Endothelial necrosis at 1h post-burn predicts progression of tissue injury

PubMed Central

Hirth, Douglas; McClain, Steve A.; Singer, Adam J.; Clark, Richard A.F.

2013-01-01

Burn injury progression has not been well characterized at the cellular level. To define burn injury progression in terms of cell death, histopathologic spatiotemporal relationships of cellular necrosis and apoptosis were investigated in a validated porcine model of vertical burn injury progression. Cell necrosis was identified by High Mobility Group Box 1 protein and apoptosis by Caspase 3a staining of tissue samples taken 1h, 24h and 7 days post-burn. Level of endothelial cell necrosis at 1h was predictive of level of apoptosis at 24h (Pearson's r=0.87) and of level of tissue necrosis at 7 days (Pearson's r=0.87). Furthermore, endothelial cell necrosis was deeper than interstitial cell necrosis at 1h (p<0.001). Endothelial cell necrosis at 1h divided the zone of injury progression (Jackson's zone of stasis) into an upper subzone with necrotic endothelial cells and initially viable adnexal and interstitial cells at 1h that progressed to necrosis by 24h, and a lower zone with initially viable endothelial cells at 1h, but necrosis and apoptosis of all cell types by 24h. Importantly, this spatiotemporal series of events and rapid progression resembles myocardial infarction and stroke, and implicates mechanisms of these injuries, ischemia, ischemia reperfusion, and programmed cell death, in burn progression. PMID:23627744

[Circulating endothelial cells: biomarkers for monitoring activity of antiangiogenic therapy].

PubMed

Farace, Françoise; Bidart, Jean-Michel

2007-07-01

Tumor vessel formation is largely dependent on the recruitment of endothelial cells. Rare in healthy individuals, circulating endothelial cells (CEC) are shed from vessel walls and enter the circulation reflecting endothelial damage or dysfunction. Increased numbers of CEC have been documented in different types of cancer. Recent studies have suggested the role for CEC in tumor angiogenesis, but whose presence could also reflect normal endothelium perturbation in cancer. Originating from the bone marrow rather than from vessel walls, endothelial progenitor cells (EPC) are mobilized following tissue ischemia and may be recruited to complement local angiogenesis supplied by existing endothelium. Recently, studies in mouse models suggest that the circulating fraction of endothelial progenitors (CEP) is involved in tumor angiogenesis but their contribution is less clear in humans. The detection of CEC and CEP is difficult and impeded by the rarity of these cells. They may have important clinical implication as novel biomarkers susceptible to predict more efficiently and rapidly the therapeutic response to anti-angiogenic treatments. However, a methodological consensus would be necessary in order to correctly evaluate the clinical interest of CEC and CEP in patients.

Lenalidomide, an anti-tumor drug, regulates retinal endothelial cell function: Implication for treating ocular neovascular disorder

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Ling-Feng; Yao, Jin; Wang, Xiao-Qun

Ocular angiogenesis is an important pathologic character of several ocular diseases, such as retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration (AMD). Inhibition of ocular angiogenesis has great therapeutic value for treating these dieses. Here we show that lenalidomide, an anti-tumor drug, has great anti-angiogenic potential in ocular diseases. Lenalidomide inhibits retinal endothelial cell viability in normal and pathological condition, and inhibits VEGF-induced endothelial cell migration and tube formation in vitro. Moreover, lenalidomide inhibits ocular angiogenesis in vivo through the reduction of angiogenesis- and inflammation-related protein expression. Collectively, lenalidomide is a promising drug for treating ocular angiogenesis through its anti-proliferative andmore » anti-inflammatory property. - Highlights: • Lenalidomide inhibits retinal endothelial cell viability in vitro. • Lenalidomide inhibits retinal endothelial cell migration and tube formation. • Lenalidomide inhibits pathological ocular angiogenesis in vivo. • Lenalidomide inhibits angiogenesis- and inflammation-related protein expression.« less

Factors Released from Endothelial Cells Exposed to Flow Impact Adhesion, Proliferation, and Fate Choice in the Adult Neural Stem Cell Lineage.

PubMed

Dumont, Courtney M; Piselli, Jennifer M; Kazi, Nadeem; Bowman, Evan; Li, Guoyun; Linhardt, Robert J; Temple, Sally; Dai, Guohao; Thompson, Deanna M

2017-08-15

The microvasculature within the neural stem cell (NSC) niche promotes self-renewal and regulates lineage progression. Previous work identified endothelial-produced soluble factors as key regulators of neural progenitor cell (NPC) fate and proliferation; however, endothelial cells (ECs) are sensitive to local hemodynamics, and the effect of this key physiological process has not been defined. In this study, we evaluated adult mouse NPC response to soluble factors isolated from static or dynamic (flow) EC cultures. Endothelial factors generated under dynamic conditions significantly increased neuronal differentiation, while those released under static conditions stimulated oligodendrocyte differentiation. Flow increases EC release of neurogenic factors and of heparin sulfate glycosaminoglycans that increase their bioactivity, likely underlying the enhanced neuronal differentiation. Additionally, endothelial factors, especially from static conditions, promoted adherent growth. Together, our data suggest that blood flow may impact proliferation, adhesion, and the neuron-glial fate choice of adult NPCs, with implications for diseases and aging that reduce flow.

An EMMPRIN–γ-catenin–Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions

PubMed Central

Moreno, Vanessa; Gonzalo, Pilar; Gómez-Escudero, Jesús; Pollán, Ángela; Acín-Pérez, Rebeca; Breckenridge, Mark; Yáñez-Mó, María; Barreiro, Olga; Orsenigo, Fabrizio; Kadomatsu, Kenji; Chen, Christopher S.; Enríquez, José A.; Dejana, Elisabetta; Sánchez-Madrid, Francisco; Arroyo, Alicia G.

2014-01-01

ABSTRACT Cell–cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly. PMID:24994937

Early outgrowth cells versus endothelial colony forming cells functions in platelet aggregation.

PubMed

Bou Khzam, Lara; Bouchereau, Olivier; Boulahya, Rahma; Hachem, Ahmed; Zaid, Younes; Abou-Saleh, Haissam; Merhi, Yahye

2015-11-09

Endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis, endothelial repair and cell-based therapies for cardiovascular diseases. We have previously shown that the recruitment of EPCs to sites of vascular lesions is facilitated by platelets where EPCs, in turn, modulate platelet function and thrombosis. However, EPCs encompass a heterogeneous population of progenitor cells that may exert different effects on platelet function. Recent evidence suggests the existence of two EPC subtypes: early outgrowth cells (EOCs) and endothelial colony-forming cells (ECFCs). We aimed at characterizing these two EPC subtypes and at identifying their role in platelet aggregation. EOCs and ECFCs were generated from human peripheral blood mononuclear cells (PBMCs) seeded in conditioned media on fibronectin and collagen, respectively. The morphological, phenotypical and functional characteristics of EOCs and ECFCs were assessed by optical and confocal laser scanning microscopes, cell surface markers expression, and Matrigel tube formation. The impact of EOCs and ECFCs on platelet aggregation was monitored in collagen-induced optical aggregometry and compared with PBMCs and human umbilical vein endothelial cells (HUVECs). The levels of the anti-platelet agents' nitric oxide (NO) and prostacyclin (PGI2) released from cultured cells as well as the expression of their respective producing enzymes NO synthases (NOS) and cyclooxygenases (COX) were also assessed. We showed that EOCs display a monocytic-like phenotype whereas ECFCs have an endothelial-like phenotype. We demonstrated that both EOCs and ECFCs and their supernatants inhibited platelet aggregation; however ECFCs were more efficient than EOCs. This could be related to the release of significantly higher amounts of NO and PGI2 from ECFCs, in comparison to EOCs. Indeed, ECFCs, like HUVECs, constitutively express the endothelial (eNOS)-and inducible (iNOS)-NOS isoforms, and COX-1 and weakly express COX-2, whereas EOCs do not constitutively express these NO and PGI2 producing enzymes. The different morphological, phenotypic and more importantly the release of the anti-aggregating agents PGI2 and NO in each EPC subtype are implicated in their respective roles in platelet function and thus, may be linked to the increased efficiency of ECFCs in inhibiting platelet aggregation as compared to EOCs.

The role of endothelial cell attachment to elastic fibre molecules in the enhancement of monolayer formation and retention, and the inhibition of smooth muscle cell recruitment.

PubMed

Williamson, Matthew R; Shuttleworth, Adrian; Canfield, Ann E; Black, Richard A; Kielty, Cay M

2007-12-01

The endothelium is an essential modulator of vascular tone and thrombogenicity and a critical barrier between the vessel wall and blood components. In tissue-engineered small-diameter vascular constructs, endothelial cell detachment in flow can lead to thrombosis and graft failure. The subendothelial extracellular matrix provides stable endothelial cell anchorage through interactions with cell surface receptors, and influences the proliferation, migration, and survival of both endothelial cells and smooth muscle cells. We have tested the hypothesis that these desired physiological characteristics can be conferred by surface coatings of natural vascular matrix components, focusing on the elastic fiber molecules, fibrillin-1, fibulin-5 and tropoelastin. On fibrillin-1 or fibulin-5-coated surfaces, endothelial cells exhibited strong integrin-mediated attachment in static conditions (82% and 76% attachment, respectively) and flow conditions (67% and 78% cell retention on fibrillin-1 or fibulin-5, respectively, at 25 dynes/cm2), confluent monolayer formation, and stable functional characteristics. Adhesion to these two molecules also strongly inhibited smooth muscle cell migration to the endothelial monolayer. In contrast, on elastin, endothelial cells attached poorly, did not spread, and had markedly impaired functional properties. Thus, fibrillin-1 and fibulin-5, but not elastin, can be exploited to enhance endothelial stability, and to inhibit SMC migration within vascular graft scaffolds. These findings have important implications for the design of vascular graft scaffolds, the clinical performance of which may be enhanced by exploiting natural cell-matrix biology to regulate cell attachment and function.

Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress.

PubMed

Chen, Feng; Qian, Li-Hua; Deng, Bo; Liu, Zhi-Min; Zhao, Ying; Le, Ying-Ying

2013-09-01

Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes. © 2013 John Wiley & Sons Ltd.

Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis.

PubMed

Suárez, Yajaira; Fernández-Hernando, Carlos; Yu, Jun; Gerber, Scott A; Harrison, Kenneth D; Pober, Jordan S; Iruela-Arispe, M Luisa; Merkenschlager, Matthias; Sessa, William C

2008-09-16

Posttranscriptional gene regulation by microRNAs (miRNAs) is important for many aspects of development, homeostasis, and disease. Here, we show that reduction of endothelial miRNAs by cell-specific inactivation of Dicer, the terminal endonuclease responsible for the generation of miRNAs, reduces postnatal angiogenic response to a variety of stimuli, including exogenous VEGF, tumors, limb ischemia, and wound healing. Furthermore, VEGF regulated the expression of several miRNAs, including the up-regulation of components of the c-Myc oncogenic cluster miR-17-92. Transfection of endothelial cells with components of the miR-17-92 cluster, induced by VEGF treatment, rescued the induced expression of thrombospondin-1 and the defect in endothelial cell proliferation and morphogenesis initiated by the loss of Dicer. Thus, endothelial miRNAs regulate postnatal angiogenesis and VEGF induces the expression of miRNAs implicated in the regulation of an integrated angiogenic response.

Identification of a Monocyte Receptor on Herpesvirus-Infected Endothelial Cells

NASA Astrophysics Data System (ADS)

Etingin, Orli R.; Silverstein, Roy L.; Hajjar, David P.

1991-08-01

The adhesion of circulating blood cells to vascular endothelium may be an initial step in atherosclerosis, inflammation, and wound healing. One mechanism for promoting cell-cell adhesion involves the expression of adhesion molecules on the surface of the target cell. Herpes simplex virus infection of endothelium induces arterial injury and has been implicated in the development of human atherosclerosis. We now demonstrate that HSV-infected endothelial cells express the adhesion molecule GMP140 and that this requires cell surface expression of HSV glycoprotein C and local thrombin generation. Monocyte adhesion to HSV-infected endothelial cells was completely inhibited by anti-GMP140 antibodies but not by antibodies to other adhesion molecules such as VCAM and ELAM-1. The induction of GMP140 expression on HSV-infected endothelium may be an important pathophysiological mechanism in virus-induced cell injury and inflammation.

Endothelial dysfunction and amyloid-β-induced neurovascular alterations

PubMed Central

Koizumi, Kenzo; Wang, Gang; Park, Laibaik

2015-01-01

Alzheimer's disease (AD) and cerebrovascular diseases share common vascular risk factors that have disastrous effects on cerebrovascular regulation. Endothelial cells, lining inner walls of cerebral blood vessels, form a dynamic interface between the blood and the brain and are critical for the maintenance of neurovascular homeostasis. Accordingly, injury in endothelial cells is regarded as one of the earliest symptoms of impaired vasoregulatory mechanisms. Extracellular buildup of amyloid-β (Aβ) is a central pathogenic factor in AD. Aβ exerts potent detrimental effects on cerebral blood vessels and impairs endothelial structure and function. Recent evidence implicates vascular oxidative stress and activation of the nonselective cationic channel transient receptor potential melastatin (TRPM)-2 on endothelial cells in the mechanisms of Aβ-induced neurovascular dysfunction. Thus, Aβ triggers opening of TRPM2 channels in endothelial cells leading to intracellular Ca2+ overload and vasomotor dysfunction. The cerebrovascular dysfunction may contribute to AD pathogenesis by reducing the cerebral blood supply, leading to increased susceptibility to vascular insufficiency, and by promoting Aβ accumulation. The recent realization that vascular factors contribute to AD pathobiology suggests new targets for the prevention and treatment of this devastating disease. PMID:26328781

Endothelial progenitor cells--an evolving story.

PubMed

Pearson, Jeremy D

2010-05-01

The first description of endothelial progenitor cells (EPC) in 1997 led rapidly to substantial changes in our understanding of angiogenesis, and within 5 years to the first clinical studies in humans using bone marrow derived EPC to enhance coronary neovascularisation and cardiac function after myocardial ischemia. However, to improve the success of this therapy a clearer understanding of the biology of EPC is needed. This article summarises recent data indicating that most EPC are not, in fact, endothelial progenitors but can be better described as angiogenic monocytes, and explores the implications this has for their future therapeutic use. Copyright 2009 Elsevier Inc. All rights reserved.

Polymeric stent materials dysregulate macrophage and endothelial cell functions: implications for coronary artery stent

PubMed Central

Wang, Xintong; Zachman, Angela L.; Chun, Young Wook; Shen, Fang-Wen; Hwang, Yu-Shik; Sung, Hak-Joon

2014-01-01

Background Biodegradable polymers have been applied as bulk or coating materials for coronary artery stents. The degradation of polymers, however, could induce endothelial dysfunction and aggravate neointimal formation. Here we use polymeric microparticles to simulate and demonstrate the effects of degraded stent materials on phagocytic activity, cell death and dysfunction of macrophages and endothelial cells. Methods Microparticles made of low molecular weight polyesters were incubated with human macrophages and coronary artery endothelial cells (ECs). Microparticle-induced phagocytosis, cytotoxicity, apoptosis, cytokine release and surface marker expression were determined by immunostaining or ELISA. Elastase expression was analyzed by ELISA and the elastase-mediated polymer degradation was assessed by mass spectrometry. Results We demonstrated poly(D,L-lactic acid) (PLLA) and polycaprolactone (PCL) microparticles induced cytotoxicity in macrophages and ECs, partially through cell apoptosis. The particle treatment alleviated EC phagocytosis, as opposed to macrophages, but enhanced the expression of vascular cell adhesion molecule-1 (VCAM) along with decreased nitric oxide production, indicating ECs were activated and lost their capacity to maintain homeostasis. The activation of both cell types induced release of elastase or elastase-like protease, which further accelerated polymer degradation. Conclusions This study revealed that low molecule weight PLLA and PCL microparticles increased cytotoxicity and dysregulated endothelial cell function, which in turn enhanced elastase release and polymer degradation. These indicate polymer or polymer-coated stents impose a risk of endothelial dysfunction after deployment which can potentially lead to delayed endothelialization, neointimal hyperplasia and late thrombosis. PMID:24820736

Sulfoglucuronosyl paragloboside promotes endothelial cell apoptosis in inflammation: Elucidation of a novel glycosphingolipid-signaling pathway

PubMed Central

Dasgupta, Somsankar; Wang, Guanghu; Yu, Robert K.

2011-01-01

Sulfoglucuronosyl paragloboside (SGPG), a minor glycosphingolipid (GSL) of endothelial cells, is a ligand for L-selectin and has been implicated in neuro-inflammatory diseases, such as Guillian-Barré syndrome. Inflammatory cytokines, such as TNFα and IL-1β, up-regulate SGPG expression by stimulating gene expression for glucuronosyltransferases, both P and S forms (GlcATp and GlcATs), and the HNK-1 sulfotransferase (HNK-1 ST). Transfection of a human cerebromicrovascular endothelial cell (SV-HCEC) line with HNK-1 ST siRNA down-regulated SGPG expression, inhibited cytokine-stimulated T cell adhesion, and offered protection against apoptosis. However, the precise mechanisms of SGPG elevation in endothelial cell death (apoptosis) and the maintenance of blood-brain or blood-nerve barrier (BBB or BNB) integrity in inflammation have not been elucidated. Blocking SGPG expression inhibited cytokine-mediated stimulation of NF-κB activity but stimulated MAP kinase (ERK) activity. Furthermore, elevation of SGPG by over-expression of GlcATp and GlcATs triggered endothelial cell apoptosis, with GlcATs being more potent than GlcATp. While SGPG-mediated endothelial cell apoptosis was preceded by inhibiting the intracellular NF-κB activity, interfering with Akt and ERK activation and stimulating caspase 3 in SV-HCECs, HNK-1ST siRNA transfection also interfered with IKB phosphorylation but stimulated ERK activation. Our data indicate that SGPG is a critical regulatory molecule for maintaining endothelial cell survival and BBB/BNB barrier function. PMID:21916893

Rapid flow-induced responses in endothelial cells

NASA Technical Reports Server (NTRS)

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

2001-01-01

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

Young endothelial cells revive aging blood.

PubMed

Chang, Vivian Y; Termini, Christina M; Chute, John P

2017-11-01

The hematopoietic system declines with age, resulting in decreased hematopoietic stem cell (HSC) self-renewal capacity, myeloid skewing, and immune cell depletion. Aging of the hematopoietic system is associated with an increased incidence of myeloid malignancies and a decline in adaptive immunity. Therefore, strategies to rejuvenate the hematopoietic system have important clinical implications. In this issue of the JCI, Poulos and colleagues demonstrate that infusions of bone marrow (BM) endothelial cells (ECs) from young mice promoted HSC self-renewal and restored immune cell content in aged mice. Additionally, delivery of young BM ECs along with HSCs following total body irradiation improved HSC engraftment and enhanced survival. These results suggest an important role for BM endothelial cells (ECs) in regulating hematopoietic aging and support further research to identify the rejuvenating factors elaborated by BM ECs that restore HSC function and the immune repertoire in aged mice.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colleselli, Daniela; Bijuklic, Klaudija; Mosheimer, Birgit A.

2006-09-10

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

PCB 126 toxicity is modulated by cross-talk between caveolae and Nrf2 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petriello, Michael C.; University of Kentucky Superfund Research Center, Lexington, KY 40536; Han, Sung Gu

2014-06-01

Environmental toxicants such as polychlorinated biphenyls (PCBs) have been implicated in the promotion of multiple inflammatory disorders including cardiovascular disease, but information regarding mechanisms of toxicity and cross-talk between relevant cell signaling pathways is lacking. To examine the hypothesis that cross-talk between membrane domains called caveolae and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways alters PCB-induced inflammation, caveolin-1 was silenced in vascular endothelial cells, resulting in a decreased PCB-induced inflammatory response. Cav-1 silencing (siRNA treatment) also increased levels of Nrf2-ARE transcriptional binding, resulting in higher mRNA levels of the antioxidant genes glutathione s-transferase and NADPH dehydrogenase quinone-1 in both vehiclemore » and PCB-treated systems. Along with this upregulated antioxidant response, Cav-1 siRNA treated cells exhibited decreased mRNA levels of the Nrf2 inhibitory protein Keap1 in both vehicle and PCB-treated samples. Silencing Cav-1 also decreased protein levels of Nrf2 inhibitory proteins Keap1 and Fyn kinase, especially in PCB-treated cells. Further, endothelial cells from wildtype and Cav-1 −/− mice were isolated and treated with PCB to better elucidate the role of functional caveolae in PCB-induced endothelial inflammation. Cav-1 −/− endothelial cells were protected from PCB-induced cellular dysfunction as evidenced by decreased vascular cell adhesion molecule (VCAM-1) protein induction. Compared to wildtype cells, Cav-1 −/− endothelial cells also allowed for a more effective antioxidant response, as observed by higher levels of the antioxidant genes. These data demonstrate novel cross-talk mechanisms between Cav-1 and Nrf2 and implicate the reduction of Cav-1 as a protective mechanism for PCB-induced cellular dysfunction and inflammation. - Highlights: • Reduction of caveolin-1 protein protects against polychlorinated biphenyl toxicity. • Decreasing caveolin-1 levels increases the Nrf2 antioxidant response. • Reducing caveolin-1 levels decreases expression of Nrf2 inhibitory proteins. • Caveolin-1/Nrf2 cross-talk is evident in mouse, human, and porcine endothelial cells.« less

Hypercholesterolemia potentiates aortic endothelial response to inhaled diesel exhaust

PubMed Central

Maresh, J. Gregory; Campen, Matthew J.; Reed, Matthew D.; Darrow, April L.; Shohet, Ralph V.

2012-01-01

Background Inhalation of diesel exhaust induces vascular effects including impaired endothelial function and increased atherosclerosis. Objective To examine the in vivo effects of subchronic diesel exhaust exposure on endothelial cell transcriptional responses in the presence of hypercholesterolemia. Methods ApoE (−/−) and ApoE (+/+) mice inhaled diesel exhaust diluted to particulate matter levels of 300 or 1000 μg/m3 vs. filtered air. After 30 days, endothelial cells were harvested from dispersed aortic cells by fluorescent-activated cell sorting (FACS). Relative mRNA abundance was evaluated by microarray analysis to measure strain-specific transcriptional responses in mice exposed to dilute diesel exhaust vs. filtered air. Results Forty-nine transcripts were significantly dysregulated by >2.8-fold in the endothelium of ApoE (−/−) mice receiving diesel exhaust at 300 or 1000 μg/m3. These included transcripts with roles in plasminogen activation, endothelial permeability, inflammation, genomic stability, and atherosclerosis; similar responses were not observed in ApoE (+/+) mice. Conclusions The potentiation of diesel exhaust-related endothelial gene regulation by hypercholesterolemia helps to explain air pollution-induced vascular effects in animals and humans. The observed regulated transcripts implicate pathways important in the acceleration of atherosclerosis by air pollution. PMID:21222557

The effect of fibrin on cultured vascular endothelial cells.

PubMed

Kadish, J L; Butterfield, C E; Folkman, J

1979-01-01

The normal cobblestone monolayer architecture of cultured vascular endothelium becomes rapidly disorganized after contact of the cell layer with a fibrin clot. The cells of a confluent endothelial monolayer separate into individual migratory cells in 4--6 hr after contact with fibrin. The effect is reversible in that removal of the fibrin clot results in resumption of the normal morphology within about 2 hr. No other cell type tested exhibits the same change in organization when exposed to fibrin. A similar morphological change in endothelium does occur after the cell layer is overlaid with a collagen fibril gel but a gel of methylcellulose has no effect. It is proposed that the change in behavior of endothelial cells in response to contact with fibrin may represent a cellular component of fibrinolysis. The implications of this finding for the pathophysiology of disease states involving intravascular fibrin deposition are discussed.

Drug-induced in vitro inhibition of neutrophil-endothelial cell adhesion.

PubMed Central

Pellegatta, F.; Lu, Y.; Radaelli, A.; Zocchi, M. R.; Ferrero, E.; Chierchia, S.; Gaja, G.; Ferrero, M. E.

1996-01-01

1. Leukocyte-endothelial cell interactions play an important role during ischaemia-reperfusion events. Adhesion molecules are specifically implicated in this interaction process. 2. Since defibrotide has been shown to be an efficient drug in reducing damage due to ischaemia-reperfusion in many experimental models, we analysed the effect of defibrotide in vitro on leukocyte adhesion to endothelial cells in basal conditions and after their stimulation. 3. In basal conditions, defibrotide (1000 micrograms ml-1) partially inhibited leukocyte adhesion to endothelial cells by 17.3% +/- 3.6 (P < 0.05), and after endothelial cell stimulation (TNF-alpha, 500 u ml-1) or after leukocyte stimulation (fMLP, 10(-7) M), it inhibited leukocyte adhesion by 26.5% +/- 3.4 and 32.4% +/- 1.8, respectively (P < 0.05). 4. In adhesion blockage experiments, the use of the monoclonal antibody anti-CD31 (5 micrograms ml-1) did not demonstrate a significant inhibitory effect whereas use of the monoclonal antibody anti-LFA-1 (5 micrograms ml-1) significantly interfered with the effect of defibrotide. 5. This result was confirmed in NIH/3T3-ICAM-1 transfected cells. 6. We conclude that defibrotide is able to interfere with leukocyte adhesion to endothelial cells mainly in activated conditions and that the ICAM-1/LFA-1 adhesion system is involved in the defibrotide mechanism of action. PMID:8762067

Antioxidant betalains from cactus pear (Opuntia ficus-indica) inhibit endothelial ICAM-1 expression.

PubMed

Gentile, C; Tesoriere, L; Allegra, M; Livrea, M A; D'Alessio, P

2004-12-01

It has been suggested that some pigments would have antioxidant properties and that their presence in dietary constituents would contribute to reduce the risk of oxidative stress-correlated diseases. Among others, inflammatory response depends on redox status and may implicate oxidative stress. Vascular endothelial cells are a direct target of oxidative stress in inflammation. We have tested the impact of the free radical scavenger and antioxidant properties of betalains from the prickle pear in an in vitro model of endothelial cells. Here we show the capacity of betalains to protect endothelium from cytokine-induced redox state alteration, through ICAM-1 inhibition.

Genetic ablation of the alpha 6-integrin subunit in Tie1Cre mice enhances tumour angiogenesis.

PubMed

Germain, Mitchel; De Arcangelis, Adèle; Robinson, Stephen D; Baker, Marianne; Tavora, Bernardo; D'Amico, Gabriela; Silva, Rita; Kostourou, Vassiliki; Reynolds, Louise E; Watson, Alan; Jones, J Louise; Georges-Labouesse, Elisabeth; Hodivala-Dilke, Kairbaan

2010-02-01

Laminins are expressed highly in blood vessel basement membranes and have been implicated in angiogenesis. alpha6beta1- and alpha6beta4-integrins are major receptors for laminins in endothelial cells, but the precise role of endothelial alpha6-integrin in tumour angiogenesis is not clear. We show that blood vessels in human invasive ductal carcinoma of the breast have decreased expression of the alpha6-integrin-subunit when compared with normal breast tissue. These data suggest that a decrease in alpha6-integrin-subunit expression in endothelial cells is associated with tumour angiogenesis. To test whether the loss of the endothelial alpha6-integrin subunit affects tumour growth and angiogenesis, we generated alpha6fl/fl-Tie1Cre+ mice and showed that endothelial deletion of alpha6-integrin is sufficient to enhance tumour size and tumour angiogenesis in both murine B16F0 melanoma and Lewis cell lung carcinoma. Mechanistically, endothelial alpha6-integrin deficiency elevated significantly VEGF-mediated angiogenesis both in vivo and ex vivo. In particular, alpha6-integrin-deficient endothelial cells displayed increased levels of VEGF-receptor 2 (VEGFR2) and VEGF-mediated downstream ERK1/2 activation. By developing the first endothelial-specific alpha6-knockout mice, we show that the expression of the alpha6-integrin subunit in endothelial cells acts as a negative regulator of angiogenesis both in vivo and ex vivo. Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Reactive Oxygen Species, Mitochondria, and Endothelial Cell Death during In Vitro Simulated Dives.

PubMed

Wang, Qiong; Guerrero, François; Mazur, Aleksandra; Lambrechts, Kate; Buzzacott, Peter; Belhomme, Marac; Theron, Michaël

2015-07-01

Excessive reactive oxygen species (ROS) is considered a consequence of hyperoxia and a major contributor to diving-derived vascular endothelial damage and decompression sickness. The aims of this work were: 1) to directly observe endothelial ROS production during simulated air dives as well as its relation with both mitochondrial activity and cell survival; and 2) to determine which ambient factor during air diving (hydrostatic pressure or oxygen and/or nitrogen partial pressure) is responsible for the observed modifications. In vitro diving simulation was performed with bovine arterial endothelial cells under real-time observation. The effects of air diving, hydrostatic, oxygen and nitrogen pressures, and N-acetylcysteine (NAC) treatment on mitochondrial ROS generation, mitochondrial membrane potential and cellular survival during simulation were investigated. Vascular endothelial cells performing air diving simulation suffered excessive mitochondrial ROS, mitochondrial depolarization, and cell death. These effects were prevented by NAC: after NAC treatment, the cells presented no difference in damage from nondiving cells. Oxygen diving showed a higher effect on ROS generation but lower impacts on mitochondrial depolarization and cell death than hydrostatic or nitrogen diving. Nitrogen diving had no effect on the inductions of ROS, mito-depolarization, or cell death. This study is the first direct observation of mitochondrial ROS production, mitochondrial membrane potential and cell survival during diving. Simulated air SCUBA diving induces excessive ROS production, which leads to mitochondrial depolarization and endothelial cell death. Oxygen partial pressure plays a crucial role in the production of ROS. Deleterious effects of hyperoxia-induced ROS are potentiated by hydrostatic pressure. These findings hold new implications for the pathogenesis of diving-derived endothelial dysfunction.

Mechanotransduction Effects on Endothelial Cell Proliferation via CD31 and VEGFR2: Implications for Immunomagnetic Separation.

PubMed

Mahajan, Kalpesh D; Nabar, Gauri M; Xue, Wei; Anghelina, Mirela; Moldovan, Nicanor I; Chalmers, Jeffrey J; Winter, Jessica O

2017-09-01

Immunomagnetic separation is used to isolate circulating endothelial cells (ECs) and endothelial progenitor cells (EPCs) for diagnostics and tissue engineering. However, potentially detrimental changes in cell properties have been observed post-separation. Here, the effect of mechanical force, which is naturally applied during immunomagnetic separation, on proliferation of human umbilical vein endothelial cells (HUVEC), kinase insert domain-positive receptor (KDR) cells, and peripheral blood mononuclear cells (PBMCs). Cells are exposed to CD31 or Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) targeted MACSi beads at varying bead to cell ratios and compared to free antibody and unconjugated beads. A vertical magnetic gradient is applied to static 2D cultures, and a magnetic cell sorter is used to analyze cells in dynamic flow. No significant difference in EC proliferation is observed for controls or VEGFR2-targeting beads, whereas CD31-conjugated beads increase proliferation in a dose dependent manner in static 2-D cultures. This effect occurs in the absence of magnetic field, but is more pronounced with magnetic force. After flow sorting, similar increases in proliferation are seen for CD31 targeting beads. Thus, the effects of targeting antibody and magnetic force applied should be considered when designing immunomagnetic separation protocols for ECs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role of the Retinal Vascular Endothelial Cell in Ocular Disease

PubMed Central

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

2012-01-01

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

Sulfoglucuronosyl paragloboside promotes endothelial cell apoptosis in inflammation: elucidation of a novel glycosphingolipid-signaling pathway.

PubMed

Dasgupta, Somsankar; Wang, Guanghu; Yu, Robert K

2011-11-01

Sulfoglucuronosyl paragloboside (SGPG), a minor glycosphingolipid of endothelial cells, is a ligand for L-selectin and has been implicated in neuro-inflammatory diseases, such as Guillian-Barré syndrome. Inflammatory cytokines, such as TNFα and IL-1β, up-regulate SGPG expression by stimulating gene expression for glucuronosyltransferases, both P and S forms (GlcATp and GlcATs), and the human natural killer antigen (HNK-1) sulfotransferase (HNK-1 ST). Transfection of a human cerebromicrovascular endothelial cell (SV-HCEC) line with HNK-1 ST siRNA down-regulated SGPG expression, inhibited cytokine-stimulated T-cell adhesion, and offered protection against apoptosis. However, the precise mechanisms of SGPG elevation in endothelial cell apoptosis and the maintenance of blood-brain or blood-nerve barrier integrity in inflammation have not been elucidated. Blocking SGPG expression inhibited cytokine-mediated stimulation of NF-κB activity but stimulated MAP kinase activity. Furthermore, elevation of SGPG by over-expression of GlcATp and GlcATs triggered endothelial cell apoptosis, with GlcATs being more potent than GlcATp. Although SGPG-mediated endothelial cell apoptosis was preceded by inhibiting the intracellular NF-κB activity, interfering with Akt and ERK activation and stimulating caspase 3 in SV-HCECs, HNK-1ST siRNA transfection also interfered with IκB phosphorylation but stimulated ERK activation. Our data indicate that SGPG is a critical regulatory molecule for maintaining endothelial cell survival and blood-brain or blood-nerve barrier function. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Nitric-oxide synthase trafficking inducer is a pleiotropic regulator of endothelial cell function and signaling

PubMed Central

2017-01-01

Endothelial nitric-oxide synthase (eNOS) and its bioactive product, nitric oxide (NO), mediate many endothelial cell functions, including angiogenesis and vascular permeability. For example, vascular endothelial growth factor (VEGF)-mediated angiogenesis is inhibited upon reduction of NO bioactivity both in vitro and in vivo. Moreover, genetic disruption or pharmacological inhibition of eNOS attenuates angiogenesis during tissue repair, resulting in delayed wound closure. These observations emphasize that eNOS-derived NO can promote angiogenesis. Intriguingly, eNOS activity is regulated by nitric-oxide synthase trafficking inducer (NOSTRIN), which sequesters eNOS, thereby attenuating NO production. This has prompted significant interest in NOSTRIN's function in endothelial cells. We show here that NOSTRIN affects the functional transcriptome of endothelial cells by down-regulating several genes important for invasion and angiogenesis. Interestingly, the effects of NOSTRIN on endothelial gene expression were independent of eNOS activity. NOSTRIN also affected the expression of secreted cytokines involved in inflammatory responses, and ectopic NOSTRIN overexpression functionally restricted endothelial cell proliferation, invasion, adhesion, and VEGF-induced capillary tube formation. Furthermore, NOSTRIN interacted directly with TNF receptor-associated factor 6 (TRAF6), leading to the suppression of NFκB activity and inhibition of AKT activation via phosphorylation. Interestingly, TNF-α-induced NFκB pathway activation was reversed by NOSTRIN. We found that the SH3 domain of NOSTRIN is involved in the NOSTRIN-TRAF6 interaction and is required for NOSTRIN-induced down-regulation of endothelial cell proteins. These results have broad biological implications, as aberrant NOSTRIN expression leading to deactivation of the NFκB pathway, in turn triggering an anti-angiogenic cascade, might inhibit tumorigenesis and cancer progression. PMID:28235804

Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.

PubMed

Ahluwalia, A; Jones, M K; Szabo, S; Tarnawski, A S

2014-04-01

In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.

Kaposi’s Sarcoma-Associated Herpesvirus Interleukin-6 Modulates Endothelial Cell Movement by Upregulating Cellular Genes Involved in Migration

PubMed Central

Giffin, Louise; West, John A.

2015-01-01

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of human Kaposi’s sarcoma, a tumor that arises from endothelial cells, as well as two B cell lymphoproliferative diseases, primary effusion lymphoma and multicentric Castleman’s disease. KSHV utilizes a variety of mechanisms to evade host immune responses and promote cellular transformation and growth in order to persist for the life of the host. A viral homolog of human interleukin-6 (hIL-6) named viral interleukin-6 (vIL-6) is encoded by KSHV and expressed in KSHV-associated cancers. Similar to hIL-6, vIL-6 is secreted, but the majority of vIL-6 is retained within the endoplasmic reticulum, where it can initiate functional signaling through part of the interleukin-6 receptor complex. We sought to determine how intracellular vIL-6 modulates the host endothelial cell environment by analyzing vIL-6’s impact on the endothelial cell transcriptome. vIL-6 significantly altered the expression of many cellular genes associated with cell migration. In particular, vIL-6 upregulated the host factor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) at the protein and message levels. CEACAM1 has been implicated in tumor invasion and metastasis and promotes migration and vascular remodeling in endothelial cells. We report that vIL-6 upregulates CEACAM1 by a STAT3-dependent mechanism and that CEACAM1 promotes vIL-6-mediated migration. Furthermore, latent and de novo KSHV infections of endothelial cells also induce CEACAM1 expression. Collectively, our data suggest that vIL-6 modulates endothelial cell migration by upregulating the expression of cellular factors, including CEACAM1. PMID:26646010

The TGFβ pathway is a key player for the endothelial-to-hematopoietic transition in the embryonic aorta.

PubMed

Lempereur, A; Canto, P Y; Richard, C; Martin, S; Thalgott, J; Raymond, K; Lebrin, F; Drevon, C; Jaffredo, T

2018-02-15

The embryonic aorta produces hematopoietic stem and progenitor cells from a hemogenic endothelium localized in the aortic floor through an endothelial to hematopoietic transition. It has been long proposed that the Bone Morphogenetic Protein (BMP)/Transforming Growth Factor ß (TGFß) signaling pathway was implicated in aortic hematopoiesis but the very nature of the signal was unknown. Here, using thorough expression analysis of the BMP/TGFß signaling pathway members in the endothelial and hematopoietic compartments of the aorta at pre-hematopoietic and hematopoietic stages, we show that the TGFß pathway is preferentially balanced with a prominent role of Alk1/TgfßR2/Smad1 and 5 on both chicken and mouse species. Functional analysis using embryonic stem cells mutated for Acvrl1 revealed an enhanced propensity to produce hematopoietic cells. Collectively, we reveal that TGFß through the Alk1/TgfßR2 receptor axis is acting on endothelial cells to produce hematopoiesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Ischemia-induced endothelial cell swelling and mitochondrial dysfunction are attenuated by cinnamtannin D1, green tea extract, and resveratrol in vitro.

PubMed

Panickar, Kiran S; Qin, Bolin; Anderson, Richard A

2015-10-01

Polyphenols possess antioxidant and anti-inflammatory properties. Oxidative stress (OS) and inflammation have been implicated in the pathogenesis of cytotoxic brain edema in cerebral ischemia. In addition, OS and pro-inflammatory cytokines also damage the endothelial cells and the neurovascular unit. Endothelial cell swelling may contribute to a leaky blood-brain barrier which may result in vasogenic edema in the continued presence of the existing cytotoxic edema. We investigated the protective effects of polyphenols on cytotoxic cell swelling in bEND3 endothelial cultures subjected to 5 hours oxygen-glucose deprivation (OGD). A polyphenol trimer from cinnamon (cinnamtannin D1), a polyphenol-rich extract from green tea, and resveratrol prevented the OGD-induced rise in mitochondrial free radicals, cell swelling, and the dissipation of the inner mitochondrial membrane potential. Monocyte chemoattractant protein (also called CCL2), a chemokine, but not tumor necrosis factor-α or interleukin-6, augmented the cell swelling. This effect of monochemoattractant protein 1-1 was attenuated by the polyphenols. Cyclosporin A, a blocker of the mitochondrial permeability transition pore, did not attenuate cell swelling but BAPTA-AM, an intracellular calcium chelator did, indicating a role of [Ca(2+)]i but not the mPT in cell swelling. These results indicate that the polyphenols reduce mitochondrial reactive oxygen species and subsequent cell swelling in endothelial cells following ischemic injury and thus may reduce brain edema and associated neural damage in ischemia. One possible mechanism by which the polyphenols may attenuate endothelial cell swelling is through the reduction in [Ca(2+)]i.

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

Ceramide-1-phosphate regulates migration of multipotent stromal cells (MSCs) and endothelial progenitor cells (EPCs) – implications for tissue regeneration

PubMed Central

Kim, ChiHwa; Schneider, Gabriela; Abdel-Latif, Ahmed; Mierzejewska, Kasia; Sunkara, Manjula; Borkowska, Sylwia; Ratajczak, Janina; Morris, Andrew J.; Kucia, Magda; Ratajczak, Mariusz Z.

2012-01-01

Ceramide-1-phosphate (C1P) is a bioactive lipid that, in contrast to ceramide, is an anti-apoptotic molecule released from cells that are damaged and “leaky”. As reported recently, C1P promotes migration of hematopoietic cells. In the current paper, we tested the hypothesis that C1P released upon tissue damage may play an underappreciated role in chemoattraction of various types of stem cells and endothelial cells involved in tissue/organ regeneration. We show for a first time that C1P is upregulated in damaged tissues and chemoattracts BM-derived multipotent stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs). Furthermore, compared to other bioactive lipids, C1P more potently chemoattracted human umbilical vein endothelial cells (HUVECs) and stimulated tube formation by these cells. C1P also promoted in vivo vascularization of Matrigel implants and stimulated secretion of stromal derived factor-1 (SDF-1) from BM-derived fibroblasts. Thus, our data demonstrate, for the first time, that C1P is a potent bioactive lipid released from damaged cells that potentially plays an important and novel role in recruitment of stem/progenitor cells to damaged organs and may promote their vascularization. PMID:23193025

Arginase-I enhances vascular endothelial inflammation and senescence through eNOS-uncoupling.

PubMed

Zhu, Cuicui; Yu, Yi; Montani, Jean-Pierre; Ming, Xiu-Fen; Yang, Zhihong

2017-02-02

Augmented arginase-II (Arg-II) is implicated in endothelial senescence and inflammation through a mutual positive regulatory circuit with S6K1. This study was conducted to investigate whether Arg-I, another isoform of arginase that has been also reported to play a role in vascular endothelial dysfunction, promotes endothelial senescence through similar mechanisms. The non-senescent human endothelial cells from umbilical veins (passage 2 to 4) were transduced with empty recombinant adenovirus vector (rAd/CMV) as control or rAd/CMV-Arg-I to overexpress Arg-I. Overexpressing Arg-I promoted eNOS-uncoupling, enhanced senescence markers including p53-S15, p21 and senescence-associated β-galactosidase (SA-β-gal) staining, and increased inflammatory vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) as well as monocyte adhesion to endothelial cells without activating S6K1. All the effects of Arg-I were inhibited by the anti-oxidant N-acetylcysteine (NAC). Our study demonstrates that Arg-I promotes endothelial senescence and inflammatory responses through eNOS-uncoupling unrelated to activation of the S6K1 pathway.

Targeted endothelial nanomedicine for common acute pathological conditions

PubMed Central

Shuvaev, Vladimir V.; Brenner, Jacob S.; Muzykantov, Vladimir R.

2017-01-01

Endothelium, a thin monolayer of specialized cells lining the lumen of blood vessels is the key regulatory interface between blood and tissues. Endothelial abnormalities are implicated in many diseases, including common acute conditions with high morbidity and mortality lacking therapy, in part because drugs and drug carriers have no natural endothelial affinity. Precise endothelial drug delivery may improve management of these conditions. Using ligands of molecules exposed to the bloodstream on the endothelial surface enables design of diverse targeted endothelial nanomedicine agents. Target molecules and binding epitopes must be accessible to drug carriers, carriers must be free of harmful effects, and targeting should provide desirable sub-cellular addressing of the drug cargo. The roster of current candidate target molecules for endothelial nanomedicine includes peptidases and other enzymes, cell adhesion molecules and integrins, localized in different domains of the endothelial plasmalemma and differentially distributed throughout the vasculature. Endowing carriers with an affinity to specific endothelial epitopes enables an unprecedented level of precision of control of drug delivery: binding to selected endothelial cell phenotypes, cellular addressing and duration of therapeutic effects. Features of nanocarrier design such as choice of epitope and ligand control delivery and effect of targeted endothelial nanomedicine agents. Pathological factors modulate endothelial targeting and uptake of nanocarriers. Selection of optimal binding sites and design features of nanocarriers are key controllable factors that can be iteratively engineered based on their performance from in vitro to pre-clinical in vivo experimental models. Targeted endothelial nanomedicine agents provide antioxidant, anti-inflammatory and other therapeutic effects unattainable by non-targeted counterparts in animal models of common acute severe human disease conditions. The results of animal studies provide the basis for the challenging translation endothelial nanomedicine into the clinical domain. PMID:26435455

Gene expression profiles of brain endothelial cells during embryonic development at bulk and single-cell levels.

PubMed

Hupe, Mike; Li, Minerva Xueting; Kneitz, Susanne; Davydova, Daria; Yokota, Chika; Kele-Olovsson, Julianna; Hot, Belma; Stenman, Jan M; Gessler, Manfred

2017-07-11

The blood-brain barrier is a dynamic interface that separates the brain from the circulatory system, and it is formed by highly specialized endothelial cells. To explore the molecular mechanisms defining the unique nature of vascular development and differentiation in the brain, we generated high-resolution gene expression profiles of mouse embryonic brain endothelial cells using translating ribosome affinity purification and single-cell RNA sequencing. We compared the brain vascular translatome with the vascular translatomes of other organs and analyzed the vascular translatomes of the brain at different time points during embryonic development. Because canonical Wnt signaling is implicated in the formation of the blood-brain barrier, we also compared the brain endothelial translatome of wild-type mice with that of mice lacking the transcriptional cofactor β-catenin ( Ctnnb1 ). Our analysis revealed extensive molecular changes during the embryonic development of the brain endothelium. We identified genes encoding brain endothelium-specific transcription factors ( Foxf2 , Foxl2 , Foxq1 , Lef1 , Ppard , Zfp551 , and Zic3 ) that are associated with maturation of the blood-brain barrier and act downstream of the Wnt-β-catenin signaling pathway. Profiling of individual brain endothelial cells revealed substantial heterogeneity in the population. Nevertheless, the high abundance of Foxf2 , Foxq1 , Ppard , or Zic3 transcripts correlated with the increased expression of genes encoding markers of brain endothelial cell differentiation. Expression of Foxf2 and Zic3 in human umbilical vein endothelial cells induced the production of blood-brain barrier differentiation markers. This comprehensive data set may help to improve the engineering of in vitro blood-brain barrier models. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

STATs MEDIATE FIBROBLAST GROWTH FACTOR INDUCED VASCULAR ENDOTHELIAL MORPHOGENESIS

PubMed Central

Yang, Xinhai; Qiao, Dianhua; Meyer, Kristy; Friedl, Andreas

2009-01-01

The fibroblast growth factors (FGFs) play diverse roles in development, wound healing and angiogenesis. The intracellular signal transduction pathways which mediate these pleiotropic activities remain incompletely understood. We show here that the proangiogenic factors FGF2 and FGF8b can activate signal transducers and activators of transcription (STATs) in mouse microvascular endothelial cells. Both FGF2 and FGF8b activate STAT5 and to a lesser extent STAT1, but not STAT3. The FGF2-dependent activation of endothelial STAT5 was confirmed in vivo with the matrigel plug angiogenesis assay. In tissue samples of human gliomas, a tumor type where FGF-induced angiogenesis is important, STAT5 is detected in tumor vessel endothelial cell nuclei, consistent with STAT5 activation. By forced expression of constitutively active or dominant-negative mutant STAT5A in mouse brain endothelial cells, we further show that STAT5 activation is both necessary and sufficient for FGF-induced cell migration, invasion and tube formation, which are key events in vascular endothelial morphogenesis and angiogenesis. In contrast, STAT5 is not required for brain endothelial cell mitogenesis. The cytoplasmic tyrosine kinases Src and Janus kinase 2 (Jak2) both appear to be involved in the activation of STAT5, as their inhibition reduces FGF2 and FGF8b induced STAT5 phosphorylation and endothelial cell tube formation. Constitutively active STAT5A partially restores tube formation in the presence of Src or Jak2 inhibitors. These observations demonstrate that FGFs utilize distinct signaling pathways to induce angiogenic phenotypes. Together, our findings implicate the FGF-Jak2/Src-STAT5 cascade as a critical angiogenic FGF signaling pathway. PMID:19176400

Aldosterone affects blood flow and vascular tone regulated by endothelium-derived NO: therapeutic implications

PubMed Central

Toda, Noboru; Nakanishi, Sadanobu; Tanabe, Shinichi

2013-01-01

Aldosterone, in doses inappropriate to the salt status, plays an important role in the development of cardiovascular injury, including endothelial dysfunction, independent of its hypertensive effects. Acute non-genomic effects of aldosterone acting on mineralocorticoid receptors are inconsistent in healthy humans: vasoconstriction or forearm blood flow decrease via endothelial dysfunction, vasodilatation mediated by increased NO actions, or no effects. However, in studies with experimental animals, aldosterone mostly enhances vasodilatation mediated by endothelium-derived NO. Chronic exposure to aldosterone, which induces genomic responses, results in impairments of endothelial function through decreased NO synthesis and action in healthy individuals, experimental animals and isolated endothelial cells. Chronic aldosterone reduces NO release from isolated human endothelial cells only when extracellular sodium is raised. Oxidative stress is involved in the impairment of endothelial function by promoting NO degradation. Aldosterone liberates endothelin-1 (ET-1) from endothelial cells, which elicits ETA receptor–mediated vasoconstriction by inhibiting endothelial NO synthesis and action and through its own direct vasoconstrictor action. Ca2+ flux through T-type Ca2+ channels activates aldosterone synthesis and thus enhances unwanted effects of aldosterone on the endothelium. Mineralocorticoid receptor inhibitors, ETA receptor antagonists and T-type Ca2+ channel blockers appear to diminish the pathophysiological participation of aldosterone in cardiovascular disease and exert beneficial actions on bioavailability of endothelium-derived NO, particularly in resistant hypertension and aldosteronism. PMID:23190073

Shear-induced endothelial mechanotransduction: the interplay between reactive oxygen species (ROS) and nitric oxide (NO) and the pathophysiological implications

PubMed Central

2014-01-01

Hemodynamic shear stress, the blood flow-generated frictional force acting on the vascular endothelial cells, is essential for endothelial homeostasis under normal physiological conditions. Mechanosensors on endothelial cells detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. Among the various shear-induced signaling molecules, reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in vascular homeostasis and diseases. In this review, we explore the molecular, cellular, and vascular processes arising from shear-induced signaling (mechanotransduction) with emphasis on the roles of ROS and NO, and also discuss the mechanisms that may lead to excessive vascular remodeling and thus drive pathobiologic processes responsible for atherosclerosis. Current evidence suggests that NADPH oxidase is one of main cellular sources of ROS generation in endothelial cells under flow condition. Flow patterns and magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady or pulsatile). ROS production is closely linked to NO generation and elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow. The low NO bioavailability is partly caused by the reaction of ROS with NO to form peroxynitrite, a key molecule which may initiate many pro-atherogenic events. This differential production of ROS and RNS (reactive nitrogen species) under various flow patterns and conditions modulates endothelial gene expression and thus results in differential vascular responses. Moreover, ROS/RNS are able to promote specific post-translational modifications in regulatory proteins (including S-glutathionylation, S-nitrosylation and tyrosine nitration), which constitute chemical signals that are relevant in cardiovascular pathophysiology. Overall, the dynamic interplay between local hemodynamic milieu and the resulting oxidative and S-nitrosative modification of regulatory proteins is important for ensuing vascular homeostasis. Based on available evidence, it is proposed that a regular flow pattern produces lower levels of ROS and higher NO bioavailability, creating an anti-atherogenic environment. On the other hand, an irregular flow pattern results in higher levels of ROS and yet lower NO bioavailability, thus triggering pro-atherogenic effects. PMID:24410814

MicroRNA regulation of endothelial homeostasis and commitment-implications for vascular regeneration strategies using stem cell therapies.

PubMed

Scott, Elizabeth; Loya, Komal; Mountford, Joanne; Milligan, Graeme; Baker, Andrew H

2013-09-01

Human embryonic (hESC) and induced pluripotent (hiPSC) stem cells have broad therapeutic potential in the treatment of a range of diseases, including those of the vascular system. Both hESCs and hiPSCs have the capacity for indefinite self-renewal, in addition to their ability to differentiate into any adult cell type. These cells could provide a potentially unlimited source of cells for transplantation and, therefore, provide novel treatments, e.g. in the production of endothelial cells for vascular regeneration. MicroRNAs are short, noncoding RNAs that act posttranscriptionally to control gene expression and thereby exert influence over a wide range of cellular processes, including maintenance of pluripotency and differentiation. Expression patterns of these small RNAs are tissue specific, and changes in microRNA levels have often been associated with disease states in humans, including vascular pathologies. Here, we review the roles of microRNAs in endothelial cell function and vascular disease, as well as their role in the differentiation of pluripotent stem cells to the vascular endothelial lineage. Furthermore, we discuss the therapeutic potential of stem cells and how knowledge and manipulation of microRNAs in stem cells may enhance their capacity for vascular regeneration. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Catabolic Effects of Endothelial Cell-Derived Microparticles on Disc Cells: Implications in Intervertebral Disc Neovascularization and Degeneration

PubMed Central

Pohl, Pedro H. I.; Lozito, Thomas P.; Cuperman, Thais; Yurube, Takashi; Moon, Hong J.; Ngo, Kevin; Tuan, Rocky S.; Croix, Claudette St.; Sowa, Gwendolyn A.; Rodrigues, Luciano M. R.; Kang, James D.; Vo, Nam V.

2017-01-01

Neovascularization of intervertebral discs, a phenomenon considered pathological since normal discs are primarily avascular structures, occurs most frequently in annulus fibrosus (AF) of degenerated discs. Endothelial cells (ECs) are involved in this process, but the mechanism of the interaction between AF and endothelial cells is unclear. In this study we evaluated the effects on matrix catabolic activity of AF cells by the extracellular endothelial microparticles (EMPs) and soluble protein factors (SUP fraction) produced from ECs. Passage 1 human AF cells grown in monolayer cultures were treated for 72 hours with 250μg of EMPs or SUP fraction isolated from culture of the microvascular endothelial cell line, HEMC-I. Live-cell imaging revealed uptake of EMPs by AF cells. RT-PCR analysis demonstrated increased mRNA expression of MMP-1 (50.3 fold), MMP-3 (4.5 fold) and MMP-13 (5.5 fold) in AF cell cultures treated with EMPs compared to untreated control. Western analysis also demonstrated increased MMP protein expression in EMP-treated AF cells. AF cells treated with the SUP fraction also exhibited a dramatic increase in MMP mRNA and protein expression. Increased MMP expression is primarily due to EMP or SUP stimulation of AF cells since EMPs or SUP fraction alone contained negligible amount of MMPs. Interestingly, MMP activity was elevated in AF cell cultures treated with EMPs but not with SUP. This study revealed enhanced matrix catabolism as a molecular consequence of action of ECs on AF cells via EMPs, which might be expected during neo-angiogenesis of degenerating disc. PMID:27246627

Dabigatran abrogates brain endothelial cell permeability in response to thrombin

PubMed Central

Hawkins, Brian Thomas; Gu, Yu-Huan; Izawa, Yoshikane; del Zoppo, Gregory John

2015-01-01

Atrial fibrillation (AF) increases the risk and severity of thromboembolic stroke. Generally, antithrombotic agents increase the hemorrhagic risk of thromboembolic stroke. However, significant reductions in thromboembolism and intracerebral hemorrhage have been shown with the antithrombin dabigatran compared with warfarin. As thrombin has been implicated in microvessel injury during cerebral ischemia, we hypothesized that dabigatran decreases the risk of intracerebral hemorrhage by direct inhibition of the thrombin-mediated increase in cerebral endothelial cell permeability. Primary murine brain endothelial cells (mBECs) were exposed to murine thrombin before measuring permeability to 4-kDa fluorescein isothiocyanate-dextran. Thrombin increased mBEC permeability in a concentration-dependent manner, without significant endothelial cell death. Pretreatment of mBECs with dabigatran completely abrogated the effect of thrombin on permeability. Neither the expressions of the endothelial cell β1-integrins nor the tight junction protein claudin-5 were affected by thrombin exposure. Oxygen-glucose deprivation (OGD) also increased permeability; this effect was abrogated by treatment with dabigatran, as was the additive effect of thrombin and OGD on permeability. Taken together, these results indicate that dabigatran could contribute to a lower risk of intracerebral hemorrhage during embolism-associated ischemia from AF by protection of the microvessel permeability barrier from local thrombin challenge. PMID:25669912

Biosensor Technology Reveals the Disruption of the Endothelial Barrier Function and the Subsequent Death of Blood Brain Barrier Endothelial Cells to Sodium Azide and Its Gaseous Products.

PubMed

Kho, Dan T; Johnson, Rebecca H; O'Carroll, Simon J; Angel, Catherine E; Graham, E Scott

2017-09-21

Herein we demonstrate the sensitive nature of human blood-brain barrier (BBB) endothelial cells to sodium azide and its gaseous product. Sodium azide is known to be acutely cytotoxic at low millimolar concentrations, hence its use as a biological preservative (e.g., in antibodies). Loss of barrier integrity was noticed in experiments using Electric Cell-substrate Impedance Sensing (ECIS) biosensor technology, to measure endothelial barrier integrity continuously in real-time. Initially the effect of sodium azide was observed as an artefact where it was present in antibodies being employed in neutralisation experiments. This was confirmed where antibody clones that were azide-free did not mediate loss of barrier function. A delayed loss of barrier function in neighbouring wells implied the influence of a liberated gaseous product. ECIS technology demonstrated that the BBB endothelial cells had a lower level of direct sensitivity to sodium azide of ~3 µM. Evidence of gaseous toxicity was consistently observed at 30 µM and above, with disrupted barrier function and cell death in neighbouring wells. We highlight the ability of this cellular biosensor technology to reveal both the direct and gaseous toxicity mediated by sodium azide. The sensitivity and temporal dimension of ECIS technology was instrumental in these observations. These findings have substantial implications for the wide use of sodium azide in biological reagents, raising issues of their application in live-cell assays and with regard to the protection of the user. This research also has wider relevance highlighting the sensitivity of brain endothelial cells to a known mitochondrial disruptor. It is logical to hypothesise that BBB endothelial dysfunction due to mitochondrial dys-regulation could have an important but underappreciated role in a range of neurological diseases.

Deregulation of tumor angiogenesis and blockade of tumor growth in PPARbeta-deficient mice.

PubMed

Müller-Brüsselbach, Sabine; Kömhoff, Martin; Rieck, Markus; Meissner, Wolfgang; Kaddatz, Kerstin; Adamkiewicz, Jürgen; Keil, Boris; Klose, Klaus J; Moll, Roland; Burdick, Andrew D; Peters, Jeffrey M; Müller, Rolf

2007-08-08

The peroxisome proliferator-activated receptor-beta (PPARbeta) has been implicated in tumorigenesis, but its precise role remains unclear. Here, we show that the growth of syngeneic Pparb wild-type tumors is impaired in Pparb(-/-) mice, concomitant with a diminished blood flow and an abundance of hyperplastic microvascular structures. Matrigel plugs containing pro-angiogenic growth factors harbor increased numbers of morphologically immature, proliferating endothelial cells in Pparb(-/-) mice, and retroviral transduction of Pparb triggers microvessel maturation. We have identified the Cdkn1c gene encoding the cell cycle inhibitor p57(Kip2) as a PPARbeta target gene and a mediator of the PPARbeta-mediated inhibition of cell proliferation, which provides a possible mechanistic explanation for the observed tumor endothelial hyperplasia and deregulation of tumor angiogenesis in Pparb(-/-) mice. Our data point to an unexpected essential role for PPARbeta in constraining tumor endothelial cell proliferation to allow for the formation of functional tumor microvessels.

Hypoxia-driven angiogenesis: role of tip cells and extracellular matrix scaffolding.

PubMed

Germain, Stéphane; Monnot, Catherine; Muller, Laurent; Eichmann, Anne

2010-05-01

Angiogenesis is a highly coordinated tissue remodeling process leading to blood vessel formation. Hypoxia triggers angiogenesis via induction of expression of growth factors such as vascular endothelial growth factor (VEGF). VEGF instructs endothelial cells to form tip cells, which lead outgrowing capillary sprouts, whereas Notch signaling inhibits sprout formation. Basement membrane deposition and mechanical cues from the extracellular matrix (ECM) induced by hypoxia may participate to coordinated vessel sprouting in conjunction with the VEGF and Notch signaling pathways. Hypoxia regulates ECM composition, deposition, posttranslational modifications and rearrangement. In particular, hypoxia-driven vascular remodeling is dynamically regulated through modulation of ECM-modifying enzyme activities that eventually affect both matricellular proteins and growth factor availability. Better understanding of the complex interplay between endothelial cells and soluble growth factors and mechanical factors from the ECM will certainly have significant implications for understanding the regulation of developmental and pathological angiogenesis driven by hypoxia.

Cortical actin nanodynamics determines nitric oxide release in vascular endothelium.

PubMed

Fels, Johannes; Jeggle, Pia; Kusche-Vihrog, Kristina; Oberleithner, Hans

2012-01-01

The release of the main vasodilator nitric oxide (NO) by the endothelial NO synthase (eNOS) is a hallmark of endothelial function. We aim at elucidating the underlying mechanism how eNOS activity depends on cortical stiffness (К(cortex)) of living endothelial cells. It is hypothesized that cortical actin dynamics determines К(cortex) and directly influences eNOS activity. By combined atomic force microscopy and fluorescence imaging we generated mechanical and optical sections of single living cells. This approach allows the discrimination between К(cortex) and bulk cell stiffness (К(bulk)) and, additionally, the simultaneous analysis of submembranous actin web dynamics. We show that К(cortex) softens when cortical F-actin depolymerizes and that this shift from a gel-like stiff cortex to a soft G-actin rich layer, triggers the stiffness-sensitive eNOS activity. The results implicate that stiffness changes in the ∼100 nm phase of the submembranous actin web, without affecting К(bulk), regulate NO release and thus determines endothelial function.

Angiopoietin–Tie signalling in the cardiovascular and lymphatic systems

PubMed Central

Eklund, Lauri; Kangas, Jaakko; Saharinen, Pipsa

2016-01-01

Endothelial cells that form the inner layer of blood and lymphatic vessels are important regulators of vascular functions and centrally involved in the pathogenesis of vascular diseases. In addition to the vascular endothelial growth factor (VEGF) receptor pathway, the angiopoietin (Ang)–Tie system is a second endothelial cell specific ligand–receptor signalling system necessary for embryonic cardiovascular and lymphatic development. The Ang–Tie system also regulates postnatal angiogenesis, vessel remodelling, vascular permeability and inflammation to maintain vascular homoeostasis in adult physiology. This system is implicated in numerous diseases where the vasculature has an important contribution, such as cancer, sepsis, diabetes, atherosclerosis and ocular diseases. Furthermore, mutations in the TIE2 signalling pathway cause defects in vascular morphogenesis, resulting in venous malformations and primary congenital glaucoma. Here, we review recent advances in the understanding of the Ang–Tie signalling system, including cross-talk with the vascular endothelial protein tyrosine phosphatase (VE-PTP) and the integrin cell adhesion receptors, focusing on the Ang–Tie system in vascular development and pathogenesis of vascular diseases. PMID:27941161

Pharmacological modulation of endothelial cell-associated adhesion molecule expression: implications for future treatment of dermatological diseases.

PubMed

Foster, C A; Dreyfuss, M; Mandak, B; Meingassner, J G; Naegeli, H U; Nussbaumer, A; Oberer, L; Scheel, G; Swoboda, E M

1994-11-01

Skin diseases with an inflammatory component, regardless of their etiology, are characterized at some point by the extravasation and subsequent infiltration of leukocytes into the dermal and/or epidermal compartments. This trafficking pattern is determined by a complex series of events whereby the leukocytes interact with cell adhesion molecules (CAM), particularly those induced on endothelial cells following activation with various inflammatory mediators. Vascular CAMs belonging to the selectin family (i.e., P-selectin and E-selectin) are thought to mediate early and reversible events involving leukocyte rolling and margination along the lumenal surface of microvascular cells (post-capillary venules). Certain members of the immunoglobulin supergene family (i.e., VCAM-1 and ICAM-1) regulate later and irreversible steps which lead to firm attachment and subsequent diapedesis of leukocytes. Accumulating evidence suggests that if one blocks the ligand-binding sites between leukocytes and endothelial cells, or inhibits vascular CAM expression, hematopoietic cell extravasation and progressive inflammatory events can be greatly diminished. To identify such inhibitors we developed a cell-based Elisa using the human microvascular cell line HMEC-1. As reported in the present paper, this approach yielded a naturally-occurring, low molecular weight compound which potently inhibits cytokine-induced adhesion molecule expression on cultured endothelial cells, without modulating "house-keeping" proteins.

Endothelial Cell Permeability during Hantavirus Infection Involves Factor XII-Dependent Increased Activation of the Kallikrein-Kinin System

PubMed Central

Taylor, Shannon L.; Wahl-Jensen, Victoria; Copeland, Anna Maria; Jahrling, Peter B.; Schmaljohn, Connie S.

2013-01-01

Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC) display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE)-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF). To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC) to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS). We show that incubation of factor XII (FXII), prekallikrein (PK), and high molecular weight kininogen (HK) plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL) and increased liberation of bradykinin (BK). Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS), we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation during hantavirus infection and could have profound implications for treatment of hantavirus infections. PMID:23874198

Effects of chlorogenic acid on intracellular calcium regulation in lysophosphatidylcholine-treated endothelial cells.

PubMed

Jung, Hye-Jin; Im, Seung-Soon; Song, Dae-Kyu; Bae, Jae-Hoon

2017-06-01

Lysophosphatidylcholine (LPC) is a major phospholipid component of oxidized low-density lipoprotein (ox-LDL) and is implicated in its atherogenic activity. This study investigated the effects of LPC on cell viability, intracellular calcium homeostasis, and the protective mechanisms of chlorogenic acid (CGA) in human umbilical vein endothelial cells (HUVECs). LPC increased intracellular calcium ([Ca 2＋ ] i ) by releasing Ca 2＋ from intracellular stores and via Ca 2＋ influx through store-operated channels (SOCs). LPC also increased the generation of reactive oxygen species (ROS) and decreased cell viability. The mRNA expression of Transient receptor potential canonical (TRPC) channel 1 was increased significantly by LPC treatment and suppressed by CGA. CGA inhibited LPC-induced Ca 2＋ influx and ROS generation, and restored cell viability. These results suggested that CGA inhibits SOC-mediated Ca 2＋ influx and ROS generation by attenuating TRPC1 expression in LPC-treated HUVECs. Therefore, CGA might protect endothelial cells against LPC injury, thereby inhibiting atherosclerosis. [BMB Reports 2017; 50(6): 323-328].

Effects of chlorogenic acid on intracellular calcium regulation in lysophosphatidylcholine-treated endothelial cells

PubMed Central

Jung, Hye-Jin; Im, Seung-Soon; Song, Dae-Kyu; Bae, Jae-Hoon

2017-01-01

Lysophosphatidylcholine (LPC) is a major phospholipid component of oxidized low-density lipoprotein (ox-LDL) and is implicated in its atherogenic activity. This study investigated the effects of LPC on cell viability, intracellular calcium homeostasis, and the protective mechanisms of chlorogenic acid (CGA) in human umbilical vein endothelial cells (HUVECs). LPC increased intracellular calcium ([Ca2+]i) by releasing Ca2+ from intracellular stores and via Ca2+ influx through store-operated channels (SOCs). LPC also increased the generation of reactive oxygen species (ROS) and decreased cell viability. The mRNA expression of Transient receptor potential canonical (TRPC) channel 1 was increased significantly by LPC treatment and suppressed by CGA. CGA inhibited LPC-induced Ca2+ influx and ROS generation, and restored cell viability. These results suggested that CGA inhibits SOC-mediated Ca2+ influx and ROS generation by attenuating TRPC1 expression in LPC-treated HUVECs. Therefore, CGA might protect endothelial cells against LPC injury, thereby inhibiting atherosclerosis. PMID:28088946

Implications of the Endothelial Cell Response in Glioblastoma to Stimulation by Mesenchymal Stem Cells and Ionizing Radiation

NASA Astrophysics Data System (ADS)

Zhao, Tansy Y.

Heightened angiogenesis is both the pathophysiologic hallmark and the potential cause of therapy resistance for glioblastoma (GBM), a deadly brain tumor. It is thought that mesenchymal stem cells (MSCs) play important roles in neovascularization and tumor progression. We postulated that MSCs protect ECs against radiotherapy, which subsequently enhances tumor angiogenesis, and promotes GBM tumor recurrence following therapy. We therefore sought to establish the in-vitro endothelial cell response to stimulation by MSC condition media and ionizing radiation (IR) treatment. We established the gene expression profiles of endothelial cells in response to IR, MSCs and the combination of both. Within the same gene profiles, we identified a unique gene signature that was highly predictive of response to Bevacizumab for GBM patients. We also demonstrated that MSC increased the viability of ECs in response to IR. Protein analysis in ECs suggested MSC-mediated cell cycle arrest as a mechanism for radio-resistance in ECs.

The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis.

PubMed

Aidoudi, Sallouha; Bujakowska, Kinga; Kieffer, Nelly; Bikfalvi, Andreas

2008-07-16

The human CXC-chemokine CXCL4 is a potent inhibitor of tumor-induced angiogenesis. Considering that CXCL4 is sequestered in platelet alpha-granules and released following platelet activation in the vicinity of vessel wall injury, we tested the hypothesis that CXCL4 might function as a ligand for integrins. Integrins are a family of adhesion receptors that play a crucial role in angiogenesis by regulating early angiogenic processes, such as endothelial cell adhesion and migration. Here, we show that CXCL4 interacts with alphavbeta3 on the surface of alphavbeta3-CHO. More importantly, human umbilical vein endothelial cells adhere to immobilized CXCL4 through alphavbeta3 integrin, and also through other integrins, such as alphavbeta5 and alpha5beta1. We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner. Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration. As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect.

The recovery time course of the endothelial-cell glycocalyx in vivo and its implications in vitro

PubMed Central

Potter, Daniel R.; Jiang, John; Damiano, Edward R.

2009-01-01

Compelling evidence continues to emerge suggesting that the glycocalyx surface layer on vascular endothelial cells plays a determining role in numerous physiological processes including inflammation, microvascular permeability, and endothelial mechanotransduction. Previous research has shown that enzymes degrade the glycocalyx, while inflammation causes shedding of the layer. To track the endogenous recovery of the glycocalyx in vivo, we used fluorescent micro-particle image velocimetry (µ-PIV) in mouse cremaster-muscle venules to estimate the hydrodynamically relevant glycocalyx thickness 1, 3, 5, and 7 days after enzymatic or cytokine-mediated degradation of the layer. Results indicate that after acute degradation of the glycocalyx, 5–7 days are required for the layer to endogenously restore itself to its native hydrodynamically relevant thickness in vivo. In light of these findings, and since demonstrable evidence has emerged that standard cell-culture conditions are not conducive to providing the environment and/or cellular conditions necessary to produce and maintain a physiologically relevant cell-surface glycocalyx in vitro, we sought to determine if merely the passage of time would be sufficient to promote the production of a hydrodynamically relevant glycocalyx on a confluent monolayer of human umbilical vein endothelial cells (HUVECs). Using µ-PIV, we found that the hydrodynamically relevant glycocalyx was substantially absent 7 days post-confluence on HUVEC-lined cylindrical collagen microchannels maintained under standard culture conditions. Thus it remains to be determined how a hydrodynamically relevant glycocalyx surface layer can be synthesized and maintained in culture before the endothelial-cell culture model can be used to elucidate glycocalyx-mediated mechanisms of endothelial-cell function. PMID:19443840

Suppressing iron oxide nanoparticle toxicity by vascular targeted antioxidant polymer nanoparticles.

PubMed

Cochran, David B; Wattamwar, Paritosh P; Wydra, Robert; Hilt, J Zach; Anderson, Kimberly W; Eitel, Richard E; Dziubla, Thomas D

2013-12-01

The biomedical use of superparamagnetic iron oxide nanoparticles has been of continued interest in the literature and clinic. Their ability to be used as contrast agents for imaging and/or responsive agents for remote actuation makes them exciting materials for a wide range of clinical applications. Recently, however, concern has arisen regarding the potential health effects of these particles. Iron oxide toxicity has been demonstrated in in vivo and in vitro models, with oxidative stress being implicated as playing a key role in this pathology. One of the key cell types implicated in this injury is the vascular endothelial cells. Here, we report on the development of a targeted polymeric antioxidant, poly(trolox ester), nanoparticle that can suppress oxidative damage. As the polymer undergoes enzymatic hydrolysis, active trolox is locally released, providing a long term protection against pro-oxidant agents. In this work, poly(trolox) nanoparticles are targeted to platelet endothelial cell adhesion molecules (PECAM-1), which are able to bind to and internalize in endothelial cells and provide localized protection against the cytotoxicity caused by iron oxide nanoparticles. These results indicate the potential of using poly(trolox ester) as a means of mitigating iron oxide toxicity, potentially expanding the clinical use and relevance of these exciting systems. Copyright © 2013 Elsevier Ltd. All rights reserved.

Selective Deletion of Leptin Signaling in Endothelial Cells Enhances Neointima Formation and Phenocopies the Vascular Effects of Diet-Induced Obesity in Mice.

PubMed

Hubert, Astrid; Bochenek, Magdalena L; Schütz, Eva; Gogiraju, Rajinikanth; Münzel, Thomas; Schäfer, Katrin

2017-09-01

Obesity is associated with elevated circulating leptin levels and hypothalamic leptin resistance. Leptin receptors (LepRs) are expressed on endothelial cells, and leptin promotes neointima formation in a receptor-dependent manner. Our aim was to examine the importance of endothelial LepR (End.LepR) signaling during vascular remodeling and to determine whether the cardiovascular consequences of obesity are because of hyperleptinemia or endothelial leptin resistance. Mice with loxP-flanked LepR alleles were mated with mice expressing Cre recombinase controlled by the inducible endothelial receptor tyrosine kinase promoter. Obesity was induced with high-fat diet. Neointima formation was examined after chemical carotid artery injury. Morphometric quantification revealed significantly greater intimal hyperplasia, neointimal cellularity, and proliferation in End.LepR knockout mice, and similar findings were obtained in obese, hyperleptinemic End.LepR wild-type animals. Analysis of primary endothelial cells confirmed abrogated signal transducer and activator of transcription-3 phosphorylation in response to leptin in LepR knockout and obese LepR wild-type mice. Quantitative PCR, ELISA, and immunofluorescence analyses revealed increased expression and release of endothelin-1 in End.LepR-deficient and LepR-resistant cells, and ET receptor A/B antagonists abrogated their paracrine effects on murine aortic smooth muscle cell proliferation. Reduced expression of peroxisome proliferator-activated receptor-γ and increased nuclear activator protein-1 staining was observed in End.LepR-deficient and LepR-resistant cells, and peroxisome proliferator-activated receptor-γ antagonization increased endothelial endothelin-1 expression. Our findings suggest that intact endothelial leptin signaling limits neointima formation and that obesity represents a state of endothelial leptin resistance. These observations and the identification of endothelin-1 as soluble mediator of the cardiovascular risk factor obesity may have relevant therapeutic implications. © 2017 American Heart Association, Inc.

Presence of MUC4 in human milk and at the luminal surfaces of blood vessels.

PubMed

Zhang, Jin; Perez, Aymee; Yasin, Mohammad; Soto, Pedro; Rong, Min; Theodoropoulos, George; Carothers Carraway, Coralie A; Carraway, Kermit L

2005-07-01

MUC4 is a heterodimeric membrane mucin, composed of a mucin subunit ASGP-1 (MUC4alpha) and a transmembrane subunit ASGP-2 (MUC4beta), which has been implicated in the protection of epithelial cell surfaces. Surprisingly, development and characterization of a new monoclonal antibody (mAb), called 1G8, against ASGP-2 demonstrated by immunohistochemistry the presence of MUC4 at the luminal surfaces of blood vessels of both normal tissues and tumors. Muc4 was detected with 1G8 and other Muc4 antibodies in blood vessels from humans, rats and mice. This expression of MUC4 in endothelial cells was confirmed by immunoblotting with 1G8 in human umbilical vein endothelial cells (HUVECs), human iliac artery endothelial cells (HIAECs), and human microvascular endothelial cells (HMVECs). MUC4 could be observed on HUVECs grown on either plastic or Matrigel. Finally, MUC4 expression in the three types of endothelial cell lines was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). These results provide, to our knowledge, the first demonstration of a member of the MUC gene family and membrane mucin in blood vessels. As a luminal surface component, the MUC4 is situated to contribute to the non-adhesive luminal surface and to act as an intrinsic protection and survival factor. (c) 2004 Wiley-Liss, Inc.

Dependence of Golgi apparatus integrity on nitric oxide in vascular cells: implications in pulmonary arterial hypertension

PubMed Central

Lee, Jason E.; Patel, Kirit; Almodóvar, Sharilyn; Tuder, Rubin M.; Flores, Sonia C.

2011-01-01

Although reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), its consequences on organellar structure and function within vascular cells is largely unexplored. We investigated the effect of reduced NO on the structure of the Golgi apparatus as assayed by giantin or GM130 immunofluorescence in human pulmonary arterial endothelial (HPAECs) and smooth muscle (HPASMCs) cells, bovine PAECs, and human EA.hy926 endothelial cells. Golgi structure was also investigated in cells in tissue sections of pulmonary vascular lesions in idiopathic PAH (IPAH) and in macaques infected with a chimeric simian immunodeficiency virus containing the human immunodeficiency virus (HIV)-nef gene (SHIV-nef) with subcellular three-dimensional (3D) immunoimaging. Compounds with NO scavenging activity including 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), methylene blue, N-acetylcysteine, and hemoglobin markedly fragmented the Golgi in all cell types evaluated as did monocrotaline pyrrole, while LY-83583, sildenafil, fasudil, Y-27632, Tiron, Tempol, or H2O2 did not. Golgi fragmentation by NO scavengers was inhibited by diethylamine NONOate, was evident in HPAECs after selective knockdown of endothelial nitric oxide synthase using small interfering RNA (siRNA), was independent of microtubule organization, required the GTPase dynamin 2, and was accompanied by depletion of α-soluble N-ethylmaleimide-sensitive factor (NSF) acceptor protein (α-SNAP) from Golgi membranes and codispersal of the SNAP receptor (SNARE) Vti1a with giantin. Golgi fragmentation was confirmed in endothelial and smooth muscle cells in pulmonary arterial lesions in IPAH and the SHIV-nef-infected macaque with subcellular 3D immunoimaging. In SHIV-nef-infected macaques Golgi fragmentation was observed in cells containing HIV-nef-bearing endosomes. The observed Golgi fragmentation suggests that NO plays a significant role in modulating global protein trafficking patterns that contribute to changes in the cell surface landscape and functional signaling in vascular cells. PMID:21217069

Recombinant tissue plasminogen activator enhances microparticle release from mouse brain-derived endothelial cells through plasmin.

PubMed

Garraud, Marie; Khacef, Kahina; Vion, Anne-Clémence; Leconte, Claire; Yin, Min; Renard, Jean-Marie; Marchand-Leroux, Catherine; Boulanger, Chantal M; Margaill, Isabelle; Beray-Berthat, Virginie

2016-11-15

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is currently the only approved pharmacological strategy for acute ischemic stroke. However, rt-PA exhibits vascular toxicity mainly due to endothelial damage. To investigate the mechanisms underlying rt-PA-induced endothelial alterations, we assessed the role of rt-PA in the generation of endothelial microparticles (EMPs), emerging biological markers and effectors of endothelial dysfunction. The mouse brain-derived endothelial cell line bEnd.3 was used. Cells were treated with rt-PA at 20, 40 or 80μg/ml for 15 or 24h, and EMPs were quantified in the culture media using Annexin-V staining coupled with flow cytometry. Rt-PA enhanced EMP release from bEnd.3 cells with a maximal increase at the 40μg/ml dose for 24h (+78% compared to controls). Using tranexamic acid and aprotinin we demonstrated that plasmin is responsible for rt-PA-induced EMP release. The p38 MAPK inhibitor SB203580 and the poly(ADP-ribose)polymerase (PARP) inhibitor PJ34 also reduced rt-PA-induced EMP production, suggesting that p38 MAPK and PARP are downstream intracellular effectors of rt-PA/plasmin. Rt-PA also altered through plasmin the morphology and the confluence of bEnd.3 cells. By contrast, these changes did not implicate p38 MAPK and PARP. This study demonstrates that rt-PA induces the production of microparticles by cerebral endothelial cells, through plasmin, p38 MAPK and PARP pathways. Determining the phenotype of these EMPs to clarify their role on the endothelium in ischemic conditions could thus be of particular interest. Copyright © 2016 Elsevier B.V. All rights reserved.

Chronic exposure to high glucose impairs bradykinin-stimulated nitric oxide production by interfering with the phospholipase-C-implicated signalling pathway in endothelial cells: evidence for the involvement of protein kinase C.

PubMed

Tang, Y; Li, G D

2004-12-01

Overwhelming evidence indicates that endothelial cell dysfunction in diabetes is characterised by diminished endothelium-dependent relaxation, but the matter of the underlying molecular mechanism remains unclear. As nitric oxide (NO) production from the endothelium is the major player in endothelium-mediated vascular relaxation, we investigated the effects of high glucose on NO production, and the possible alterations of signalling pathways implicated in this scenario. NO production and intracellular Ca(2+) levels ([Ca(2+)](i)) were assessed using the fluorescent probes 4,5-diaminofluorescein diacetate and fura-2 respectively. Exposure of cultured bovine aortic endothelial cells to high glucose for 5 or 10 days significantly reduced NO production induced by bradykinin (but not by Ca(2+) ionophore) in a time- and dose-dependent manner. This was probably due to an attenuation in bradykinin-induced elevations of [Ca(2+)](i) under these conditions, since a close correlation between [Ca(2+)](i) increases and NO generation was observed in intact bovine aortic endothelial cells. Both bradykinin-promoted intracellular Ca(2+) mobilisation and extracellular Ca(2+) entry were affected. Moreover, bradykinin-induced formation of Ins(1,4,5)P(3), a phospholipase C product leading to increases in [Ca(2+)](i), was also inhibited following high glucose culture. This abnormality was not attributable to a decrease in inositol phospholipids, but possibly to a reduction in the number of bradykinin receptors. The alterations in NO production, the increases in [Ca(2+)](i), and the bradykinin receptor number due to high glucose could be largely reversed by protein kinase C inhibitors and D: -alpha-tocopherol (antioxidant). Chronic exposure to high glucose reduces NO generation in endothelial cells, probably by impairing phospholipase-C-mediated Ca(2+) signalling due to excess protein kinase C activation. This defect in NO release may contribute to the diminished endothelium-dependent relaxation and thus to the development of cardiovascular diseases in diabetes.

Osteocalcin expression by circulating endothelial progenitor cells in patients with coronary atherosclerosis.

PubMed

Gössl, Mario; Mödder, Ulrike I; Atkinson, Elizabeth J; Lerman, Amir; Khosla, Sundeep

2008-10-14

This study was designed to test whether patients with coronary atherosclerosis have increases in circulating endothelial progenitor cells (EPCs) expressing an osteogenic phenotype. Increasing evidence indicates a link between bone and the vasculature, and bone marrow and circulating osteogenic cells have been identified by staining for the osteoblastic marker, osteocalcin (OCN). Endothelial progenitor cells contribute to vascular repair, but repair of vascular injury may result in calcification. Using cell surface markers (CD34, CD133, kinase insert domain receptor [KDR]) to identify EPCs, we examined whether patients with coronary atherosclerosis had increases in the percentage of EPCs expressing OCN. We studied 72 patients undergoing invasive coronary assessment: control patients (normal coronary arteries and no endothelial dysfunction, n = 21) versus 2 groups with coronary atherosclerosis-early coronary atherosclerosis (normal coronary arteries but with endothelial dysfunction, n = 22) and late coronary atherosclerosis (severe, multivessel coronary artery disease, n = 29). Peripheral blood mononuclear cells were analyzed using flow cytometry. Compared with control patients, patients with early or late coronary atherosclerosis had significant increases (approximately 2-fold) in the percentage of CD34+/KDR+ and CD34+/CD133+/KDR+ cells costaining for OCN. Even larger increases were noted in the early and late coronary atherosclerosis patients in the percentage of CD34+/CD133-/KDR+ cells costaining for OCN (5- and 2-fold, p < 0.001 and 0.05, respectively). A higher percentage of EPCs express OCN in patients with coronary atherosclerosis compared with subjects with normal endothelial function and no structural coronary artery disease. These findings have potential implications for the mechanisms of vascular calcification and for the development of novel markers for coronary atherosclerosis.

Therapeutically targeting mitochondrial redox signalling alleviates endothelial dysfunction in preeclampsia.

PubMed

McCarthy, Cathal; Kenny, Louise C

2016-09-08

Aberrant placentation generating placental oxidative stress is proposed to play a critical role in the pathophysiology of preeclampsia. Unfortunately, therapeutic trials of antioxidants have been uniformly disappointing. There is provisional evidence implicating mitochondrial dysfunction as a source of oxidative stress in preeclampsia. Here we provide evidence that mitochondrial reactive oxygen species mediates endothelial dysfunction and establish that directly targeting mitochondrial scavenging may provide a protective role. Human umbilical vein endothelial cells exposed to 3% plasma from women with pregnancies complicated by preeclampsia resulted in a significant decrease in mitochondrial function with a subsequent significant increase in mitochondrial superoxide generation compared to cells exposed to plasma from women with uncomplicated pregnancies. Real-time PCR analysis showed increased expression of inflammatory markers TNF-α, TLR-9 and ICAM-1 respectively in endothelial cells treated with preeclampsia plasma. MitoTempo is a mitochondrial-targeted antioxidant, pre-treatment of cells with MitoTempo protected against hydrogen peroxide-induced cell death. Furthermore MitoTempo significantly reduced mitochondrial superoxide production in cells exposed to preeclampsia plasma by normalising mitochondrial metabolism. MitoTempo significantly altered the inflammatory profile of plasma treated cells. These novel data support a functional role for mitochondrial redox signaling in modulating the pathogenesis of preeclampsia and identifies mitochondrial-targeted antioxidants as potential therapeutic candidates.

Carcinoembryonic antigen-stimulated THP-1 macrophages activate endothelial cells and increase cell-cell adhesion of colorectal cancer cells.

PubMed

Aarons, Cary B; Bajenova, Olga; Andrews, Charles; Heydrick, Stanley; Bushell, Kristen N; Reed, Karen L; Thomas, Peter; Becker, James M; Stucchi, Arthur F

2007-01-01

The liver is the most common site for metastasis by colorectal cancer, and numerous studies have shown a relationship between serum carcinoembryonic antigen (CEA) levels and metastasis to this site. CEA activates hepatic macrophages or Kupffer cells via binding to the CEA receptor (CEA-R), which results in the production of cytokines and the up-regulation of endothelial adhesion molecules, both of which are implicated in hepatic metastasis. Since tissue macrophages implicated in the metastatic process can often be difficult to isolate, the aim of this study was to develop an in vitro model system to study the complex mechanisms of CEA-induced macrophage activation and metastasis. Undifferentiated, human monocytic THP-1 (U-THP) cells were differentiated (D-THP) to macrophages by exposure to 200 ng/ml phorbol myristate acetate (PMA) for 18 h. Immunohistochemistry showed two CEA-R isoforms present in both U- and D-THP cells. The receptors were localized primarily to the nucleus in U-THP cells, while a significant cell-surface presence was observed following PMA-differentiation. Incubation of D-THP-1 cells with CEA resulted in a significant increase in tumor necrosis factor-alpha (TNF-alpha) release over 24 h compared to untreated D-THP-1 or U-THP controls confirming the functionality of these cell surface receptors. U-THP cells were unresponsive to CEA. Attachment of HT-29 cells to human umbilical vein endothelial cells significantly increased at 1 h after incubation with both recombinant TNF-alpha and conditioned media from CEA stimulated D-THP cells by six and eightfold, respectively. This study establishes an in vitro system utilizing a human macrophage cell line expressing functional CEA-Rs to study activation and signaling mechanisms of CEA that facilitate tumor cell attachment to activated endothelial cells. Utilization of this in vitro system may lead to a more complete understanding of the expression and function of CEA-R and facilitate the design of anti-CEA-R therapeutic modalities that may significantly diminish the metastatic potential of CEA overexpressing colorectal tumors.

Catabolic effects of endothelial cell-derived microparticles on disc cells: Implications in intervertebral disc neovascularization and degeneration.

PubMed

Pohl, Pedro H I; Lozito, Thomas P; Cuperman, Thais; Yurube, Takashi; Moon, Hong J; Ngo, Kevin; Tuan, Rocky S; St Croix, Claudette; Sowa, Gwendolyn A; Rodrigues, Luciano M R; Kang, James D; Vo, Nam V

2016-08-01

Neovascularization of intervertebral discs, a phenomenon considered pathological since normal discs are primarily avascular structures, occurs most frequently in annulus fibrosus (AF) of degenerated discs. Endothelial cells (ECs) are involved in this process, but the mechanism of the interaction between AF and endothelial cells is unclear. In this study, we evaluated the effects on matrix catabolic activity of AF cells by the extracellular endothelial microparticles (EMPs) and soluble protein factors (SUP fraction) produced from ECs. Passage 1 human AF cells grown in monolayer cultures were treated for 72 h with 250 µg of EMPs or SUP fraction isolated from culture of the microvascular endothelial cell line, HEMC-I. Live-cell imaging revealed uptake of EMPs by AF cells. RT-PCR analysis demonstrated increased mRNA expression of MMP-1 (50.3-fold), MMP-3 (4.5-fold) and MMP-13 (5.5-fold) in AF cell cultures treated with EMPs compared to untreated control. Western analysis also demonstrated increased MMP protein expression in EMP-treated AF cells. AF cells treated with the SUP fraction also exhibited a dramatic increase in MMP mRNA and protein expression. Increased MMP expression is primarily due to EMP or SUP stimulation of AF cells since EMPs or SUP fraction alone contained negligible amount of MMPs. Interestingly, MMP activity was elevated in AF cell cultures treated with EMPs but not with SUP. This study revealed enhanced matrix catabolism as a molecular consequence of action of ECs on AF cells via EMPs, which might be expected during neo-angiogenesis of degenerating disc. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1466-1474, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Uterine Spiral Artery Remodeling Involves Endothelial Apoptosis Induced by Extravillous Trophoblasts Through Fas/FasL Interactions

PubMed Central

Ashton, Sandra V.; Whitley, Guy St. J.; Dash, Philip R.; Wareing, Mark; Crocker, Ian P.; Baker, Philip N.; Cartwright, Judith E.

2014-01-01

Objective Invasion of uterine spiral arteries by extravillous trophoblasts in the first trimester of pregnancy results in loss of endothelial and musculoelastic layers. This remodeling is crucial for an adequate blood supply to the fetus with a failure to remodel implicated in the etiology of the hypertensive disorder preeclampsia. The mechanism by which trophoblasts induce this key process is unknown. This study gives the first insights into the potential mechanisms involved. Methods and Results Spiral arteries were dissected from nonplacental bed biopsies obtained at Caesarean section, and a novel model was used to mimic in vivo events. Arteries were cultured with trophoblasts in the lumen, and apoptotic changes in the endothelial layer were detected after 20 hours, leading to loss of endothelium by 96 hours. In vitro, coculture experiments showed that trophoblasts stimulated apoptosis of primary decidual endothelial cells and an endothelial cell line. This was blocked by caspase inhibition and NOK2, a FasL blocking antibody. NOK2 also abrogated trophoblast-induced endothelial apoptosis in the vessel model. Conclusions Extravillous trophoblast induction of endothelial apoptosis is a possible mechanism by which the endothelium is removed, and vascular remodeling may occur in uterine spiral arteries. Fas/FasL interactions have an important role in trophoblast-induced endothelial apoptosis. PMID:15499040

Lysophosphatidic acid receptor, LPA6, regulates endothelial blood-brain barrier function: Implication for hepatic encephalopathy.

PubMed

Masago, Kayo; Kihara, Yasuyuki; Yanagida, Keisuke; Hamano, Fumie; Nakagawa, Shinsuke; Niwa, Masami; Shimizu, Takao

2018-07-02

Cerebral edema is a life-threatening neurological condition characterized by brain swelling due to the accumulation of excess fluid both intracellularly and extracellularly. Fulminant hepatic failure (FHF) develops cerebral edema by disrupting blood-brain barrier (BBB). However, the mechanisms by which mediator induces brain edema in FHF remain to be elucidated. Here, we assessed a linkage between brain edema and lysophosphatidic acid (LPA) signaling by utilizing an animal model of FHF and in vitro BBB model. Azoxymethane-treated mice developed FHF and hepatic encephalopathy, associated with higher autotaxin (ATX) activities in serum than controls. Using in vitro BBB model, LPA disrupted the structural integrity of tight junction proteins including claudin-5, occludin, and ZO-1. Furthermore, LPA decreased transendothelial electrical resistances in in vitro BBB model, and induced cell contraction in brain endothelial monolayer cultures, both being inhibited by a Rho-associated protein kinase inhibitor, Y-27632. The brain capillary endothelial cells predominantly expressed LPA 6 mRNA, whose knockdown blocked the LPA-induced endothelial cell contraction. Taken together, the up-regulation of serum ATX in hepatic encephalopathy may activate the LPA-LPA 6 -G 12/13 -Rho pathway in brain capillary endothelial cells, leading to enhancement of BBB permeability and brain edema. Copyright © 2018 Elsevier Inc. All rights reserved.

Endothelial actions of atrial and B-type natriuretic peptides.

PubMed

Kuhn, Michaela

2012-05-01

The cardiac hormone atrial natriuretic peptide (ANP) is critically involved in the maintenance of arterial blood pressure and intravascular volume homeostasis. Its cGMP-producing GC-A receptor is densely expressed in the microvascular endothelium of the lung and systemic circulation, but the functional relevance is controversial. Some studies reported that ANP stimulates endothelial cell permeability, whereas others described that the peptide attenuates endothelial barrier dysfunction provoked by inflammatory agents such as thrombin or histamine. Many studies in vitro addressed the effects of ANP on endothelial proliferation and migration. Again, both pro- and anti-angiogenic properties were described. To unravel the role of the endothelial actions of ANP in vivo, we inactivated the murine GC-A gene selectively in endothelial cells by homologous loxP/Cre-mediated recombination. Our studies in these mice indicate that ANP, via endothelial GC-A, increases endothelial albumin permeability in the microcirculation of the skin and skeletal muscle. This effect is critically involved in the endocrine hypovolaemic, hypotensive actions of the cardiac hormone. On the other hand the homologous GC-A-activating B-type NP (BNP), which is produced by cardiac myocytes and many other cell types in response to stressors such as hypoxia, possibly exerts more paracrine than endocrine actions. For instance, within the ischaemic skeletal muscle BNP released from activated satellite cells can improve the regeneration of neighbouring endothelia. This review will focus on recent advancements in our understanding of endothelial NP/GC-A signalling in the pulmonary versus systemic circulation. It will discuss possible mechanisms accounting for the discrepant observations made for the endothelial actions of this hormone-receptor system and distinguish between (patho)physiological and pharmacological actions. Lastly it will emphasize the potential therapeutical implications derived from the actions of NPs on endothelial permeability and regeneration. © 2012 The Author. British Journal of Pharmacology © 2012 The British Pharmacological Society.

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

PubMed Central

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

2011-01-01

Background Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. Methodology/Principal Findings Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. Conclusions Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was required to bring about matrix-endothelial interactions and for xenografted hMSC -BD11 cells to optimally recruit host vasculature. PMID:21779348

Effect and possible mechanism of monocyte-derived VEGF on monocyte-endothelial cellular adhesion after electrical burns.

PubMed

Ruan, Qiongfang; Zhao, Chaoli; Ye, Ziqing; Ruan, Jingjing; Xie, Qionghui; Xie, Weiguo

2015-06-01

One of the major obstacles in the treatment of severe electrical burns is properly handling the resulting uncontrolled inflammation. Such inflammation often causes secondary injury and necrosis, thus complicating patient outcomes. Vascular endothelial grow factor (VEGF) has emerged as an important mediator for the recruitment of monocytes to the site inflammation. This study was designed to explore the effects and possible mechanism of VEGF on monocyte-endothelial cellular adhesion. To do so, we used a cultured human monocytic cell line (THP-1) that was stimulated with serum derived from rats that had received electrical burns. Serum was obtained from rats that had received electrical burns. Both the VEGF and soluble flt-1 (sflt-1) concentrations of the serum were determined by double-antibody sandwich ELISA. The concentrations of VEGF, sflt-1, and TNF-α obtained from the cell-free cultured supernatant of THP-1 cells that had been exposed to the serum were then determined by double-antibody sandwich ELISA. Serum-stimulated THP-1 cells were added to wells with a monolayer of endothelial cells to detect the level of monocyte-endothelial cells adhesion. Finally, the state of phosphorylation of AKT was determined by Western blotting. Both in vivo and in vitro studies showed that compared to controls, the levels of VEGF were significantly increased after electrical burns. This increased was accompanied by a reduction of sflt-1 levels. Furthermore, the serum of rats that had received electrical burns was able to both activate monocytes to secrete TNF-α and enhance monocyte-endothelial cell adhesion. Treatment with the serum also resulted in an up-regulation of the phosphorylation of AKT, but had no effect on the total levels of AKT. Phosphatidylinositide 3-kinases (PI3K) inhibition decreased the number of THP-1 cells that were adhered to endothelial cells. Finally, sequestering VEGF with sflt-1 was able to reduce the effect on monocyte-endothelial cells adhesion by blocking the PI3K signaling pathway. Our results indicate that VEGF is implicated in the pathogenesis of inflammation after electrical burns. Inhibition of VEGF activity could attenuate monocyte-endothelial cells adhesion by suppressing the state of phosphorylation of AKT, which is downstream of the PI3K signaling pathway. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity.

PubMed

Dai, Haibin; Yu, Zhanyang; Fan, Xiang; Liu, Ning; Yan, Min; Chen, Zhong; Lo, Eng H; Hajjar, Katherine A; Wang, Xiaoying

2013-06-01

Hyperglycaemia impairs fibrinolytic activity on the surface of endothelial cells, but the underlying mechanisms are not fully understood. In this study, we tested the hypothesis that hyperglycaemia causes dysfunction of the endothelial membrane protein annexin A2, thereby leading to an overall reduction of fibrinolytic activity. Hyperglycaemia for 7 days significantly reduced cell surface fibrinolytic activity in human brain microvascular endothelial cells (HBMEC). Hyperglycaemia also decreased tissue type plasminogen activator (t-PA), plasminogen, and annexin A2 mRNA and protein expression, while increasing plasminogen activator inhibitor-1 (PAI-1). No changes in p11 mRNA or protein expression were detected. Hyperglycaemia significantly increased AGE-modified forms of total cellular and membrane annexin A2. The hyperglycemia-associated reduction in fibrinolytic activity was fully restored upon incubation with recombinant annexin A2 (rA2), but not AGE-modified annexin A2 or exogenous t-PA. Hyperglycaemia decreased t-PA, upregulated PAI-1 and induced AGE-related disruption of annexin A2 function, all of which contributed to the overall reduction in endothelial cell surface fibrinolytic activity. Further investigations to elucidate the underlying molecular mechanisms and pathophysiological implications of A2 derivatisation might ultimately lead to a better understanding of mechanisms of impaired vascular fibrinolysis, and to development of new interventional strategies for the thrombotic vascular complications in diabetes.

Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity

PubMed Central

Dai, Haibin; Yu, Zhanyang; Fan, Xiang; Liu, Ning; Yan, Min; Chen, Zhong; Lo, Eng H.; Hajjar, Katherine A.; Wang, Xiaoying

2014-01-01

Summary Hyperglycaemia impairs fibrinolytic activity on the surface of endothelial cells, but the underlying mechanisms are not fully understood. In this study, we tested the hypothesis that hyperglycaemia causes dysfunction of the endothelial membrane protein annexin A2, thereby leading to an overall reduction of fibrinolytic activity. Hyperglycaemia for 7 days significantly reduced cell surface fibrinolytic activity in human brain microvascular endothelial cells (HBMEC). Hyperglycaemia also decreased tissue type plasminogen activator (t-PA), plasminogen, and annexin A2 mRNA and protein expression, while increasing plasminogen activator inhibitor-1 (PAI-1). No changes in p11 mRNA or protein expression were detected. Hyperglycaemia significantly increased AGE-modified forms of total cellular and membrane annexin A2. The hyperglycemia-associated reduction in fibrinolytic activity was fully restored upon incubation with recombinant annexin A2 (rA2), but not AGE-modified annexin A2 or exogenous t-PA. Hyperglycaemia decreased t-PA, upregulated PAI-1 and induced AGE-related disruption of annexin A2 function, all of which contributed to the overall reduction in endothelial cell surface fibrinolytic activity. Further investigations to elucidate the underlying molecular mechanisms and pathophysiological implications of A2 derivatisation might ultimately lead to a better understanding of mechanisms of impaired vascular fibrinolysis, and to development of new interventional strategies for the thrombotic vascular complications in diabetes. PMID:23572070

Mechanisms mediating Nitroglycerin-induced Delayed Onset Hyperalgesia in the Rat

PubMed Central

Ferrari, Luiz F.; Levine, Jon D.; Green, Paul G.

2016-01-01

Nitroglycerin (glycerol trinitrate, GTN) induces headache in migraineurs, an effect that has been used both diagnostically and in the study of the pathophysiology of this neurovascular pain syndrome. An important feature of this headache is a delay from the administration of GTN to headache onset that, because of GTN’s very rapid metabolism, cannot be due to its pharmacokinetic profile. It has recently been suggested that activation of perivascular mast cells, which has been implicated in the pathophysiology of migraine, may contribute to this delay. We reported that hyperalgesia induced by intradermal GTN has a delay to onset of ~30 min in male and ~45 min in female rats. This hyperalgesia was greater in females, was prevented by pretreatment with the anti-migraine drug, sumatriptan, as well as by chronic pretreatment with the mast cell degranulator, compound 48/80. The acute administration of GTN and compound 48/80 both induced hyperalgesia that was prevented by pretreatment with octoxynol-9, which attenuates endothelial function, suggesting that GTN and mast cell-mediated hyperalgesia are endothelial cell-dependent. Furthermore, A-317491, a P2X3 antagonist, which inhibits endothelial cell-dependent hyperalgesia, also prevents GTN and mast cell-mediated hyperalgesia. We conclude that delayed onset mechanical hyperalgesia induced by GTN is mediated by activation of mast cells, which in turn release mediators that stimulate endothelial cells to release ATP, to act on P2X3, a ligand-gated ion channel, in perivascular nociceptors. A role of the mast and endothelial cell in GTN-induced hyperalgesia suggest potential novel risk factors and targets for the treatment of migraine. PMID:26779834

Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression.

PubMed

Chabot, Andréanne; Hertig, Vanessa; Boscher, Elena; Nguyen, Quang Trinh; Boivin, Benoît; Chebli, Jasmine; Bissonnette, Lyse; Villeneuve, Louis; Brochiero, Emmanuelle; Dupuis, Jocelyn; Calderone, Angelino

2016-07-01

Endothelial and epithelial cell transition to a mesenchymal phenotype was identified as cellular paradigms implicated in the appearance of fibroblasts and development of reactive fibrosis in interstitial lung disease. The intermediate filament protein nestin was highly expressed in fibrotic tissue, detected in fibroblasts and participated in proliferation and migration. The present study tested the hypothesis that the transition of endothelial and epithelial cells to a mesenchymal phenotype was delineated by nestin expression. Three weeks following hypobaric hypoxia, adult male Sprague-Dawley rats characterized by alveolar and perivascular lung fibrosis were associated with increased nestin protein and mRNA levels and marked appearance of nestin/collagen type I((+))-fibroblasts. In the perivascular region of hypobaric hypoxic rats, displaced CD31((+))-endothelial cells were detected, exhibited a mesenchymal phenotype and co-expressed nestin. Likewise, epithelial cells in the lungs of hypobaric hypoxic rats transitioned to a mesenchymal phenotype distinguished by the co-expression of E-cadherin and collagen. Following the removal of FBS from primary passage rat alveolar epithelial cells, TGF-β1 was detected in the media and a subpopulation acquired a mesenchymal phenotype characterized by E-cadherin downregulation and concomitant induction of collagen and nestin. Bone morphogenic protein-7 treatment of alveolar epithelial cells prevented E-cadherin downregulation, suppressed collagen induction but partially inhibited nestin expression. These data support the premise that the transition of endothelial and epithelial cells to a mesenchymal cell may have contributed in part to the appearance nestin/collagen type I((+))-fibroblasts and the reactive fibrotic response in the lungs of hypobaric hypoxic rats. © 2015 Wiley Periodicals, Inc.

Advanced Glycation End-Products Induce Apoptosis in Pancreatic Islet Endothelial Cells via NF-κB-Activated Cyclooxygenase-2/Prostaglandin E2 Up-Regulation

PubMed Central

Lan, Kuo-Cheng; Chiu, Chen-Yuan; Kao, Chia-Wei; Huang, Kuo-How; Wang, Ching-Chia; Huang, Kuo-Tong; Tsai, Keh-Sung

2015-01-01

Microvascular complications eventually affect nearly all patients with diabetes. Advanced glycation end-products (AGEs) resulting from hyperglycemia are a complex and heterogeneous group of compounds that accumulate in the plasma and tissues in diabetic patients. They are responsible for both endothelial dysfunction and diabetic vasculopathy. The aim of this study was to investigate the cytotoxicity of AGEs on pancreatic islet microvascular endothelial cells. The mechanism underlying the apoptotic effect of AGEs in pancreatic islet endothelial cell line MS1 was explored. The results showed that AGEs significantly decreased MS1 cell viability and induced MS1 cell apoptosis in a dose-dependent manner. AGEs dose-dependently increased the expressions of cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase in MS1 cells. Treatment of MS1 cells with AGEs also resulted in increased nuclear factor (NF)-κB-p65 phosphorylation and cyclooxygenase (COX)-2 expression. However, AGEs did not affect the expressions of endoplasmic reticulum (ER) stress-related molecules in MS1 cells. Pretreatment with NS398 (a COX-2 inhibitor) to inhibit prostaglandin E2 (PGE2) production reversed the induction of cleaved caspase-3, cleaved PARP, and MS1 cell viability. Moreover, AGEs significantly increased the receptor for AGEs (RAGE) protein expression in MS1 cells, which could be reversed by RAGE neutralizing antibody. RAGE Neutralizing antibody could also reverse the induction of cleaved caspase-3 and cleaved PARP and decreased cell viability induced by AGEs. These results implicate the involvement of NF-κB-activated COX-2/PGE2 up-regulation in AGEs/RAGE-induced islet endothelial cell apoptosis and cytotoxicity. These findings may provide insight into the pathological processes within the pancreatic islet microvasculature induced by AGEs accumulation. PMID:25898207

Mitochondria-targeted esculetin alleviates mitochondrial dysfunction by AMPK-mediated nitric oxide and SIRT3 regulation in endothelial cells: potential implications in atherosclerosis.

PubMed

Karnewar, Santosh; Vasamsetti, Sathish Babu; Gopoju, Raja; Kanugula, Anantha Koteswararao; Ganji, Sai Krishna; Prabhakar, Sripadi; Rangaraj, Nandini; Tupperwar, Nitin; Kumar, Jerald Mahesh; Kotamraju, Srigiridhar

2016-04-11

Mitochondria-targeted compounds are emerging as a new class of drugs that can potentially alter the pathophysiology of those diseases where mitochondrial dysfunction plays a critical role. We have synthesized a novel mitochondria-targeted esculetin (Mito-Esc) with an aim to investigate its effect during oxidative stress-induced endothelial cell death and angiotensin (Ang)-II-induced atherosclerosis in ApoE(-/-) mice. Mito-Esc but not natural esculetin treatment significantly inhibited H2O2- and Ang-II-induced cell death in human aortic endothelial cells by enhancing NO production via AMPK-mediated eNOS phosphorylation. While L-NAME (NOS inhibitor) significantly abrogated Mito-Esc-mediated protective effects, Compound c (inhibitor of AMPK) significantly decreased Mito-Esc-mediated increase in NO production. Notably, Mito-Esc promoted mitochondrial biogenesis by enhancing SIRT3 expression through AMPK activation; and restored H2O2-induced inhibition of mitochondrial respiration. siSIRT3 treatment not only completely reversed Mito-Esc-mediated mitochondrial biogenetic marker expressions but also caused endothelial cell death. Furthermore, Mito-Esc administration to ApoE(-/-) mice greatly alleviated Ang-II-induced atheromatous plaque formation, monocyte infiltration and serum pro-inflammatory cytokines levels. We conclude that Mito-Esc is preferentially taken up by the mitochondria and preserves endothelial cell survival during oxidative stress by modulating NO generation via AMPK. Also, Mito-Esc-induced SIRT3 plays a pivotal role in mediating mitochondrial biogenesis and perhaps contributes to its anti-atherogenic effects.

Circulating endothelial cells as marker of endothelial damage in male hypogonadism.

PubMed

Milardi, Domenico; Grande, Giuseppe; Giampietro, Antonella; Vendittelli, Francesca; Palumbo, Sara; Tartaglione, Linda; Marana, Riccardo; Pontecorvi, Alfredo; de Marinis, Laura; Zuppi, Cecilia; Capoluongo, Ettore

2012-01-01

Testosterone deficiency has become a frequently diagnosed condition in today's society affected by epidemic obesity, and is associated with cardiovascular risk. Recent studies have established the importance of altered vascular endothelium function in cardiovascular disease. The damage to the endothelium might also cause endothelial cell detachment, resulting in increased numbers of circulating endothelial cells (CEC) within the bloodstream. To evaluate whether hypogonadism could modify CEC count in peripheral bloodstream, we investigated peripheral blood CEC count using the CellSearch System, a semiautomatic method to accurately and reliably enumerate CECs, which are sorted based on a CD146(+), CD105(+), DAPI(+), CD45(-) phenotype, in a population of 20 patients with hypogonadism. The control group comprised 10 age- and sex-matched healthy participants. CEC count per milliliter was significantly increased in patients with hypogonadism vs the control group. In the group with hypogonadism, an inverse exponential correlation was present between testosterone levels and CEC count per milliliter. A direct linear correlation was present between waist circumference and CECs and between body mass index and CECs. The regression analysis showed that testosterone was the significant independent determinant of CECs. Our results underline that male hypogonadism is associated with endothelial dysfunction. The correlation between CEC and waist circumference underlines that visceral obesity may be synergically implicated in this regulation. Future studies are required to unveil the mechanisms involved in the pathogenesis of testosterone-induced endothelial disfunction, which may provide novel therapeutic targets to be incorporated in the management of hypogonadism.

Deregulation of tumor angiogenesis and blockade of tumor growth in PPARβ-deficient mice

PubMed Central

Müller-Brüsselbach, Sabine; Kömhoff, Martin; Rieck, Markus; Meissner, Wolfgang; Kaddatz, Kerstin; Adamkiewicz, Jürgen; Keil, Boris; Klose, Klaus J; Moll, Roland; Burdick, Andrew D; Peters, Jeffrey M; Müller, Rolf

2007-01-01

The peroxisome proliferator-activated receptor-β (PPARβ) has been implicated in tumorigenesis, but its precise role remains unclear. Here, we show that the growth of syngeneic Pparb wild-type tumors is impaired in Pparb−/− mice, concomitant with a diminished blood flow and an abundance of hyperplastic microvascular structures. Matrigel plugs containing pro-angiogenic growth factors harbor increased numbers of morphologically immature, proliferating endothelial cells in Pparb−/− mice, and retroviral transduction of Pparb triggers microvessel maturation. We have identified the Cdkn1c gene encoding the cell cycle inhibitor p57Kip2 as a PPARβ target gene and a mediator of the PPARβ-mediated inhibition of cell proliferation, which provides a possible mechanistic explanation for the observed tumor endothelial hyperplasia and deregulation of tumor angiogenesis in Pparb−/− mice. Our data point to an unexpected essential role for PPARβ in constraining tumor endothelial cell proliferation to allow for the formation of functional tumor microvessels. PMID:17641685

Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis.

PubMed

Tampo, Yoshiko; Kotamraju, Srigiridhar; Chitambar, Christopher R; Kalivendi, Shasi V; Keszler, Agnes; Joseph, Joy; Kalyanaraman, B

2003-01-10

Dichlorodihydrofluorescein (DCFH) is one of the most frequently used probes for detecting intracellular oxidative stress. In this study, we report that H2O2-dependent intracellular oxidation of DCFH to a green fluorescent product, 2',7'-dichlorofluorescein (DCF), required the uptake of extracellular iron transported through a transferrin receptor (TfR) in endothelial cells. H2O2-induced DCF fluorescence was inhibited by the monoclonal IgA-class anti-TfR antibody (42/6) that blocked TfR endocytosis and the iron uptake. H2O2-mediated inactivation of cytosolic aconitase was responsible for activation of iron regulatory protein-1 and increased expression of TfR, resulting in an increased iron uptake into endothelial cells. H2O2-mediated caspase-3 proteolytic activation was inhibited by anti-TfR antibody. Similar results were obtained in the presence of a lipid hydroperoxide. We conclude that hydroperoxide-induced DCFH oxidation and endothelial cell apoptosis required the uptake of extracellular iron by the TfR-dependent iron transport mechanism and that the peroxide-induced iron signaling, in general, has broader implications in oxidative vascular biology.

The CXC-Chemokine CXCL4 Interacts with Integrins Implicated in Angiogenesis

PubMed Central

Aidoudi, Sallouha; Bujakowska, Kinga; Kieffer, Nelly; Bikfalvi, Andreas

2008-01-01

The human CXC-chemokine CXCL4 is a potent inhibitor of tumor-induced angiogenesis. Considering that CXCL4 is sequestered in platelet α-granules and released following platelet activation in the vicinity of vessel wall injury, we tested the hypothesis that CXCL4 might function as a ligand for integrins. Integrins are a family of adhesion receptors that play a crucial role in angiogenesis by regulating early angiogenic processes, such as endothelial cell adhesion and migration. Here, we show that CXCL4 interacts with αvβ3 on the surface of αvβ3-CHO. More importantly, human umbilical vein endothelial cells adhere to immobilized CXCL4 through αvβ3 integrin, and also through other integrins, such as αvβ5 and α5β1. We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner. Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration. As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect. PMID:18648521

Vascular endothelial cells mediate mechanical stimulation-induced enhancement of endothelin hyperalgesia via activation of P2X2/3 receptors on nociceptors.

PubMed

Joseph, Elizabeth K; Green, Paul G; Bogen, Oliver; Alvarez, Pedro; Levine, Jon D

2013-02-13

Endothelin-1 (ET-1) is unique among a broad range of hyperalgesic agents in that it induces hyperalgesia in rats that is markedly enhanced by repeated mechanical stimulation at the site of administration. Antagonists to the ET-1 receptors, ET(A) and ET(B), attenuated both initial as well as stimulation-induced enhancement of hyperalgesia (SIEH) by endothelin. However, administering antisense oligodeoxynucleotide to attenuate ET(A) receptor expression on nociceptors attenuated ET-1 hyperalgesia but had no effect on SIEH, suggesting that this is mediated via a non-neuronal cell. Because vascular endothelial cells are both stretch sensitive and express ET(A) and ET(B) receptors, we tested the hypothesis that SIEH is dependent on endothelial cells by impairing vascular endothelial function with octoxynol-9 administration; this procedure eliminated SIEH without attenuating ET-1 hyperalgesia. A role for protein kinase Cε (PKCε), a second messenger implicated in the induction and maintenance of chronic pain, was explored. Intrathecal antisense for PKCε did not inhibit either ET-1 hyperalgesia or SIEH, suggesting no role for neuronal PKCε; however, administration of a PKCε inhibitor at the site of testing selectively attenuated SIEH. Compatible with endothelial cells releasing ATP in response to mechanical stimulation, P2X(2/3) receptor antagonists eliminated SIEH. The endothelium also appears to contribute to hyperalgesia in two ergonomic pain models (eccentric exercise and hindlimb vibration) and in a model of endometriosis. We propose that SIEH is produced by an effect of ET-1 on vascular endothelial cells, sensitizing its release of ATP in response to mechanical stimulation; ATP in turn acts at the nociceptor P2X(2/3) receptor.

Ceruloplasmin reduces the adhesion and scavenges superoxide during the interaction of activated polymorphonuclear leukocytes with endothelial cells.

PubMed Central

Broadley, C.; Hoover, R. L.

1989-01-01

The plasma protein, ceruloplasmin, has been implicated as an anti-inflammatory agent, although this property has not been demonstrated unequivocally in vivo. The role of this protein in an in vitro system of cultured endothelial cells and polymorphonuclear leukocytes (PMNs) was investigated. One of the initial steps in an inflammatory response is increased adhesion between PMNs and the endothelial lining of the blood vessels. The results showed that ceruloplasmin interferes with this process and reduces the number of phorbol myristate acetate-activated leukocytes that adhere to endothelium. Preincubation of either the activated PMNs or the endothelium with ceruloplasmin did not produce the same results, suggesting that the continuous presence of ceruloplasmin is required. During attachment PMNs become activated and release a variety of substances, including toxic oxygen species such as superoxide and hydrogen peroxide. In the in vitro system used in this study no injury occurred to the endothelial cells, as measured by 51Cr release, when activated PMNs were added with ceruloplasmin. The data show that ceruloplasmin reduced, in a dose dependent manner, the levels of superoxide produced by the activated PMNs, further supporting ceruloplasmin's previously reported role as a scavenger of superoxide. Ceruloplasmin also reduced the levels of superoxide when activated PMNs were in contact with endothelial cells. Although ceruloplasmin interfered with the copper-dependent scavenger enzyme, superoxide dismutase (SOD), in a cell-free system, ceruloplasmin had no effect on SOD in intact endothelial cells. These results suggest that ceruloplasmin may act as an anti-inflammatory agent by reducing the number of PMNs attaching to endothelium and by acting as an extracellular scavenger of superoxide. PMID:2552811

Ascorbate protects endothelial barrier function during septic insult: Role of protein phosphatase type 2A.

PubMed

Han, Min; Pendem, Suresh; Teh, Suet Ling; Sukumaran, Dinesh K; Wu, Feng; Wilson, John X

2010-01-01

Endothelial barrier dysfunction contributes to morbidity in sepsis. We tested the hypothesis that raising the intracellular ascorbate concentration protects the endothelial barrier from septic insult by inhibiting protein phosphatase type 2A. Monolayer cultures of microvascular endothelial cells were incubated with ascorbate, dehydroascorbic acid (DHAA), the NADPH oxidase inhibitors apocynin and diphenyliodonium, or the PP2A inhibitor okadaic acid and then were exposed to septic insult (lipopolysaccharide and interferon-gamma). Under standard culture conditions that depleted intracellular ascorbate, septic insult stimulated oxidant production and PP2A activity, dephosphorylated phosphoserine and phosphothreonine residues in the tight junction-associated protein occludin, decreased the abundance of occludin at cell borders, and increased monolayer permeability to albumin. NADPH oxidase inhibitors prevented PP2A activation and monolayer leak, showing that these changes required reactive oxygen species. Okadaic acid, at a concentration that inhibited PP2A activity and monolayer leak, prevented occludin dephosphorylation and redistribution, implicating PP2A in the response of occludin to septic insult. Incubation with ascorbate or DHAA raised intracellular ascorbate concentrations and mitigated the effects of septic insult. In conclusion, ascorbate acts within microvascular endothelial cells to inhibit septic stimulation of oxidant production by NADPH oxidase and thereby prevents PP2A activation, PP2A-dependent dephosphorylation and redistribution of occludin, and disruption of the endothelial barrier. Copyright 2009 Elsevier Inc. All rights reserved.

Biological properties of mud extracts derived from various spa resorts.

PubMed

Spilioti, Eliana; Vargiami, Margarita; Letsiou, Sophia; Gardikis, Konstantinos; Sygouni, Varvara; Koutsoukos, Petros; Chinou, Ioanna; Kassi, Eva; Moutsatsou, Paraskevi

2017-08-01

Spa resorts are known for thousands of years for their healing properties and have been empirically used for the treatment of many inflammatory conditions. Mud is one of the most often used natural materials for preventive, healing and cosmetic reasons and although it has been used since the antiquity, little light has been shed on its physical, chemical and biological properties. In this study we examined the effect of mud extracts on the expression of adhesion molecules (CAMs) by endothelial cells as well as their effects on monocyte adhesion to activated endothelial cells. Most of mud extracts inhibited the expression of VCAM-1 by endothelial cells and reduced monocyte adhesion to activated endothelial cells, indicating a potent anti-inflammatory activity. Furthermore, the mud extracts were tested for their antimicrobial activity; however, most of them appeared inactive against S. aureus and S. epidermidis. One of the mud extracts (showing the best stabilization features) increased significantly the expression of genes involved in cell protection, longevity and hydration of human keratinocytes, such as, collagen 6A1, forkhead box O3, sirtuin-1, superoxide dismutase 1 and aquaporin-3. The present study reveals that mud exerts important beneficial effects including anti-inflammatory and anti-aging activity as well as moisturizing effects, implicating important cosmeceutical applications.

Pdgfrα functions in endothelial-derived cells to regulate neural crest cells and the development of the great arteries.

PubMed

Aghajanian, Haig; Cho, Young Kuk; Rizer, Nicholas W; Wang, Qiaohong; Li, Li; Degenhardt, Karl; Jain, Rajan

2017-09-01

Originating as a single vessel emerging from the embryonic heart, the truncus arteriosus must septate and remodel into the aorta and pulmonary artery to support postnatal life. Defective remodeling or septation leads to abnormalities collectively known as conotruncal defects, which are associated with significant mortality and morbidity. Multiple populations of cells must interact to coordinate outflow tract remodeling, and the cardiac neural crest has emerged as particularly important during this process. Abnormalities in the cardiac neural crest have been implicated in the pathogenesis of multiple conotruncal defects, including persistent truncus arteriosus, double outlet right ventricle and tetralogy of Fallot. However, the role of the neural crest in the pathogenesis of another conotruncal abnormality, transposition of the great arteries, is less well understood. In this report, we demonstrate an unexpected role of Pdgfra in endothelial cells and their derivatives during outflow tract development. Loss of Pdgfra in endothelium and endothelial-derived cells results in double outlet right ventricle and transposition of the great arteries. Our data suggest that loss of Pdgfra in endothelial-derived mesenchyme in the outflow tract endocardial cushions leads to a secondary defect in neural crest migration during development. © 2017. Published by The Company of Biologists Ltd.

The regulation of Jmjd3 upon the expression of NF-κB downstream inflammatory genes in LPS activated vascular endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Shaoqing; Graduate School of Medicine, Nanchang University, Nanchang; Chen, Xia

Inflammatory mediators and adhesion molecules have been implicated in a variety of diseases including atherosclerosis. As both the mediator-releasing and targeted cells, vascular endothelial cells play key role in pathological processes. NF-κB signaling regulates a cluster of inflammatory factors in LPS-activated vascular endothelial cells but the underlying mechanisms remain largely unknown. Here, we investigated the epigenetic regulation of LPS upon the expression of inflammatory mediators and adhesion molecules. We found that LPS treatment promoted jmjd3 expression, enhanced Jmjd3 nuclear accumulation in human vascular endothelial cells. In addition, LPS enhanced the demethylation of H3K27me3, a specific substrate of Jmjd3. LPS treatmentmore » recruited Jmjd3 and NF-κB to the promoter region of target genes, suggesting Jmjd3 synergizes with NF-κB to activate the expression of target genes. We further found that Jmjd3 attenuated the methylation status in promoter region of target genes, culminating in target gene expression. Our findings unveil epigenetic regulations of LPS upon NF-κB pathway and identify Jmjd3 as a critical modulator of NF-κB pathway and potential therapeutic target for NF-κB related diseases including atherosclerosis.« less

Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells: IMPLICATIONS IN VEGF-DEPENDENT ANGIOGENESIS AND DIABETIC WOUND HEALING.

PubMed

Zhang, Yixuan; Li, Qiang; Youn, Ji Youn; Cai, Hua

2017-01-13

The VEGF/VEGFR2/Akt/eNOS/NO pathway is essential to VEGF-induced angiogenesis. We have previously discovered a novel role of calpain in mediating VEGF-induced PI3K/AMPK/Akt/eNOS activation through Ezrin. Here, we sought to identify possible feedback regulation of VEGFR2 by calpain via its substrate protein phosphotyrosine phosphatase 1B (PTP1B), and the relevance of this pathway to VEGF-induced angiogenesis, especially in diabetic wound healing. Overexpression of PTP1B inhibited VEGF-induced VEGFR2 and Akt phosphorylation in bovine aortic endothelial cells, while PTP1B siRNA increased both, implicating negative regulation of VEGFR2 by PTP1B. Calpain inhibitor ALLN induced VEGFR2 activation, which can be completely blocked by PTP1B overexpression. Calpain activation induced by overexpression or Ca/A23187 resulted in PTP1B cleavage, which can be blocked by ALLN. Moreover, calpain activation inhibited VEGF-induced VEGFR2 phosphorylation, which can be restored by PTP1B siRNA. These data implicate calpain/PTP1B negative feedback regulation of VEGFR2, in addition to the primary signaling pathway of VEGF/VEGFR2/calpain/PI3K/AMPK/Akt/eNOS. We next examined a potential role of PTP1B in VEGF-induced angiogenesis. Endothelial cells transfected with PTP1B siRNA showed faster wound closure in response to VEGF. Aortic discs isolated from PTP1B siRNA-transfected mice also had augmented endothelial outgrowth. Importantly, PTP1B inhibition and/or calpain overexpression significantly accelerated wound healing in STZ-induced diabetic mice. In conclusion, our data for the first time demonstrate a calpain/PTP1B/VEGFR2 negative feedback loop in the regulation of VEGF-induced angiogenesis. Modulation of local PTP1B and/or calpain activities may prove beneficial in the treatment of impaired wound healing in diabetes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of CD22 Ligands on Bone Marrow Sinusoidal Endothelium Implicated in CD22-dependent Homing of Recirculating B Cells

PubMed Central

Nitschke, Lars; Floyd, Helen; Ferguson, David J.P.; Crocker, Paul R.

1999-01-01

CD22 is a B cell–specific transmembrane protein known to function as a negative regulator of B cell signaling. It has also been implicated in cell adhesion through recognition of α2,6-linked sialic acids on glycans of target cells. Previous studies showed that CD22-deficient mice had a strongly reduced population of mature recirculating B cells in the bone marrow despite normal B cell development. Using a soluble recombinant form of the receptor (CD22-Fc), we demonstrate here that sialylated ligands for CD22 are expressed on sinusoidal endothelial cells of murine bone marrow but not on endothelial cells in other tissues examined. Injection of CD22-Fc revealed that the CD22 ligands in the bone marrow were accessible to the circulation. Treatment of mice with either CD22-Fc or affinity-purified anti-CD22 antibody led to an ∼50% reduction in mature recirculating B cells in the bone marrow without affecting numbers in the spleen. Finally, consistent with the notion that CD22 is a homing receptor, we show that compared with wild-type mice, CD22-deficient animals have a lower number of immunoglobulin M–secreting plasma cells in the bone marrow. PMID:10224292

Mechanisms mediating nitroglycerin-induced delayed-onset hyperalgesia in the rat.

PubMed

Ferrari, L F; Levine, J D; Green, P G

2016-03-11

Nitroglycerin (glycerol trinitrate, GTN) induces headache in migraineurs, an effect that has been used both diagnostically and in the study of the pathophysiology of this neurovascular pain syndrome. An important feature of this headache is a delay from the administration of GTN to headache onset that, because of GTN's very rapid metabolism, cannot be due to its pharmacokinetic profile. It has recently been suggested that activation of perivascular mast cells, which has been implicated in the pathophysiology of migraine, may contribute to this delay. We reported that hyperalgesia induced by intradermal GTN has a delay to onset of ∼ 30 min in male and ∼ 45 min in female rats. This hyperalgesia was greater in females, was prevented by pretreatment with the anti-migraine drug, sumatriptan, as well as by chronic pretreatment with the mast cell degranulator, compound 48/80. The acute administration of GTN and compound 48/80 both induced hyperalgesia that was prevented by pretreatment with octoxynol-9, which attenuates endothelial function, suggesting that GTN and mast cell-mediated hyperalgesia are endothelial cell-dependent. Furthermore, A-317491, a P2X3 antagonist, which inhibits endothelial cell-dependent hyperalgesia, also prevents GTN and mast cell-mediated hyperalgesia. We conclude that delayed-onset mechanical hyperalgesia induced by GTN is mediated by activation of mast cells, which in turn release mediators that stimulate endothelial cells to release ATP, to act on P2X3, a ligand-gated ion channel, in perivascular nociceptors. A role of the mast and endothelial cell in GTN-induced hyperalgesia suggests potential novel risk factors and targets for the treatment of migraine. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Impact of calcium signaling during infection of Neisseria meningitidis to human brain microvascular endothelial cells.

PubMed

Asmat, Tauseef M; Tenenbaum, Tobias; Jonsson, Ann-Beth; Schwerk, Christian; Schroten, Horst

2014-01-01

The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.

Regulation of Dipeptidyl Peptidase IV in the Post-stroke Rat Brain and In Vitro Ischemia: Implications for Chemokine-Mediated Neural Progenitor Cell Migration and Angiogenesis.

PubMed

Wesley, Umadevi V; Hatcher, James F; Ayvaci, Emine R; Klemp, Abby; Dempsey, Robert J

2017-09-01

Cerebral ischemia evokes abnormal release of proteases in the brain microenvironment that spatiotemporally impact angio-neurogenesis. Dipeptidyl peptidase IV (DPPIV), a cell surface and secreted protease, has been implicated in extracellular matrix remodeling by regulating cell adhesion, migration, and angiogenesis through modifying the functions of the major chemokine stromal-derived factor, SDF1. To elucidate the possible association of DPPIV in ischemic brain, we examined the expression of DPPIV in the post-stroke rat brain and under in vitro ischemia by oxygen glucose deprivation (OGD). We further investigated the effects of DPPIV on SDF1 mediated in vitro chemotactic and angiogenic functions. DPPIV protein and mRNA levels were significantly upregulated during repair phase in the ischemic cortex of the rat brain, specifically in neurons, astrocytes, and endothelial cells. In vitro exposure of Neuro-2a neuronal cells and rat brain endothelial cells to OGD resulted in upregulation of DPPIV. In vitro functional analysis showed that DPPIV decreases the SDF1-mediated angiogenic potential of rat brain endothelial cells and inhibits the migration of Neuro-2a and neural progenitor cells. Western blot analyses revealed decreased levels of phosphorylated ERK1/2 and AKT in the presence of DPPIV. DPPIV inhibitor restored the effects of SDF1. Proteome profile array screening further revealed that DPPIV decreases matrix metalloproteinase-9, a key downstream effector of ERK-AKT signaling pathways. Overall, delayed induction of DPPIV in response to ischemia/reperfusion suggests that DPPIV may play an important role in endogenous brain tissue remodeling and repair processes. This may be mediated through modulation of SDF1-mediated cell migration and angiogenesis.

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

PubMed Central

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

2015-01-01

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

Fibroblast TGF-Beta Signaling in Breast Development and Cancer

DTIC Science & Technology

2012-09-01

occurrence of a field effect for gene silencing in the stroma by cancer cells through hypermetylation. Among the genes implicated are GSTP1 , APC, RASSF1A...endothelial cells display GSTP1 and RARbeta2 promoter methylation in human prostate cancer. J Transl Med 2006;4:13. [19] Krop I, Player A, Tablante A

Brain mesenchymal stem cells: physiology and pathological implications.

PubMed

Pombero, Ana; Garcia-Lopez, Raquel; Martinez, Salvador

2016-06-01

Mesenchymal stem cells (MSCs) are defined as progenitor cells that give rise to a number of unique, differentiated mesenchymal cell types. This concept has progressively evolved towards an all-encompassing concept including multipotent perivascular cells of almost any tissue. In central nervous system, pericytes are involved in blood-brain barrier, and angiogenesis and vascular tone regulation. They form the neurovascular unit (NVU) together with endothelial cells, astrocytes and neurons. This functional structure provides an optimal microenvironment for neural proliferation in the adult brain. Neurovascular niche include both diffusible signals and direct contact with endothelial and pericytes, which are a source of diffusible neurotrophic signals that affect neural precursors. Therefore, MSCs/pericyte properties such as differentiation capability, as well as immunoregulatory and paracrine effects make them a potential resource in regenerative medicine. © 2016 Japanese Society of Developmental Biologists.

Theoretical estimates of mechanical properties of the endothelial cell cytoskeleton.

PubMed Central

Satcher, R L; Dewey, C F

1996-01-01

Current modeling of endothelial cell mechanics does not account for the network of F-actin that permeates the cytoplasm. This network, the distributed cytoplasmic structural actin (DCSA), extends from apical to basal membranes, with frequent attachments. Stress fibers are intercalated within the network, with similar frequent attachments. The microscopic structure of the DCSA resembles a foam, so that the mechanical properties can be estimated with analogy to these well-studied systems. The moduli of shear and elastic deformations are estimated to be on the order of 10(5) dynes/cm2. This prediction agrees with experimental measurements of the properties of cytoplasm and endothelial cells reported elsewhere. Stress fibers can potentially increase the modulus by a factor of 2-10, depending on whether they act in series or parallel to the network in transmitting surface forces. The deformations produced by physiological flow fields are of insufficient magnitude to disrupt cell-to-cell or DCSA cross-linkages. The questions raised by this paradox, and the ramifications of implicating the previously unreported DCSA as the primary force transmission element are discussed. Images FIGURE 2 PMID:8804594

The evidence for and against different modes of tumour cell extravasation in the lung: diapedesis, capillary destruction, necroptosis, and endothelialization.

PubMed

Paku, Sándor; Laszlo, Viktoria; Dezso, Katalin; Nagy, Peter; Hoda, Mir Alireza; Klepetko, Walter; Renyi-Vamos, Ferenc; Timar, Jozsef; Reynolds, Andrew R; Dome, Balazs

2017-03-01

The development of lung metastasis is a significant negative prognostic factor for cancer patients. The extravasation phase of lung metastasis involves interactions of tumour cells with the pulmonary endothelium. These interactions may have broad biological and medical significance, with potential clinical implications ranging from the discovery of lung metastasis biomarkers to the identification of targets for intervention in preventing lung metastases. Because of the potential significance, the mechanisms of tumour cell extravasation require cautious, systematic studies. Here, we discuss the literature pertaining to the proposed mechanisms of extravasation and critically compare a recently proposed mechanism (tumour cell-induced endothelial necroptosis) with the already described extravasation mechanisms in the lung. We also provide novel data that may help to explain the underlying physiological basis for endothelialization as a mechanism of tumour cell extravasation in the lung. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

VX680/MK-0457, a potent and selective Aurora kinase inhibitor, targets both tumor and endothelial cells in clear cell renal cell carcinoma

PubMed Central

Li, Yan; Zhang, Zhong-Fa; Chen, Jindong; Huang, Dan; Ding, Yan; Tan, Min-Han; Qian, Chao-Nan; Resau, James H; Kim, Hyung; Teh, Bin Tean

2010-01-01

Aurora kinases are key regulators of cell mitosis and have been implicated in the process of tumorigenesis. In recent years, the Aurora kinases have attracted much interest as promising targets for cancer treatment. Here we report on the roles of Aurora A and Aurora B kinases in clear cell renal cell carcinoma (ccRCC). Using genomewide expression array analysis of 174 patient samples of ccRCC, we found that expression levels of Aurora A and B were significantly elevated in ccRCC compared to normal kidney samples. High expression levels of Aurora A and Aurora B were significantly associated with advanced tumor stage and poor patient survival. Inhibition of Aurora kinase activity with the drug VX680 (also referred to as MK-0457) inhibited ccRCC cell growth in vitro and led to ccRCC cell accumulation in the G2/M phase and apoptosis. Growth of ccRCC xenograft tumors was also inhibited by VX680 treatment, accompanied by a reduction of tumor microvessel density. Analysis of endothelial cell lines demonstrated that VX680 inhibits endothelial cell growth with effects similar to that seen in ccRCC cells. Our findings suggest that VX680 inhibits the growth of ccRCC tumors by targeting the proliferation of both ccRCC tumor cells and tumor-associated endothelial cells. Aurora kinases and their downstream cell cycle proteins have an important role in ccRCC and may be potent prognostic markers and therapy targets for this disease. PMID:20589168

Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS1

PubMed Central

Zhang, Hong-Hai; Lechuga, Thomas J.; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

2016-01-01

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with “light” (L-12C614N4-Arg and L-12C614N2-Lys) or “heavy” (L-13C615N4-Arg and L-13C615N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. PMID:27075618

Intra-islet endothelial cell and β-cell crosstalk: Implication for islet cell transplantation

PubMed Central

Narayanan, Siddharth; Loganathan, Gopalakrishnan; Dhanasekaran, Maheswaran; Tucker, William; Patel, Ankit; Subhashree, Venugopal; Mokshagundam, SriPrakash; Hughes, Michael G; Williams, Stuart K; Balamurugan, Appakalai N

2017-01-01

The intra-islet microvasculature is a critical interface between the blood and islet endocrine cells governing a number of cellular and pathophysiological processes associated with the pancreatic tissue. A growing body of evidence indicates a strong functional and physical interdependency of β-cells with endothelial cells (ECs), the building blocks of islet microvasculature. Intra-islet ECs, actively regulate vascular permeability and appear to play a role in fine-tuning blood glucose sensing and regulation. These cells also tend to behave as “guardians”, controlling the expression and movement of a number of important immune mediators, thereby strongly contributing to the physiology of islets. This review will focus on the molecular signalling and crosstalk between the intra-islet ECs and β-cells and how their relationship can be a potential target for intervention strategies in islet pathology and islet transplantation. PMID:28507914

Pregnancy Augments VEGF-Stimulated In Vitro Angiogenesis and Vasodilator (NO and H2S) Production in Human Uterine Artery Endothelial Cells.

PubMed

Zhang, Hong-Hai; Chen, Jennifer C; Sheibani, Lili; Lechuga, Thomas J; Chen, Dong-Bao

2017-07-01

Augmented uterine artery (UA) production of vasodilators, including nitric oxide (NO) and hydrogen sulfide (H2S), has been implicated in pregnancy-associated and agonist-stimulated rise in uterine blood flow that is rate-limiting to pregnancy health. Developing a human UA endothelial cell (hUAEC) culture model from main UAs of nonpregnant (NP) and pregnant (P) women for testing a hypothesis that pregnancy augments endothelial NO and H2S production and endothelial reactivity to vascular endothelial growth factor (VEGF). Main UAs from NP and P women were used for developing hUAEC culture models. Comparisons were made between NP- and P-hUAECs in in vitro angiogenesis, activation of cell signaling, expression of endothelial NO synthase (eNOS) and H2S-producing enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase, and NO/H2S production upon VEGF stimulation. NP- and P-hUAECs displayed a typical cobblestone-like shape in culture and acetylated low-density lipoprotein uptake, stained positively for endothelial and negatively for smooth muscle markers, maintained key signaling proteins during passage, and had statistically significant greater eNOS and CBS proteins in P- vs NP-hUAECs. Treatment with VEGF stimulated in vitro angiogenesis and eNOS protein and NO production only in P-hUEACs and more robust cell signaling in P- vs NP-hUAECs. VEGF stimulated CBS protein expression, accounting for VEGF-stimulated H2S production in hUAECs. Comparisons between NP- and P-hUAECs reveal that pregnancy augments VEGF-stimulated in vitro angiogenesis and NO/H2S production in hUAECs, showing that the newly established hUAEC model provides a critical in vitro tool for understanding human uterine hemodynamics. Copyright © 2017 Endocrine Society

Elevated expression of NEU1 sialidase in idiopathic pulmonary fibrosis provokes pulmonary collagen deposition, lymphocytosis, and fibrosis.

PubMed

Luzina, Irina G; Lockatell, Virginia; Hyun, Sang W; Kopach, Pavel; Kang, Phillip H; Noor, Zahid; Liu, Anguo; Lillehoj, Erik P; Lee, Chunsik; Miranda-Ribera, Alba; Todd, Nevins W; Goldblum, Simeon E; Atamas, Sergei P

2016-05-15

Idiopathic pulmonary fibrosis (IPF) poses challenges to understanding its underlying cellular and molecular mechanisms and the development of better therapies. Previous studies suggest a pathophysiological role for neuraminidase 1 (NEU1), an enzyme that removes terminal sialic acid from glycoproteins. We observed increased NEU1 expression in epithelial and endothelial cells, as well as fibroblasts, in the lungs of patients with IPF compared with healthy control lungs. Recombinant adenovirus-mediated gene delivery of NEU1 to cultured primary human cells elicited profound changes in cellular phenotypes. Small airway epithelial cell migration was impaired in wounding assays, whereas, in pulmonary microvascular endothelial cells, NEU1 overexpression strongly impacted global gene expression, increased T cell adhesion to endothelial monolayers, and disrupted endothelial capillary-like tube formation. NEU1 overexpression in fibroblasts provoked increased levels of collagen types I and III, substantial changes in global gene expression, and accelerated degradation of matrix metalloproteinase-14. Intratracheal instillation of NEU1 encoding, but not control adenovirus, induced lymphocyte accumulation in bronchoalveolar lavage samples and lung tissues and elevations of pulmonary transforming growth factor-β and collagen. The lymphocytes were predominantly T cells, with CD8(+) cells exceeding CD4(+) cells by nearly twofold. These combined data indicate that elevated NEU1 expression alters functional activities of distinct lung cell types in vitro and recapitulates lymphocytic infiltration and collagen accumulation in vivo, consistent with mechanisms implicated in lung fibrosis.

Comparative Biology of Decellularized Lung Matrix: Implications of Species Mismatch in Regenerative Medicine

PubMed Central

Balestrini, Jenna L.; Gard, Ashley L.; Gerhold, Kristin A.; Wilcox, Elise C.; Liu, Angela; Schwan, Jonas; Le, Andrew V.; Baevova, Pavlina; Dimitrievska, Sashka; Zhao, Liping; Sundaram, Sumati; Sun, Huanxing; Rittié, Laure; Dyal, Rachel; Broekelmann, Tom J.; Mecham, Robert P.; Schwartz, Martin A.; Niklason, Laura E.; White, Eric S.

2016-01-01

Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration. PMID:27344365

Leptin promotes endothelial dysfunction in chronic kidney disease through AKT/GSK3β and β-catenin signals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Nannan; Liu, Bing; Song, Jiaguang

Endothelial dysfunction (ED) is a well-recognized instigator of cardiovascular diseases and develops in chronic kidney disease (CKD) with high rate. Recent studies have implicated that leptin is associated with endothelial dysfunction. We investigated the relationship between leptin and markers of ED in CKD patients and how leptin contributed to endothelial damage. 140 CKD patients and 140 healthy subjects were studied. Serum leptin levels were significantly higher in CKD than in controls and displayed significantly positive association with the increase levels of sICAM-1 and sVCAM-1 but negative correlation with flow-mediated dilatation (FMD) reduction in patients. Our in vitro study demonstrated that leptinmore » induced overexpression of ICAM-1 and VCAM-1, led to f-actin reorganization and vinculin assembly, increased endothelial monolayer permeability for FITC-dextran, and accelerated endothelial cell migration; these changes were markedly reversed when the cells were transfected with AKT or β-catenin shRNA vectors. Notably, high leptin resulted in hyper-phosphorylation of AKT and GSK3β, along with nuclear accumulation of β-catenin. In conclusion, serum leptin was elevated in CKD patients and it might contribute to endothelial dysfunction by disarrangement of f-actin cytoskeleton via a mechanism involving the AKT/GSK3β and β-catenin pathway. - Highlights: • Serum leptin was elevated in CKD patients and it was associated with endothelial dysfunction. • Leptin induced endothelial dysfunction by remodeling cytoskeleton in HUVECs. • Leptin promoted endothelial dysfunction via a mechanism involving the AKT/GSK3β and β-catenin signals.« less

Endothelial cellular senescence is inhibited by nitric oxide: Implications in atherosclerosis associated with menopause and diabetes

PubMed Central

Hayashi, Toshio; Matsui-Hirai, Hisako; Miyazaki-Akita, Asaka; Fukatsu, Akiko; Funami, Jun; Ding, Qun-Fang; Kamalanathan, Sumitra; Hattori, Yuichi; Ignarro, Louis J.; Iguchi, Akihisa

2006-01-01

Senescence may contribute to the pathogenesis of atherosclerosis. Although the bioavailability of nitric oxide (NO) is limited in senescence, the effect of NO on senescence and its relationship to cardiovascular risk factors have not been investigated fully. We studied these factors by investigating senescence-associated β-galactosidase (SA-β-gal) and human telomerase activity in human umbilical venous endothelial cells (HUVECs). Treatment with NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-aminoethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) and transfection with endothelial NO synthase (eNOS) into HUVECs each decreased the number of SA-β-gal positive cells and increased telomerase activity. The NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) abolished the effect of eNOS transfection. The physiological concentration of 17β-estradiol activated hTERT, decreased SA-β-gal-positive cells, and caused cell proliferation. However, ICI 182780, an estrogen receptor-specific antagonist, and l-NAME each inhibited these effects. Finally, we investigated the effect of NO bioavailability on high glucose-promoted cellular senescence of HUVECs. Inhibition by eNOS transfection of this cellular senescence under high glucose conditions was less pronounced. Treatment with l-arginine or l-citrulline of eNOS-transfected cells partially inhibited, and combination of l-arginine and l-citrulline with antioxidants strongly prevented, high glucose-induced cellular senescence. These data demonstrate that NO can prevent endothelial senescence, thereby contributing to the anti-senile action of estrogen. The ingestion of NO-boosting substances, including l-arginine, l-citrulline, and antioxidants, can delay endothelial senescence under high glucose. We suggest that the delay in endothelial senescence through NO and/or eNOS activation may have clinical utility in the treatment of atherosclerosis in the elderly. PMID:17075048

Endothelial glycocalyx dysfunction in disease: albuminuria and increased microvascular permeability.

PubMed

Salmon, Andrew H J; Satchell, Simon C

2012-03-01

Appreciation of the glomerular microcirculation as a specialized microcirculatory bed, rather than as an entirely separate entity, affords important insights into both glomerular and systemic microvascular pathophysiology. In this review we compare regulation of permeability in systemic and glomerular microcirculations, focusing particularly on the role of the endothelial glycocalyx, and consider the implications for disease processes. The luminal surface of vascular endothelium throughout the body is covered with endothelial glycocalyx, comprising surface-anchored proteoglycans, supplemented with adsorbed soluble proteoglycans, glycosaminoglycans and plasma constituents. In both continuous and fenestrated microvessels, this endothelial glycocalyx provides resistance to the transcapillary escape of water and macromolecules, acting as an integral component of the multilayered barrier provided by the walls of these microvessels (ie acting in concert with clefts or fenestrae across endothelial cell layers, basement membranes and pericytes). Dysfunction of any of these capillary wall components, including the endothelial glycocalyx, can disrupt normal microvascular permeability. Because of its ubiquitous nature, damage to the endothelial glycocalyx alters the permeability of multiple capillary beds: in the glomerulus this is clinically apparent as albuminuria. Generalized damage to the endothelial glycocalyx can therefore manifest as both albuminuria and increased systemic microvascular permeability. This triad of altered endothelial glycocalyx, albuminuria and increased systemic microvascular permeability occurs in a number of important diseases, such as diabetes, with accumulating evidence for a similar phenomenon in ischaemia-reperfusion injury and infectious disease. The detection of albuminuria therefore has implications for the function of the microcirculation as a whole. The importance of the endothelial glycocalyx for other aspects of vascular function/dysfunction, such as mechanotransduction, leukocyte-endothelial interactions and the development of atherosclerosis, indicate that alterations in the endothelial glycocalyx may also be playing a role in the dysfunction of other organs observed in these disease states. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Sickle red cell adhesion: many issues and some answers.

PubMed

Kaul, D K

2008-01-01

Among multiple pathologies associated with sickle cell disease, sickle red cell-endothelial interaction has been implicated as a potential initiating mechanism in vaso-occlusive events that characterize this disease. Vast literature exists on various aspects of sickle red cell adhesion, but many issues remain unresolved, especially pertaining to the role of sickle red cell heterogeneity, the relative role of multiple adhesion mechanisms and targets of antiadhesive therapy. This review briefly analyzes these issues.

A plant-based diet, atherogenesis, and coronary artery disease prevention.

PubMed

Tuso, Phillip; Stoll, Scott R; Li, William W

2015-01-01

A plant-based diet is increasingly becoming recognized as a healthier alternative to a diet laden with meat. Atherosclerosis associated with high dietary intake of meat, fat, and carbohydrates remains the leading cause of mortality in the US. This condition results from progressive damage to the endothelial cells lining the vascular system, including the heart, leading to endothelial dysfunction. In addition to genetic factors associated with endothelial dysfunction, many dietary and other lifestyle factors, such as tobacco use, high meat and fat intake, and oxidative stress, are implicated in atherogenesis. Polyphenols derived from dietary plant intake have protective effects on vascular endothelial cells, possibly as antioxidants that prevent the oxidation of low-density lipoprotein. Recently, metabolites of L-carnitine, such as trimethylamine-N-oxide, that result from ingestion of red meat have been identified as a potential predictive marker of coronary artery disease (CAD). Metabolism of L-carnitine by the intestinal microbiome is associated with atherosclerosis in omnivores but not in vegetarians, supporting CAD benefits of a plant-based diet. Trimethylamine-N-oxide may cause atherosclerosis via macrophage activation. We suggest that a shift toward a plant-based diet may confer protective effects against atherosclerotic CAD by increasing endothelial protective factors in the circulation while reducing factors that are injurious to endothelial cells. The relative ratio of protective factors to injurious endothelial exposure may be a novel approach to assessing an objective dietary benefit from a plant-based diet. This review provides a mechanistic perspective of the evidence for protection by a plant-based diet against atherosclerotic CAD.

Kaposi's Sarcoma-Associated Herpesvirus Interleukin-6 Modulates Endothelial Cell Movement by Upregulating Cellular Genes Involved in Migration.

PubMed

Giffin, Louise; West, John A; Damania, Blossom

2015-12-08

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of human Kaposi's sarcoma, a tumor that arises from endothelial cells, as well as two B cell lymphoproliferative diseases, primary effusion lymphoma and multicentric Castleman's disease. KSHV utilizes a variety of mechanisms to evade host immune responses and promote cellular transformation and growth in order to persist for the life of the host. A viral homolog of human interleukin-6 (hIL-6) named viral interleukin-6 (vIL-6) is encoded by KSHV and expressed in KSHV-associated cancers. Similar to hIL-6, vIL-6 is secreted, but the majority of vIL-6 is retained within the endoplasmic reticulum, where it can initiate functional signaling through part of the interleukin-6 receptor complex. We sought to determine how intracellular vIL-6 modulates the host endothelial cell environment by analyzing vIL-6's impact on the endothelial cell transcriptome. vIL-6 significantly altered the expression of many cellular genes associated with cell migration. In particular, vIL-6 upregulated the host factor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) at the protein and message levels. CEACAM1 has been implicated in tumor invasion and metastasis and promotes migration and vascular remodeling in endothelial cells. We report that vIL-6 upregulates CEACAM1 by a STAT3-dependent mechanism and that CEACAM1 promotes vIL-6-mediated migration. Furthermore, latent and de novo KSHV infections of endothelial cells also induce CEACAM1 expression. Collectively, our data suggest that vIL-6 modulates endothelial cell migration by upregulating the expression of cellular factors, including CEACAM1. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked with the development of three human malignancies, Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. KSHV expresses many factors that enable the virus to manipulate the host environment in order to persist and induce disease. The viral interleukin-6 (vIL-6) produced by KSHV is structurally and functionally homologous to the human cytokine interleukin-6, except that vIL-6 is secreted slowly and functions primarily from inside the host cell. To investigate the unique intracellular role of vIL-6, we analyzed the impact of vIL-6 on endothelial cell gene expression. We report that vIL-6 significantly alters the expression of genes associated with cell movement, including that for CEACAM1. The gene for CEACAM1 was upregulated by vIL-6 and by latent and primary KSHV infection and promotes vIL-6-mediated endothelial cell migration. This work advances the field's understanding of vIL-6 function and its contribution to KSHV pathogenesis. Copyright © 2015 Giffin et al.

Endothelial Progenitor Cells and Kidney Diseases.

PubMed

Ozkok, Abdullah; Yildiz, Alaattin

2018-05-10

Endothelial progenitor cells (EPC) are bone marrow derived or tissue-resident cells that play major roles in the maintenance of vascular integrity and repair of endothelial damage. Although EPCs may be capable of directly engrafting and regenerating the endothelium, the most important effects of EPCs seem to be depended on paracrine effects. In recent studies, specific microvesicles and mRNAs have been found to mediate the pro-angiogenic and regenerative effects of EPCs on endothelium. EPC counts have important prognostic implications in cardiovascular diseases (CVD). Uremia and inflammation are associated with lower EPC counts which probably contribute to increased CVD risks in patients with chronic kidney disease. Beneficial effects of the EPC therapies have been shown in studies performed on different models of CVD and kidney diseases such as acute and chronic kidney diseases and glomerulonephritis. However, lack of a clear definition and specific marker of EPCs is the most important problem causing difficulties in interpretation of the results of the studies investigating EPCs. © 2018 The Author(s). Published by S. Karger AG, Basel.

Caveolae: a regulatory platform for nutritional modulation of inflammatory diseases

PubMed Central

Layne, Joseph; Majkova, Zuzana; Smart, Eric J.; Toborek, Michal; Hennig, Bernhard

2010-01-01

Dietary intervention strategies have proven to be an effective means of decreasing several risk factors associated with the development of atherosclerosis. Endothelial cell dysfunction influences vascular inflammation and is involved in promoting the earliest stages of lesion formation. Caveolae are lipid raft microdomains abundant within the plasma membrane of endothelial cells and are responsible for mediating receptor-mediated signal transduction. Caveolae have been implicated in the regulation of enzymes associated with several key signaling pathways capable of determining intracellular redox status. Diet and plasma-derived nutrients may modulate an inflammatory outcome by interacting with and altering caveolae-associated cellular signaling. For example, omega-3 fatty acids and several polyphenolics have been shown to improve endothelial cell function by decreasing the formation of ROS and increasing NO bioavailability, events associated with altered caveolae composition. Thus, nutritional modulation of caveolae-mediated signaling events may provide an opportunity to ameliorate inflammatory signaling pathways capable of promoting the formation of vascular diseases, including atherosclerosis. PMID:21292468

Paracrine interactions of cancer-associated fibroblasts, macrophages and endothelial cells: tumor allies and foes.

PubMed

Ronca, Roberto; Van Ginderachter, Jo A; Turtoi, Andrei

2018-01-01

Tumor stroma is composed of many cellular subtypes, of which the most abundant are fibroblasts, macrophages and endothelial cells. During the process of tissue injury, these three cellular subtypes must coordinate their activity to efficiently contribute to tissue regeneration. In tumor, this mechanism is hijacked by cancer cells, which rewire the interaction of stromal cells to benefit tumor development. The present review aims at summarizing most relevant information concerning both pro-tumorigenic and anti-tumorigenic actions implicating the three stromal cell subtypes as well as their mutual interactions. Although stromal cells are generally regarded as tumor-supportive and at will manipulated by cancer cells, several novel studies point at many defaults in cancer cell-mediated stromal reprograming. Indeed, parts of initial tissue-protective and homeostatic functions of the stromal cells remain in place even after tumor development. Both tumor-supportive and tumor-suppressive functions have been well described for macrophages, whereas similar results are emerging for fibroblasts and endothelial cells. Recent success of immunotherapies have finally brought the long awaited proof that stroma is key for efficient tumor targeting. However, a better understanding of paracrine stromal interactions is needed in order to encourage drug development not only aiming at disruption of tumor-supportive communication but also re-enforcing, existing, tumor-suppressive mechanisms.

Targeting Endothelial Function to Treat Heart Failure with Preserved Ejection Fraction: The Promise of Exercise Training

PubMed Central

Lemmens, Katrien; Vrints, Christiaan J.

2017-01-01

Although the burden of heart failure with preserved ejection fraction (HFpEF) is increasing, there is no therapy available that improves prognosis. Clinical trials using beta blockers and angiotensin converting enzyme inhibitors, cardiac-targeting drugs that reduce mortality in heart failure with reduced ejection fraction (HFrEF), have had disappointing results in HFpEF patients. A new “whole-systems” approach has been proposed for designing future HFpEF therapies, moving focus from the cardiomyocyte to the endothelium. Indeed, dysfunction of endothelial cells throughout the entire cardiovascular system is suggested as a central mechanism in HFpEF pathophysiology. The objective of this review is to provide an overview of current knowledge regarding endothelial dysfunction in HFpEF. We discuss the molecular and cellular mechanisms leading to endothelial dysfunction and the extent, presence, and prognostic importance of clinical endothelial dysfunction in different vascular beds. We also consider implications towards exercise training, a promising therapy targeting system-wide endothelial dysfunction in HFpEF. PMID:28706575

Development of the blood-brain barrier: a historical point of view.

PubMed

Ribatti, Domenico; Nico, Beatrice; Crivellato, Enrico; Artico, Marco

2006-01-01

Although there has been considerable controversy since the observation by Ehrlich more than 100 years ago that the brain did not take up dyes from the vascular system, the concept of an endothelial blood-brain barrier (BBB) was confirmed by the unequivocal demonstration that the passage of molecules from blood to brain and vice versa was prevented by endothelial tight junctions (TJs). There are three major functions implicated in the term "BBB": protection of the brain from the blood milieu, selective transport, and metabolism or modification of blood- or brain-borne substances. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of complex TJs and a number of specific transport and enzyme systems that regulate molecular traffic across the endothelial cells. The development of the BBB is a complex process that leads to endothelial cells with unique permeability characteristics due to high electrical resistance and the expression of specific transporters and metabolic pathways. This review article summarizes the historical background underlying our current knowledge of the cellular and molecular mechanisms involved in the development and maintenance of the BBB. (c) 2006 Wiley-Liss, Inc.

Mice with targeted inactivation of ppap2b in endothelial and hematopoietic cells display enhanced vascular inflammation and permeability.

PubMed

Panchatcharam, Manikandan; Salous, Abdel K; Brandon, Jason; Miriyala, Sumitra; Wheeler, Jessica; Patil, Pooja; Sunkara, Manjula; Morris, Andrew J; Escalante-Alcalde, Diana; Smyth, Susan S

2014-04-01

Lipid phosphate phosphatase 3 (LPP3), encoded by the PPAP2B gene, is an integral membrane enzyme that dephosphorylates, and thereby terminates, the G-protein-coupled receptor-mediated signaling actions of lysophosphatidic acid (LPA) and sphingosine-1-phosphate. LPP3 is essential for normal vascular development in mice, and a common PPAP2B polymorphism is associated with increased risk of coronary artery disease in humans. Herein, we investigate the function of endothelial LPP3 to understand its role in the development and human disease. We developed mouse models with selective LPP3 deficiency in endothelial and hematopoietic cells. Tyrosine kinase Tek promoter-mediated inactivation of Ppap2b resulted in embryonic lethality because of vascular defects. LPP3 deficiency in adult mice, achieved using a tamoxifen-inducible Cre transgene under the control of the Tyrosine kinase Tek promoter, enhanced local and systemic inflammatory responses. Endothelial, but not hematopoietic, cell LPP3 deficiency led to significant increases in vascular permeability at baseline and enhanced sensitivity to inflammation-induced vascular leak. Endothelial barrier function was restored by pharmacological or genetic inhibition of either LPA production by the circulating lysophospholipase D autotaxin or of G-protein-coupled receptor-dependent LPA signaling. Our results identify a role for the autotaxin/LPA-signaling nexus as a mediator of endothelial permeability in inflammation and demonstrate that LPP3 limits these effects. These findings have implications for therapeutic targets to maintain vascular barrier function in inflammatory states.

Blood vessel endothelium-directed tumor cell streaming in breast tumors requires the HGF/C-Met signaling pathway

PubMed Central

Leung, E; Xue, A; Wang, Y; Rougerie, P; Sharma, V P; Eddy, R; Cox, D; Condeelis, J

2017-01-01

During metastasis to distant sites, tumor cells migrate to blood vessels. In vivo, breast tumor cells utilize a specialized mode of migration known as streaming, where a linear assembly of tumor cells migrate directionally towards blood vessels on fibronectin-collagen I-containing extracellular matrix (ECM) fibers in response to chemotactic signals. We have successfully reconstructed tumor cell streaming in vitro by co-plating tumors cells, macrophages and endothelial cells on 2.5 μm thick ECM-coated micro-patterned substrates. We found that tumor cells and macrophages, when plated together on the micro-patterned substrates, do not demonstrate sustained directional migration in only one direction (sustained directionality) but show random bi-directional walking. Sustained directionality of tumor cells as seen in vivo was established in vitro when beads coated with human umbilical vein endothelial cells were placed at one end of the micro-patterned ‘ECM fibers' within the assay. We demonstrated that these endothelial cells supply the hepatocyte growth factor (HGF) required for the chemotactic gradient responsible for sustained directionality. Using this in vitro reconstituted streaming system, we found that directional streaming is dependent on, and most effectively blocked, by inhibiting the HGF/C-Met signaling pathway between endothelial cells and tumor cells. Key observations made with the in vitro reconstituted system implicating C-Met signaling were confirmed in vivo in mammary tumors using the in vivo invasion assay and intravital multiphoton imaging of tumor cell streaming. These results establish HGF/C-Met as a central organizing signal in blood vessel-directed tumor cell migration in vivo and highlight a promising role for C-Met inhibitors in blocking tumor cell streaming and metastasis in vivo, and for use in human trials. PMID:27893712

TRAIL death receptor 4 signaling via lysosome fusion and membrane raft clustering in coronary arterial endothelial cells: evidence from ASM knockout mice.

PubMed

Li, Xiang; Han, Wei-Qing; Boini, Krishna M; Xia, Min; Zhang, Yang; Li, Pin-Lan

2013-01-01

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4 (DR4), have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MR) clustering and the formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and co-localized with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1 (+/+)) mice. Furthermore, TRAIL triggered ASM translocation, ceramide production, and NADPH oxidase aggregation in MR clusters in Smpd1 ( +/+ ) CAECs, whereas these observations were not found in Smpd1 (-/-) CAECs. Moreover, ASM deficiency reduced TRAIL-induced O(2) (-[Symbol: see text]) production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer, we found that Lamp-1 (lysosome membrane marker protein) and ganglioside G(M1) (MR marker) were trafficking together in Smpd1 (+/+) CAECs, which was absent in Smpd1 (-/-) CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1 (-/-) CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking, membrane fusion and formation of MR redox signaling platforms, which may play an important role in DR4-mediated redox signaling in CAECs and consequently endothelial dysfunction.

Tumor microenvironment conditions alter Akt and Na+/H+ exchanger NHE1 expression in endothelial cells more than hypoxia alone: implications for endothelial cell function in cancer.

PubMed

Pedersen, A K; Mendes Lopes de Melo, J; Mørup, N; Tritsaris, K; Pedersen, S F

2017-08-14

Chronic angiogenesis is a hallmark of most tumors and takes place in a hostile tumor microenvironment (TME) characterized by hypoxia, low nutrient and glucose levels, elevated lactate and low pH. Despite this, most studies addressing angiogenic signaling use hypoxia as a proxy for tumor conditions. Here, we compared the effects of hypoxia and TME conditions on regulation of the Na + /H + exchanger NHE1, Ser/Thr kinases Akt1-3, and downstream effectors in endothelial cells. Human umbilical vein endothelial cells (HUVEC) and Ea.hy926 endothelial cells were exposed to simulated TME (1% hypoxia, low serum, glucose, pH, high lactate) or 1% hypoxia for 24 or 48 h, with or without NHE1 inhibition or siRNA-mediated knockdown. mRNA and protein levels of NHE1, Akt1-3, and downstream effectors were assessed by qPCR and Western blotting, vascular endothelial growth factor (VEGF) release by ELISA, and motility by scratch assay. Within 24 h, HIF-1α level and VEGF mRNA level were increased robustly by TME and modestly by hypoxia alone. The NHE1 mRNA level was decreased by both hypoxia and TME, and NHE1 protein was reduced by TME in Ea.hy926 cells. Akt1-3 mRNA was detected in HUVEC and Ea.hy926 cells, Akt1 most abundantly. Akt1 protein expression was reduced by TME yet unaffected by hypoxia, while Akt phosphorylation was increased by TME. The Akt loss was partly reversed by MCF-7 human breast cancer cell conditioned medium, suggesting that in vivo, the cancer cell secretome may compensate for adverse effects of TME on endothelial cells. TME, yet not hypoxia, reduced p70S6 kinase activity and ribosomal protein S6 phosphorylation and increased eIF2α phosphorylation, consistent with inhibition of protein translation. Finally, TME reduced Retinoblastoma protein phosphorylation and induced poly-ADP-ribose polymerase (PARP) cleavage consistent with inhibition of proliferation and induction of apoptosis. NHE1 knockdown, mimicking the effect of TME on NHE1 expression, reduced Ea.hy926 migration. TME effects on HIF-1α, VEGF, Akt, translation, proliferation or apoptosis markers were unaffected by NHE1 knockdown/inhibition. NHE1 and Akt are downregulated by TME conditions, more potently than by hypoxia alone. This inhibits endothelial cell migration and growth in a manner likely modulated by the cancer cell secretome.

Leukocyte-inspired biodegradable particles that selectively and avidly adhere to inflamed endothelium in vitro and in vivo

NASA Astrophysics Data System (ADS)

Sakhalkar, Harshad S.; Dalal, Milind K.; Salem, Aliasger K.; Ansari, Ramin; Fu, Jie; Kiani, Mohammad F.; Kurjiaka, David T.; Hanes, Justin; Shakesheff, Kevin M.; Goetz, Douglas J.

2003-12-01

We exploited leukocyte-endothelial cell adhesion chemistry to generate biodegradable particles that exhibit highly selective accumulation on inflamed endothelium in vitro and in vivo. Leukocyte-endothelial cell adhesive particles exhibit up to 15-fold higher adhesion to inflamed endothelium, relative to noninflamed endothelium, under in vitro flow conditions similar to that present in blood vessels, a 6-fold higher adhesion to cytokine inflamed endothelium relative to non-cytokine-treated endothelium in vivo, and a 10-fold enhancement in adhesion to trauma-induced inflamed endothelium in vivo due to the addition of a targeting ligand. The leukocyte-inspired particles have adhesion efficiencies similar to that of leukocytes and were shown to target each of the major inducible endothelial cell adhesion molecules (E-selectin, P-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1) that are up-regulated at sites of pathological inflammation. The potential for targeted drug delivery to inflamed endothelium has significant implications for the improved treatment of an array of pathologies, including cardiovascular disease, arthritis, inflammatory bowel disease, and cancer.

Inhibition of cell migration by focal adhesion kinase: Time-dependent difference in integrin-induced signaling between endothelial and hepatoblastoma cells.

PubMed

Yu, Hongchi; Gao, Min; Ma, Yunlong; Wang, Lijuan; Shen, Yang; Liu, Xiaoheng

2018-05-01

angiogenesis plays an important role in the development and progression of tumors, and it involves a series of signaling pathways contributing to the migration of endothelial cells for vascularization and to the invasion of cancer cells for secondary tumor formation. Among these pathways, the focal adhesion kinase (FAK) signaling cascade has been implicated in a variety of human cancers in connection with cell adhesion and migration events leading to tumor angiogenