenatal diagnostic procedures. Cells were then exposed to distinct differentiation treatments and distinguished according to morphology, immunophenotype and molecular markers. Human StC obtained from adult bone marrow (BMStC) were used as control. CVStC cultured either in MM or AM presented stromal morphology and immunophenotype, were negative for pluripotency factors (Nanog, Oct-4 and Sox-2), lacked detectable telomerase activity and retained high genomic stability. In AM, however, CVStC exhibited a faster proliferation rate compared to BMStC or CVStC kept in MM. During differentiation, CVStC were less efficient than BMStC in acquiring adipocytes and osteocytes features; the cardiomyogenic conversion occurred at low efficiency in both cell types. Remarkably, in the presence of pro-angiogenic factors, CVStC reprogrammed toward an endothelial-like phenotype at significantly higher efficiency than BMStC. This effect was particularly evident in CVStC expanded in AM. Mechanistically, the reduced CVStC expression of anti-angiogenic microRNA could support this process. The present study demonstrates that, despite of fetal origin, CVStC exhibit restricted plasticity, distinct from that of BMStC and predominantly directed toward the endothelial lineage.

  4. HSPA12B inhibits lipopolysaccharide-induced inflammatory response in human umbilical vein endothelial cells

    PubMed Central

    Wu, Jun; Li, Xuehan; Huang, Lei; Jiang, Surong; Tu, Fei; Zhang, Xiaojin; Ma, He; Li, Rongrong; Li, Chuanfu; Li, Yuehua; Ding, Zhengnian; Liu, Li

    2015-01-01

    Heat shock protein A12B (HSPA12B) is a newly discovered member of the HSP70 protein family. This study investigated the effects of HSPA12B on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) and the possible mechanisms involved. A HUVECs inflammatory model was induced by LPS. Overexpression of HSPA12B in HUVECs was achieved by infection with recombinant adenoviruses encoding green fluorescence protein-HSPA12B. Knockdown of HSPA12B was achieved by siRNA technique. Twenty four hours after virus infection or siRNA transfection, HUVECs were stimulated with 1 μg/ml LPS for 4 hrs. Endothelial cell permeability ability was determined by transwell permeability assay. The binding rate of human neutrophilic polymorphonuclear leucocytes (PMN) with HUVECs was examined using myeloperoxidase assay. Cell migrating ability was determined by the wound-healing assay. The mRNA and protein expression levels of interested genes were analyzed by RT-qPCR and Western blot, respectively. The release of cytokines interleukin-6 and tumour necrosis factor-α was measured by ELISA. HSPA12B suppressed LPS-induced HUVEC permeability and reduced PMN adhesion to HUVECs. HSPA12B also inhibited LPS-induced up-regulation of adhesion molecules and inflammatory cytokine expression. By contrast, knockdown of HSPA12B enhanced LPS-induced increases in the expression of adhesion molecules and inflammatory cytokines. Moreover, HSPA12B activated PI3K/Akt signalling pathway and pharmacological inhibition of this pathway by Wortmannin completely abrogated the protection of HSPA12B against inflammatory response in HUVECs. Our results suggest that HSPA12B attenuates LPS-induced inflammatory responses in HUVECs via activation of PI3K/Akt signalling pathway. PMID:25545050

  5. Expression of vascular endothelial growth factor-b in human astrocytoma.

    PubMed Central

    Gollmer, J. C.; Ladoux, A.; Gioanni, J.; Paquis, P.; Dubreuil, A.; Chatel, M.; Frelin, C.

    2000-01-01

    Growth of human malignant gliomas is stringently dependent on an angiogenic process that probably involves vascular endothelial growth factor (VEGF). Expressions of mRNA coding for the different forms of VEGF were analyzed in surgical specimens from human astrocytomas. Low levels of placental growth factor (PGF) and VEGFC mRNA were observed in polymerase chain reaction, but not in Northern blot experiments. VEGF mRNA was found in some but not all grade and grade IV astrocytomas. VEGFB mRNA was observed in all tissue samples analyzed irrespective of the tumor grade. A new splice variant of VEGFB (VEGFB155) that lacks exons 5 and 6 is described. Expressions of VEGF mRNA in cultured glioblastomas cells were upregulated by hypoxia, but the sensitivity of the cells to hypoxia was reduced as compared with normal rat astrocytes. VEGF expression was depressed by dexamethasone. Expressions of VEGFB mRNA were affected neither by hypoxia nor by dexamethasone. The results indicate a coexpression of VEGF mRNA and VEGFB mRNA in human astrocytomas. Expression of VEGFB is markedly different from that of VEGF. Possible roles of VEGFB as a cofactor for hypoxia-induced angiogenesis in human astrocytomas are discussed. PMID:11303624

  6. Degradation of endothelial basement membrane by human breast cancer cell lines

    SciTech Connect

    Yee, C.; Shiu, R.P.

    1986-04-01

    During metastasis, it is believed that tumor cells destroy the basement membrane (BM) of blood vessels in order to disseminate through the circulatory system. By radioactively labeling the extracellular matrix produced by primary endothelial cells in vitro, the ability of human breast cancer cells to degrade BM components was studied. We found that T-47D, a human breast cancer line, was able to degrade significant amounts of (35S)methionine-labeled and (3H)proline-labeled BM, but not 35SO4-labeled BM. Six other tumor cell lines of human breast origin were assayed in the same manner and were found to degrade BM to varying degrees. Several non-tumor cell lines tested showed relatively little degrading activity. The use of serum-free medium greatly enhanced degradation of the BM by tumor cells, suggesting a role for naturally occurring enzyme inhibitors in the serum. Direct cell contact with the BM was required for BM degradation, suggesting that the active enzymes are cell associated. The addition of hormones implicated in the etiology of breast cancer did not significantly alter the ability of T-47D cells to degrade the BM. The use of this assay affords future studies on the mechanism of invasion and metastasis of human breast cancer.

  7. Random positions of dendritic spines in human cerebral cortex.

    PubMed

    Morales, Juan; Benavides-Piccione, Ruth; Dar, Mor; Fernaud, Isabel; Rodríguez, Angel; Anton-Sanchez, Laura; Bielza, Concha; Larrañaga, Pedro; DeFelipe, Javier; Yuste, Rafael

    2014-07-23

    Dendritic spines establish most excitatory synapses in the brain and are located in Purkinje cell's dendrites along helical paths, perhaps maximizing the probability to contact different axons. To test whether spine helixes also occur in neocortex, we reconstructed >500 dendritic segments from adult human cortex obtained from autopsies. With Fourier analysis and spatial statistics, we analyzed spine position along apical and basal dendrites of layer 3 pyramidal neurons from frontal, temporal, and cingulate cortex. Although we occasionally detected helical positioning, for the great majority of dendrites we could not reject the null hypothesis of spatial randomness in spine locations, either in apical or basal dendrites, in neurons of different cortical areas or among spines of different volumes and lengths. We conclude that in adult human neocortex spine positions are mostly random. We discuss the relevance of these results for spine formation and plasticity and their functional impact for cortical circuits.

  8. Random Positions of Dendritic Spines in Human Cerebral Cortex

    PubMed Central

    Morales, Juan; Benavides-Piccione, Ruth; Dar, Mor; Fernaud, Isabel; Rodríguez, Angel; Anton-Sanchez, Laura; Bielza, Concha; Larrañaga, Pedro; DeFelipe, Javier

    2014-01-01

    Dendritic spines establish most excitatory synapses in the brain and are located in Purkinje cell's dendrites along helical paths, perhaps maximizing the probability to contact different axons. To test whether spine helixes also occur in neocortex, we reconstructed >500 dendritic segments from adult human cortex obtained from autopsies. With Fourier analysis and spatial statistics, we analyzed spine position along apical and basal dendrites of layer 3 pyramidal neurons from frontal, temporal, and cingulate cortex. Although we occasionally detected helical positioning, for the great majority of dendrites we could not reject the null hypothesis of spatial randomness in spine locations, either in apical or basal dendrites, in neurons of different cortical areas or among spines of different volumes and lengths. We conclude that in adult human neocortex spine positions are mostly random. We discuss the relevance of these results for spine formation and plasticity and their functional impact for cortical circuits. PMID:25057209

  9. Random positions of dendritic spines in human cerebral cortex.

    PubMed

    Morales, Juan; Benavides-Piccione, Ruth; Dar, Mor; Fernaud, Isabel; Rodríguez, Angel; Anton-Sanchez, Laura; Bielza, Concha; Larrañaga, Pedro; DeFelipe, Javier; Yuste, Rafael

    2014-07-23

    Dendritic spines establish most excitatory synapses in the brain and are located in Purkinje cell's dendrites along helical paths, perhaps maximizing the probability to contact different axons. To test whether spine helixes also occur in neocortex, we reconstructed >500 dendritic segments from adult human cortex obtained from autopsies. With Fourier analysis and spatial statistics, we analyzed spine position along apical and basal dendrites of layer 3 pyramidal neurons from frontal, temporal, and cingulate cortex. Although we occasionally detected helical positioning, for the great majority of dendrites we could not reject the null hypothesis of spatial randomness in spine locations, either in apical or basal dendrites, in neurons of different cortical areas or among spines of different volumes and lengths. We conclude that in adult human neocortex spine positions are mostly random. We discuss the relevance of these results for spine formation and plasticity and their functional impact for cortical circuits. PMID:25057209

  10. Autocrine control of angiogenesis by endogenous acetylcholine in an in vitro model using human endothelial cells: evidence for an autocrine cholinergic system in endothelial cells.

    PubMed

    Dhein, Stefan; Wermke, Alice; von Salisch, Sandy; Schlegel, Franziska; Stepan, Holger; Dohmen, Pascal Maria; Kostelka, Martin; Mohr, Friedrich-Wilhelm

    2015-05-01

    We wanted to elucidate whether acetylcholine as the endogenous ligand at cholinoceptors (ChRs) may have effects on angiogenesis and whether they are transduced through muscarinic or nicotinic ChRs. Human umbilical vein endothelial cells were cultured until confluence and thereafter seeded in Matrigel in vitro angiogenesis assays for 18 hours. During the entire cell culture and angiogenesis period, cells were treated with vehicle, eserine (1 μM), in the absence or presence of additional atropine (1 μM) or mecamylamine (1 μM). Finally, the resulting angiogenetic network was investigated histologically. Eserine significantly enhanced acetylcholine formation. When acetylcholine acted through muscarinic ChRs (eserine + mecamylamine), we observed enhanced complexity of the angiogenic network pattern with increased tube length and cell number. In contrast, when acting through nicotinic ChRs (eserine + atropine), we found reduced complexity of pattern with less branches, shorter tubes, and reduced cell number. If acting on both types of ChRs (eserine alone), there were only very small effects. Using α-bungarotoxin, lobeline, and dihydro-β-erythroidine, we also could show that these effects to various degrees involve α7, α3/β2, and α4/β2 n-ChRs. In conclusion, our results support the hypothesis that human umbilical vein endothelial cells possess an autocrine nonneuronal cholinergic system regulating angiogenesic branch formation through the partially opposing effects of n-ChRs and m-ChRs.

  11. Development and function of human cerebral cortex neural networks from pluripotent stem cells in vitro.

    PubMed

    Kirwan, Peter; Turner-Bridger, Benita; Peter, Manuel; Momoh, Ayiba; Arambepola, Devika; Robinson, Hugh P C; Livesey, Frederick J

    2015-09-15

    A key aspect of nervous system development, including that of the cerebral cortex, is the formation of higher-order neural networks. Developing neural networks undergo several phases with distinct activity patterns in vivo, which are thought to prune and fine-tune network connectivity. We report here that human pluripotent stem cell (hPSC)-derived cerebral cortex neurons form large-scale networks that reflect those found in the developing cerebral cortex in vivo. Synchronised oscillatory networks develop in a highly stereotyped pattern over several weeks in culture. An initial phase of increasing frequency of oscillations is followed by a phase of decreasing frequency, before giving rise to non-synchronous, ordered activity patterns. hPSC-derived cortical neural networks are excitatory, driven by activation of AMPA- and NMDA-type glutamate receptors, and can undergo NMDA-receptor-mediated plasticity. Investigating single neuron connectivity within PSC-derived cultures, using rabies-based trans-synaptic tracing, we found two broad classes of neuronal connectivity: most neurons have small numbers (<10) of presynaptic inputs, whereas a small set of hub-like neurons have large numbers of synaptic connections (>40). These data demonstrate that the formation of hPSC-derived cortical networks mimics in vivo cortical network development and function, demonstrating the utility of in vitro systems for mechanistic studies of human forebrain neural network biology.

  12. 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

  13. Sialyl Lewis X mimetics attenuate E-selectin-mediated adhesion of leukocytes to irradiated human endothelial cells.

    PubMed

    Hallahan, D E; Kuchibhotla, J; Wyble, C

    1997-01-01

    Ionizing radiation causes histological changes in normal tissues that resemble those resulting from the inflammatory response. Inflammation is a multistep process requiring expression of adhesion molecules on the surface of endothelial cells which results in leukocyte extravasation. E-selectin is an adhesion molecule that mediates leukocyte "rolling" on the endothelium and is required for the inflammatory response. We quantified E-selectin expression and selectin-dependent adhesion of leukocytes to human endothelial cells after X irradiation to determine whether E-selectin participates in the radiation-mediated inflammation-like response. Immunofluorescence staining of irradiated endothelial cells demonstrated expression of E-selectin on the cell surface similar to that elicited by treatment with interleukin-1 (IL-1). Radiation-mediated expression of E-selectin was dependent on dose and time and occurred at doses as low as 0.5 Gy. Furthermore, the increased adhesion of leukocytes to irradiated endothelial cells was prevented by an E-selectin-blocking antibody. Sialyl Lewis X is one of the molecules on the surface of leukocytes that adheres to E-selectin. The anti-inflammatory agents glycyrrhizin and carminic acid, which are structural analogues of sialyl Lewis X, attenuated adhesion of leukocytes to endothelial cells treated with X rays or IL-1. These data implicate a new class of anti-inflammatory agents in the prevention of adhesions of leukocytes to the irradiated vascular endothelium. PMID:8989368

  14. Endothelial Cells Enhance Human Immunodeficiency Virus Type 1 Replication in Macrophages through a C/EBP-Dependent Mechanism

    PubMed Central

    Lee, Eileen S.; Zhou, Huiyu; Henderson, Andrew J.

    2001-01-01

    Macrophages are early targets of human immunodeficiency virus type 1 (HIV-1) infection and serve as potential reservoirs for long-term infection. Through inflammatory mediators and direct cell contact, infected macrophages interact with neighboring cell populations, such as the endothelium, which create a microenvironment favorable for HIV-1 replication. We hypothesize that the transcriptional activator C/EBPβ is critical for macrophages to respond to endothelial cell-derived signals. We show that endothelial cells significantly enhance C/EBPβ binding activity and HIV-1 replication in macrophages. This increase in HIV-1 transcription is due to cell-cell contact as well as the production of soluble factors, mediated in part by ICAM-1 and interleukin 6, respectively. Furthermore, C/EBP factors are necessary for endothelial cell-dependent activation of HIV-1 transcription in macrophages, and HIV-1 induction can be inhibited by a C/EBP dominant-negative protein. In addition, C/EBP binding sites are necessary for efficient LTR activity and HIV-1 replication in the presence of endothelial cells. Taken together, these results indicate that endothelial cells, through the activation of C/EBPβ, provide a microenvironment that supports HIV-1 replication in monocytes/macrophages. PMID:11559803

  15. Augmentation of Neovascularizaiton in Hindlimb Ischemia by Combined Transplantation of Human Embryonic Stem Cells-Derived Endothelial and Mural Cells

    PubMed Central

    Itoh, Hiroshi; Yamashita, Jun K.; Yurugi-Kobayashi, Takami; Homma, Koichiro; Chao, Ting-Hsing; Miyashita, Kazutoshi; Park, Kwijun; Oyamada, Naofumi; Sawada, Naoya; Taura, Daisuke; Fukunaga, Yasutomo; Tamura, Naohisa; Nakao, Kazuwa

    2008-01-01

    Background We demonstrated that mouse embryonic stem (ES) cells-derived vascular endothelial growth factor receptor-2 (VEGF-R2) positive cells could differentiate into both endothelial cells (EC) and mural cells (MC), and termed them as vascular progenitor cells (VPC). Recently, we have established a method to expand monkey and human ES cells-derived VPC with the proper differentiation stage in a large quantity. Here we investigated the therapeutic potential of human VPC-derived EC and MC for vascular regeneration. Methods and Results After the expansion of human VPC-derived vascular cells, we transplanted these cells to nude mice with hindlimb ischemia. The blood flow recovery and capillary density in ischemic hindlimbs were significantly improved in human VPC-derived EC-transplanted mice, compared to human peripheral and umbilical cord blood-derived endothelial progenitor cells (pEPC and uEPC) transplanted mice. The combined transplantation of human VPC-derived EC and MC synergistically improved blood flow of ischemic hindlimbs remarkably, compared to the single cell transplantations. Transplanted VPC-derived vascular cells were effectively incorporated into host circulating vessels as EC and MC to maintain long-term vascular integrity. Conclusions Our findings suggest that the combined transplantation of human ES cells-derived EC and MC can be used as a new promising strategy for therapeutic vascular regeneration in patients with tissue ischemia. PMID:18301744

  16. Cytokines and Adhesion Molecules Expression in the Brain in Human Cerebral Malaria

    PubMed Central

    Armah, Henry; Wiredu, Edwin Kwame; Dodoo, Alfred Kofi; Adjei, Andrew Anthony; Tettey, Yao; Gyasi, Richard

    2005-01-01

    Although the role of systemic proinflammatory cytokines, IL-1β and TNF-α, and their up-regulation of adhesion molecules, ICAM-1, VCAM-1 and E-Selectin, in the pathogenesis of cerebral malaria (CM) is well established, the role of local cytokine release remain unclear. Immunohistochemistry (IHC) was used to compare the expression of ICAM-1, VCAM-1, E-Selectin, IL-1β, TNF-α and TGF- β at light microscopic level in cerebral, cerebellar and brainstem postmortem cryostat sections from 10 CM, 5 severe malarial anemia (SMA), 1 purulent bacterial meningitis (PBM), 2 non-central nervous system infections (NCNSI) and 3 non-infections (NI) deaths in Ghanaian children. Fatal malaria and Salmonella sepsis showed significantly higher vascular expression of all 3 adhesion molecules, with highly significant co-localization with sequestration in the malaria cases. However, there was negligible difference between CM and SMA. TGF-β showed intravascular and perivascular distribution in all cases, but expression was most intense in the PBM case and CM group. TNF-α and IL-1β showed prominent brain parenchymal staining, in addition to intravascular and perivascular staining, in only the PBM case and CM group. The maximal expression of all 6 antigens studied was in the cerebellar sections of the malaria cases. Endothelial activation is a feature of fatal malaria and Salmonella sepsis, with adhesion molecule expression being highly correlated with sequestration. IL-1β and TNF-α are upregulated in only cases with neurodegenerative lesions, whilst TGF-β is present in all cases. Both cytokines and adhesion molecules were maximally upregulated in the cerebellar sections of the malaria cases. PMID:16705810

  17. Induction of oxidative stress, lysosome activation and autophagy by nanoparticles in human brain-derived endothelial cells.

    PubMed

    Halamoda Kenzaoui, Blanka; Chapuis Bernasconi, Catherine; Guney-Ayra, Seher; Juillerat-Jeanneret, Lucienne

    2012-02-01

    Different types of NPs (nanoparticles) are currently under development for diagnostic and therapeutic applications in the biomedical field, yet our knowledge about their possible effects and fate in living cells is still limited. In the present study, we examined the cellular response of human brain-derived endothelial cells to NPs of different size and structure: uncoated and oleic acid-coated iron oxide NPs (8-9 nm core), fluorescent 25 and 50 nm silica NPs, TiO2 NPs (21 nm mean core diameter) and PLGA [poly(lactic-co-glycolic acid)]-PEO [poly(ethylene oxide)] polymeric NPs (150 nm). We evaluated their uptake by the cells, and their localization, generation of oxidative stress and DNA-damaging effects in exposed cells. We show that NPs are internalized by human brain-derived endothelial cells; however, the extent of their intracellular uptake is dependent on the characteristics of the NPs. After their uptake by human brain-derived endothelial cells NPs are transported into the lysosomes of these cells, where they enhance the activation of lysosomal proteases. In brain-derived endothelial cells, NPs induce the production of an oxidative stress after exposure to iron oxide and TiO2 NPs, which is correlated with an increase in DNA strand breaks and defensive mechanisms that ultimately induce an autophagy process in the cells. PMID:22026563

  18. Isolation and growth of endothelial cells from the microvessels of the newborn human foreskin in cell culture.

    PubMed

    Davison, P M; Bensch, K; Karasek, M A

    1980-10-01

    A procedure for the isolation and in vitro cultivation of endothelial cells from the microvessels of the newborn human foreskin dermis is described. The epidermis was removed from foreskin tissue using a Castroviejo keratotome (0.1 mm shim). Endothelial cells were released from the dermal vessels by trypsinization of 5 mm2 sections of dermis at 37 degrees C for 40 min. Cells were expressed into Minimal Essential Medium (MEM) containing 10% pooled human serum, collected by centrifugation and plated onto either a plain plastic or a fibronectin treated culture surface. In primary culture the rate of endothelial cell proliferation was dependent upon serum type and concentration being optimal in 50% pooled human serum. High serum concentration in combination with pretreatment of the culture surface with fibronectin was required for maximal proliferation rate, for the cells to achieve confluence and for subcultivation. Primary and subcultured cells were characterized as endothelial by light microscopic, immunofluorescent (Factor VIII associated protein) and ultrastructural (Weibel-Palade body) criteria.

  19. Functional and Transcriptional Characterization of Human Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Myocardial Infarction

    PubMed Central

    Li, Zongjin; Wilson, Kitchener D.; Smith, Bryan; Kraft, Daniel L.; Jia, Fangjun; Huang, Mei; Xie, Xiaoyan; Robbins, Robert C.; Gambhir, Sanjiv S.; Weissman, Irving L.; Wu, Joseph C.

    2009-01-01

    Background Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However, the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product, both of which can limit the future clinical application of hESC-ECs. Moreover, to fully understand the beneficial effects of stem cell therapy, investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time. Methodology In this study, we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray, and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover, our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods. Conclusion Taken together, we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes, form functional vessels in vivo, and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future. PMID:20046878

  20. Inhibitory effects of three diketopiperazines from marine-derived bacteria on endothelial protein C receptor shedding in human endothelial cells and mice.

    PubMed

    Lee, Wonhwa; Ku, Sae-Kwang; Choi, Hyukjae; Bae, Jong-Sup

    2016-04-01

    Diketopiperazine is a natural products found from bacteria, fungi, marine sponges, gorgonian and red algae. They are cyclic dipeptides possessing relatively simple and rigid structures with chiral nature and various side chains. The compounds in this structure class have been known to possess diverse bioactivities including antibiotic activity, anti-cancer activity, neuroprotective activity, and anti-inflammatory activity. The endothelial cell protein C receptor (EPCR) plays an important role in the cytoprotective pathway and in the activation of protein C. Endothelial cell protein C receptor (EPCR) can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of diketopiperazine on EPCR shedding. We investigated this issue by monitoring the effects of diketopiperazine on phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β-induced EPCR shedding in human umbilical vein endothelial cells (HUVECs), and cecal ligation and puncture (CLP)-mediated EPCR shedding in mice and underlying mechanism. Here, three (1-3) of diketopiperazines were isolated from two strains of marine-derived bacteria and 1-3 induced potent inhibition of PMA-, TNF-α-, IL-1β (in HUVECs), and CLP-induced EPCR shedding (in mice) via inhibition of phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38, janus kinase (JNK), and extracellular signal-regulated kinase (ERK) 1/2. 1-3 also inhibited the expression and activity of PMA-induced TACE in HUVECs suggesting that p38, ERK1/2, and JNK could be molecular targets of 1-3. These results demonstrate the potential of 1-3 as an anti-EPCR shedding reagent against PMA-mediated and CLP-mediated EPCR shedding. PMID:27012760

  1. Inhibitory effects of three diketopiperazines from marine-derived bacteria on endothelial protein C receptor shedding in human endothelial cells and mice.

    PubMed

    Lee, Wonhwa; Ku, Sae-Kwang; Choi, Hyukjae; Bae, Jong-Sup

    2016-04-01

    Diketopiperazine is a natural products found from bacteria, fungi, marine sponges, gorgonian and red algae. They are cyclic dipeptides possessing relatively simple and rigid structures with chiral nature and various side chains. The compounds in this structure class have been known to possess diverse bioactivities including antibiotic activity, anti-cancer activity, neuroprotective activity, and anti-inflammatory activity. The endothelial cell protein C receptor (EPCR) plays an important role in the cytoprotective pathway and in the activation of protein C. Endothelial cell protein C receptor (EPCR) can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of diketopiperazine on EPCR shedding. We investigated this issue by monitoring the effects of diketopiperazine on phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β-induced EPCR shedding in human umbilical vein endothelial cells (HUVECs), and cecal ligation and puncture (CLP)-mediated EPCR shedding in mice and underlying mechanism. Here, three (1-3) of diketopiperazines were isolated from two strains of marine-derived bacteria and 1-3 induced potent inhibition of PMA-, TNF-α-, IL-1β (in HUVECs), and CLP-induced EPCR shedding (in mice) via inhibition of phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38, janus kinase (JNK), and extracellular signal-regulated kinase (ERK) 1/2. 1-3 also inhibited the expression and activity of PMA-induced TACE in HUVECs suggesting that p38, ERK1/2, and JNK could be molecular targets of 1-3. These results demonstrate the potential of 1-3 as an anti-EPCR shedding reagent against PMA-mediated and CLP-mediated EPCR shedding.

  2. Oxidized low-density lipoprotein induces apoptotic insults to mouse cerebral endothelial cells via a Bax-mitochondria-caspase protease pathway

    SciTech Connect

    Chen, T.-G.; Chen, T.-L.; Chang, H.-C.; Tai, Y.-T.; Cherng, Y.-G.; Chang, Y.-T.; Chen, R.-M. . E-mail: rmchen@tmu.edu.tw

    2007-02-15

    Cerebral endothelial cells (CECs) are crucial components of the blood-brain barrier. Oxidized low-density lipoprotein (oxLDL) can induce cell injuries. In this study, we attempted to evaluate the effects of oxLDL on mouse CECs and its possible mechanisms. Mouse CECs were isolated from brain tissues and identified by immunocytochemical staining of vimentin and Factor VIII. oxLDL was prepared from LDL oxidation by copper sulfate. Exposure of mouse CECs to oxLDL decreased cell viability in concentration- and time-dependent manners. oxLDL time-dependently caused shrinkage of cell morphologies. Administration of oxLDL to CECs induced DNA fragmentation in concentration- and time-dependent manners. Analysis of the cell cycle revealed that oxLDL concentration- and time-dependently increased the proportion of CECs which underwent apoptosis. Analysis of confocal microscopy and immunoblot revealed that oxLDL significantly increased cellular and mitochondrial Bax levels as well as the translocation of this proapoptotic protein from the cytoplasm to mitochondria. In parallel with the increase in the levels and translocation of Bax, oxLDL time-dependently decreased the mitochondrial membrane potential. Exposure of mouse CECs to oxLDL decreased the amounts of mitochondrial cytochrome c, but enhanced cytosolic cytochrome c levels. The amounts of intracellular reactive oxygen species were significantly augmented after oxLDL administration. Sequentially, oxLDL increased activities of caspase-9, -3, and -6 in time-dependent manners. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, significantly decreased caspase-6 activity and the oxLDL-induced DNA fragmentation and cell apoptosis. This study showed that oxLDL induces apoptotic insults to CECs via signal-transducing events, including enhancing Bax translocation, mitochondrial dysfunction, cytochrome c release, increases in intracellular reactive oxygen species, and cascade activation of caspase-9, -3, and -6. Therefore, ox

  3. Oxidized low-density lipoprotein induces apoptotic insults to mouse cerebral endothelial cells via a Bax-mitochondria-caspase protease pathway.

    PubMed

    Chen, Tyng-Guey; Chen, Ta-Liang; Chang, Huai-Chia; Tai, Yu-Ting; Cherng, Yih-Giun; Chang, Ya-Ting; Chen, Ruei-Ming

    2007-02-15

    Cerebral endothelial cells (CECs) are crucial components of the blood-brain barrier. Oxidized low-density lipoprotein (oxLDL) can induce cell injuries. In this study, we attempted to evaluate the effects of oxLDL on mouse CECs and its possible mechanisms. Mouse CECs were isolated from brain tissues and identified by immunocytochemical staining of vimentin and Factor VIII. oxLDL was prepared from LDL oxidation by copper sulfate. Exposure of mouse CECs to oxLDL decreased cell viability in concentration- and time-dependent manners. oxLDL time-dependently caused shrinkage of cell morphologies. Administration of oxLDL to CECs induced DNA fragmentation in concentration- and time-dependent manners. Analysis of the cell cycle revealed that oxLDL concentration- and time-dependently increased the proportion of CECs which underwent apoptosis. Analysis of confocal microscopy and immunoblot revealed that oxLDL significantly increased cellular and mitochondrial Bax levels as well as the translocation of this proapoptotic protein from the cytoplasm to mitochondria. In parallel with the increase in the levels and translocation of Bax, oxLDL time-dependently decreased the mitochondrial membrane potential. Exposure of mouse CECs to oxLDL decreased the amounts of mitochondrial cytochrome c, but enhanced cytosolic cytochrome c levels. The amounts of intracellular reactive oxygen species were significantly augmented after oxLDL administration. Sequentially, oxLDL increased activities of caspase-9, -3, and -6 in time-dependent manners. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, significantly decreased caspase-6 activity and the oxLDL-induced DNA fragmentation and cell apoptosis. This study showed that oxLDL induces apoptotic insults to CECs via signal-transducing events, including enhancing Bax translocation, mitochondrial dysfunction, cytochrome c release, increases in intracellular reactive oxygen species, and cascade activation of caspase-9, -3, and -6. Therefore, ox

  4. Functional Mineralocorticoid Receptors in Human Vascular Endothelial Cells Regulate ICAM-1 Expression and Promote Leukocyte Adhesion

    PubMed Central

    Caprio, Massimiliano; Newfell, Brenna G.; la Sala, Andrea; Baur, Wendy; Fabbri, Andrea; Rosano, Giuseppe; Mendelsohn, Michael E.; Jaffe, Iris Z.

    2008-01-01

    In clinical trials, aldosterone antagonists decrease cardiovascular mortality and ischemia by unknown mechanisms. The steroid hormone aldosterone acts by binding to the mineralocorticoid receptor (MR), a ligand-activated transcription factor. In humans, aldosterone causes MR-dependent endothelial cell (EC) dysfunction and in animal models, aldosterone increases vascular macrophage infiltration and atherosclerosis. MR antagonists inhibit these effects without changing blood pressure, suggesting a direct role for vascular MR in EC function and atherosclerosis. Whether human vascular EC express functional MR is not known. Here we show that human coronary artery and aortic EC express MR mRNA and protein and that EC MR mediates aldosterone-dependent gene transcription. Human EC also express the enzyme 11-beta hydroxysteroid dehydrogenase-2(11βHSD2) and inhibition of 11βHSD2 in aortic EC enhances gene transactivation by cortisol, supporting that EC 11βHSD2 is functional. Furthermore, aldosterone stimulates transcription of the proatherogenic leukocyte-EC adhesion molecule Intercellular Adhesion Molecule-1(ICAM1) gene and protein expression on human coronary artery EC, an effect inhibited by the MR antagonist spironolactone and by MR knock-down with siRNA. Cell adhesion assays demonstrate that aldosterone promotes leukocyte-EC adhesion, an effect that is inhibited by spironolactone and ICAM1 blocking antibody, supporting that aldosterone induction of EC ICAM1 surface expression via MR mediates leukocyte-EC adhesion. These data show that aldosterone activates endogenous EC MR and proatherogenic gene expression in clinically important human EC. These studies describe a novel mechanism by which aldosterone may influence ischemic cardiovascular events and support a new explanation for the decrease in ischemic events in patients treated with aldosterone antagonists. PMID:18467630

  5. APP Metabolism Regulates Tau Proteostasis in Human Cerebral Cortex Neurons

    PubMed Central

    Moore, Steven; Evans, Lewis D.B.; Andersson, Therese; Portelius, Erik; Smith, James; Dias, Tatyana B.; Saurat, Nathalie; McGlade, Amelia; Kirwan, Peter; Blennow, Kaj; Hardy, John; Zetterberg, Henrik; Livesey, Frederick J.

    2015-01-01

    Summary Accumulation of Aβ peptide fragments of the APP protein and neurofibrillary tangles of the microtubule-associated protein tau are the cellular hallmarks of Alzheimer’s disease (AD). To investigate the relationship between APP metabolism and tau protein levels and phosphorylation, we studied human-stem-cell-derived forebrain neurons with genetic forms of AD, all of which increase the release of pathogenic Aβ peptides. We identified marked increases in intracellular tau in genetic forms of AD that either mutated APP or increased its dosage, suggesting that APP metabolism is coupled to changes in tau proteostasis. Manipulating APP metabolism by β-secretase and γ-secretase inhibition, as well as γ-secretase modulation, results in specific increases and decreases in tau protein levels. These data demonstrate that APP metabolism regulates tau proteostasis and suggest that the relationship between APP processing and tau is not mediated solely through extracellular Aβ signaling to neurons. PMID:25921538

  6. Isolation and characterization of lymphatic endothelial cells from human glossal lymphangioma.

    PubMed

    You, Lanying; Wu, Mingfu; Chen, Ying; Xu, Gang; Wei, Juncheng; Li, Qiong; Song, Anping; Zhao, Liangpin; Li, Shuang; Han, Zhiqiang; Zhou, Jianfeng; Lu, Yunpin; Wang, Shixuan; Ma, Ding; Meng, Li

    2010-01-01

    Abnormal lymphangiogenesis is associated with several diseases such as tumor metastasis and lymphangioma. Human lymphangioma originated from the transformation of lymphatic endothelium is a benign malformation of lymphatic vessels and its pathogenesis has up to date not been illuminated and its cell model has also not been established. An optimized method was used to isolate lymphatic endothelial cells from human glossal lymphangioma (GL-LECs) and GL-LECs were further primarily cultured and expanded. GL-LECs were of typical cobblestone appearance when they reached confluence. The weible-palade body was observed in the GL-LECs cytoplasm. Almost all GL-LECs were strongly positive for specific lymphatic markers FLT-4, LYVE-1 and prox-1 by immunocytochemistry. Furthermore, three-dimension tube-like capillaries of GL-LECs resembled the lymphatic system in vivo, and the GL-LECs spheroids sprouted radically out to form three-dimensional buds when embedded in the cultured BME. These results indicated that high purity GL-LECs were successfully isolated and expanded. They had the abilities of tube formation and differentiation in vitro, which provide a favorable cell model for further uncovering the pathogenesis of human lymphangiomas.

  7. Distinct innate immune responses in human macrophages and endothelial cells infected with shrew-borne hantaviruses.

    PubMed

    Shin, Ok Sarah; Yanagihara, Richard; Song, Jin-Won

    2012-12-01

    Although hantaviruses have been previously considered as rodent-borne pathogens, recent studies demonstrate genetically distinct hantaviruses in evolutionarily distant non-rodent reservoirs, including shrews, moles and bats. The immunological responses to these newfound hantaviruses in humans are unknown. We compared the innate immune responses to Imjin virus (MJNV) and Thottapalayam virus (TPMV), two shrew-borne hantaviruses, with that toward two rodent-borne hantaviruses, pathogenic Hantann virus (HTNV) and nonpathogenic Prospect Hill virus (PHV). Infection of human macrophages and endothelial cells with either HTNV or MJNV triggered productive viral replication and up-regulation of anti-viral responsive gene expression from day 1 to day 3 postinfection, compared with PHV and TPMV. Furthermore, HTNV, MJNV and TPMV infection led to prolonged increased production of pro-inflammatory cytokines from days 3 to 7 postinfection. By contrast, PHV infection failed to induce pro-inflammatory responses. Distinct patterns of innate immune activation caused by MJNV suggest that it might be pathogenic to humans. PMID:22944108

  8. A monoclonal antibody to OspA inhibits association of Borrelia burgdorferi with human endothelial cells.

    PubMed Central

    Comstock, L E; Fikrig, E; Shoberg, R J; Flavell, R A; Thomas, D D

    1993-01-01

    Previously, it has been shown that polyclonal antibodies to Borrelia burgdorferi and some monoclonal antibodies (MAbs) to borrelia major surface proteins caused inhibition of adherence of the bacteria to cultured human umbilical vein endothelial (HUVE) cells. In this study, fragment antigen binding (Fab) molecules generated from the immunoglobulin G fraction of rabbit anti-recombinant OspA serum were found to inhibit the adherence of B. burgdorferi to HUVE cells by 73%. Subsequently, MAbs were generated for use in determining whether or how B. burgdorferi outer surface proteins (Osps) A and/or B are involved in mediating attachment to, and/or invasion of, HUVE cells by B. burgdorferi. Twenty-two MAbs were generated to borrelial proteins with apparent molecular masses (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 19, 31 (OspA), 34 (OspB), and 35 kDa. Fab molecules from one anti-OspA MAb, 9B3D, demonstrated an inhibitory effect on bacterial association with HUVE cells. None of the other MAbs, including the other anti-OspA MAbs, showed an inhibitory effect on cell association of greater than 5%. This effect of Fab 9B3D was concentration dependent and plateaued at approximately 6 micrograms of Fab per ml (nearly 80% inhibition of the bacterial association with the monolayer). Penetration assays and cell association experiments performed by using immunofluorescence also suggested that the inhibitory action of 9B3D occurs at the level of adherence. MAb 9B3D recognized the OspA of every North American strain tested (n = 19) but only 3 [corrected] of 20 strains from western Europe, Russia, and Japan, suggesting that the North American strains and strains from other parts of the world may use different molecules and/or different OspA epitopes to interact with endothelial cells. Immunoblots of Escherichia coli expressing different OspA fusion peptides suggested that the 9B3D epitope resides in the carboxy-terminal half of OspA. MAb 9B3D

  9. The Influence of C-Ions and X-rays on Human Umbilical Vein Endothelial Cells.

    PubMed

    Helm, Alexander; Lee, Ryonfa; Durante, Marco; Ritter, Sylvia

    2016-01-01

    Damage to the endothelium of blood vessels, which may occur during radiotherapy, is discussed as a potential precursor to the development of cardiovascular disease. We thus chose human umbilical vein endothelial cells as a model system to examine the effect of low- and high-linear energy transfer (LET) radiation. Cells were exposed to 250 kV X-rays or carbon ions (C-ions) with the energies of either 9.8 MeV/u (LET = 170 keV/μm) or 91 MeV/u (LET = 28 keV/μm). Subculture of cells was performed regularly up to 46 days (~22 population doublings) post-irradiation. Immediately after exposure, cells were seeded for the colony forming assay. Additionally, at regular intervals, mitochondrial membrane potential (MMP) (JC-1 staining) and cellular senescence (senescence-associated β-galactosidase staining) were assessed. Cytogenetic damage was investigated by the micronucleus assay and the high-resolution multiplex fluorescence in situ hybridization (mFISH) technique. Analysis of radiation-induced damage shortly after exposure showed that C-ions are more effective than X-rays with respect to cell inactivation or the induction of cytogenetic damage (micronucleus assay) as observed in other cell systems. For 9.8 and 91 MeV/u C-ions, relative biological effectiveness values of 2.4 and 1.5 were obtained for cell inactivation. At the subsequent time points, the number of micronucleated cells decreased to the control level. Analysis of chromosomal damage by mFISH technique revealed aberrations frequently involving chromosome 13 irrespective of dose or radiation quality. Disruption of the MMP was seen only a few days after exposure to X-rays or C-ions. Cellular senescence was not altered by radiation at any time point investigated. Altogether, our data indicate that shortly after exposure C-ions were more effective in damaging endothelial cells than X-rays. However, late damage to endothelial cells was not found for the applied conditions and endpoints.

  10. Desflurane Preconditioning Induces Oscillation of NF-κB in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Miao, Changhong; Tang, Jianguo; Zhu, Biao

    2013-01-01

    Background Nuclear factor kappa B (NF-κB) has been implicated in anesthetic preconditioning (APC) induced protection against anoxia and reoxygenation (A/R) injury. The authors hypothesized that desflurane preconditioning would induce NF-κB oscillation and prevent endothelial cells apoptosis. Methods A human umbilical vein endothelial cells (HUVECs) A/R injury model was used. A 30 minute desflurane treatment was initiated before anoxia. NF-κB inhibitor BAY11-7082 was administered in some experiments before desflurane preconditioning. Cells apoptosis was analyzed by flow cytometry using annexin V–fluorescein isothiocyanate staining and cell viability was evaluated by modified tertrozalium salt (MTT) assay. The cellular superoxide dismutases (SOD) activitiy were tested by water-soluble tetrazolium salt (WST-1) assay. NF-κB p65 subunit nuclear translocation was detected by immunofluorescence staining. Expression of inhibitor of NF-κB-α (IκBα), NF-κB p65 and cellular inhibitor of apoptosis 1 (c-IAP1), B-cell leukemia/lymphoma 2 (Bcl-2), cysteine containing aspartate specific protease 3 (caspases-3) and second mitochondrial-derived activator of caspase (SMAC/DIABLO) were determined by western blot. Results Desflurane preconditioning caused phosphorylation and nuclear translocation of NF-κB before anoxia, on the contrary, induced the synthesis of IκBα and inhibition of NF-κB after reoxygenation. Desflurane preconditioning up-regulated the expression of c-IAP1 and Bcl-2, blocked the cleavage of caspase-3 and reduced SMAC release, and decreased the cell death of HUVECs after A/R. The protective effect was abolished by BAY11-7082 administered before desflurane. Conclusions The results demonstrated that desflurane activated NF-κB during the preconditioning period and inhibited excessive activation of NF-κB in reperfusion. And the oscillation of NF-κB induced by desflurane preconditioning finally up-regulated antiapoptotic proteins expression and protected

  11. P2 receptor expression profiles in human vascular smooth muscle and endothelial cells.

    PubMed

    Wang, Lingwei; Karlsson, Lena; Moses, Sara; Hultgårdh-Nilsson, Anna; Andersson, Maria; Borna, Catharina; Gudbjartsson, Tomas; Jern, Sverker; Erlinge, David

    2002-12-01

    P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP, and UDP, regulating several physiologic responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. P2 receptor characterization has been hampered by the lack of selective antagonists. The aim of the current study was to investigate the mRNA and protein expression of P2X and P2Y receptors in human SMC and in endothelial cells (EC). Smooth muscle cells were obtained from human mammary artery and EC from human umbilical vein. Using real-time PCR, the authors established quantitative mRNA assays. Protein expression was studied using Western blotting with recently developed antibodies. The P2X1 receptor was highly specific for human SMC, while the P2X4 was the highest expressed receptor in EC. The P2Y2 receptor was present in both SMC and EC. UTP-mediated effects in these cells are likely to be mediated by P2Y2 and not P2Y4 receptors since the latter had considerably lower expression. The P2Y6 receptor was expressed in both SMC and EC. The P2Y1 and surprisingly the P2Y11 receptors were the most abundantly expressed P2Y receptors in the endothelium. Overall, Western blotting confirmed the mRNA findings in most aspects, and most interestingly, indicated oligomerization of the P2Y1 receptor that may be important for its function. In conclusion, P2X1, P2Y2, and P2Y6 are the most expressed P2 receptors in SMC and are thus probably mediating the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1, and P2Y2 are the most expressed P2 receptors in EC, and are most likely mediating release of nitric oxide, endothelium-dependent hyperpolarizing factor (EDHF), and t-PA induced by extracellular nucleotides. These findings will help to direct future cardiovascular drug development against the large P2 receptor family.

  12. Screening bioactive compounds from Ligusticum chuanxiong by high density immobilized human umbilical vein endothelial cells.

    PubMed

    Li, Qian; Wang, Jing; Liu, Guangxin; Sun, Huanmei; Bian, Liujiao; Zhao, Xinfeng; Zheng, Xiaohui

    2015-07-01

    High throughput screening methodologies play a very important role in screening bioactive compounds from complex media. In this work, a new strategy for attaching cells onto amino microspheres using human umbilical vein endothelial cells (HUVECs) as a probe was developed. The immobilization depended on the specific affinity between integrin on the cells and the RGD peptide, which was coated on poly[oligo (ethylene glycol) methacrylate] by atom transfer radical polymerization. Validated application of the stationary phase was performed in the analysis of Ligusticum chuanxiong extraction by high performance affinity chromatography-mass spectrometry. Three compounds were screened as the bioactive compounds of Ligusticum chuanxiong. Two of them were identified as 3-butyl-hexahydroisobenzofuran-1(3H)-one and tetramethylpyrazine (TMP), whereas the other one remains indistinct. The association constant of vascular endothelial growth factor (VEGF) and TMP binding to VEGF receptor (VEGFR) on HUVECs were calculated to be (1.04 ± 0.08) × 10(11) M(-1) and (9.84 ± 1.11) × 10(8) M(-1) by zonal elution. Molecular docking showed that one hydrogen bond was formed between N atom of TMP and 3-N atom of imidazole group in histidine(223) of VEGFR. Both zonal elution and molecular docking indicated that TMP and VEGF bind to the same site of VEGFR on HUVECs. It is possible to become a promising tool for high throughput screening of the bioactive compounds binding to HUVECs through broad application of the stationary phase. PMID:26018629

  13. MiR-29a modulates the angiogenic properties of human endothelial cells

    SciTech Connect

    Yang, Zeran; Wu, Lingjiao; Zhu, Xiuming; Xu, Jie; Jin, Rong; Li, Guohong; Wu, Fusheng

    2013-04-26

    Highlights: •miR-29a may be stimulated by hypoxia in HUVEC. •miR-29a regulates cell cycle, proliferation and tube network formation of HUVEC. •HMG box-containing protein-1(HBP1) is a direct target of miR-29a. •miR-29a has a potential value for treating angiogenesis-associated diseases. -- Abstract: Although extensive investigation has been made on miR-29a in relation to malignancies, only a little information has been provided about the angiogenic property of this miRNA so far. Herein, we sought to investigate the role of miR-29a in regulating cell cycle and angiogenic phenotype of endothelial cells. The results showed that miR-29a is highly expressed and upregulated by hypoxia-mimicking reagents in human umbilical vein endothelial cells (HUVEC). Consistent with this preliminary finding, introduction of exogenous agomiR-29a, or Antagomir-29a altered cell cycle progression and promoted, or repressed the proliferation and tube formation of HUVEC, respectively. Furthermore, by using luciferase reporter assay, the expression of HBP1, a suppressor transcription factor was directly regulated by miR-29a through 3′-UTR. Increased or decreased HBP1 protein level was associated with the inhibition or overexpression of miR-29a, respectively. We conclude that miR-29a has a significant role in regulating cell cycle, proliferation and angiogenic properties of HUVEC, and this function is likely mediated through HBP1 protein at the post-transcriptional level. As a novel molecular target, miR-29a may have a potential value for the treatment of angiogenesis-associated diseases such as cardiovascular diseases and cancers.

  14. Sulodexide suppresses inflammation in human endothelial cells and prevents glucose cytotoxicity.

    PubMed

    Ciszewicz, Marta; Polubinska, Alicja; Antoniewicz, Artur; Suminska-Jasinska, Katarzyna; Breborowicz, Andrzej

    2009-03-01

    Sulodexide is a mixture of heparin and dermatan sulfate with antithrombotic and profibrynolytic activity. Individual reports suggest the anti-inflammatory action of sulodexin. The goal of this study was to evaluate the effect of sulodexide on the release of the inflammatory mediators from endothelium in normal conditions and in cells chronically exposed to glucose. The experiments were performed on in vitro cultured human umbilical endothelial cells kept for 7 days in standard medium or in the same medium but supplemented with glucose 30 mmol/L. Sulodexide was added to the culture medium in concentrations of 0.125 lipase releasing unit (LRU)/mL, 0.25 LRU/mL, and 0.5 LRU/mL Spontaneous generation of oxygen-derived free radicals and the release of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) from the studied cells was evaluated. Additionally, the healing of the injured mesothelium was studied in the presence of sulodexide and glucose. Sulodexide caused the inhibition of the intracellular generation of free radicals in a dose-dependent manner (maximally by 32%, P < 0.01), as well as the inhibition of MCP-1 (maximally by 60%, P < 0.001) and IL-6 (maximally by 69%, P < 0.01). Cells cultured in a medium with glucose 30 mmol/L generated more free radicals (+20%, P < 0.05) and released more MCP-1 (+113%, P < 0.001) and IL-6 (+26%, P < 0.05). Cell monolayers treated with glucose had a decreased ability to heal after mechanical injury (-28%, P < 0.001). All these glucose effects were reversed when cells were exposed to sulodexide simultaneously. The results of our study demonstrate a significant anti-inflammatory action of sulodexide in the endothelial cells and a protective effect of that drug against glucose cytotoxicity. PMID:19218094

  15. Bradykinin Preconditioning Improves Therapeutic Potential of Human Endothelial Progenitor Cells in Infarcted Myocardium

    PubMed Central

    Li, Yefei; Yan, Fengdi; Huang, Jie; Ma, Genshan

    2013-01-01

    Objectives Stem cell preconditioning (PC) is a powerful approach in reducing cell death after transplantation. We hypothesized that PC human endothelial progenitor cells (hEPCs) with bradykinin (BK) enhance cell survival, inhibit apoptosis and repair the infarcted myocardium. Methods The hEPCs were preconditioned with or without BK. The hEPCs apoptosis induced by hypoxia along with serum deprivation was determined by annexin V-fluorescein isothiocyanate/ propidium iodide staining. Cleaved caspase-3, Akt and eNOS expressions were determined by Western blots. Caspase-3 activity and vascular endothelial growth factor (VEGF) levels were assessed in hEPCs. For in vivo studies, the survival and cardiomyocytes apoptosis of transplanted hEPCs were assessed using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodi- carbocyanine,4-chlorobenzenesul-fonate salt labeled hEPCs and TUNEL staining. Infarct size and cardiac function were measured at 10 days after transplantation, and the survival of transplanted hEPCs were visualized using near-infrared optical imaging. Results In vitro data showed a marked suppression in cell apoptosis following BK PC. The PC reduced caspase-3 activation, increased the Akt, eNOS phosphorylation and VEGF levels. In vivo data in preconditioned group showed a robust cell anti-apoptosis, reduction in infarct size, and significant improvement in cardiac function. The effects of BK PC were abrogated by the B2 receptor antagonist HOE140, the Akt and eNOS antagonists LY294002 and L-NAME, respectively. Conclusions The activation of B2 receptor-dependent PI3K/Akt/eNOS pathway by BK PC promotes VEGF secretion, hEPC survival and inhibits apoptosis, thereby improving cardiac function in vivo. The BK PC hEPC transplantation for stem cell-based therapies is a novel approach that has potential for clinical used. PMID:24312554

  16. Discovery of molecular markers to discriminate corneal endothelial cells in the human body.

    PubMed

    Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji

    2015-01-01

    The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.

  17. Discovery of molecular markers to discriminate corneal endothelial cells in the human body.

    PubMed

    Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji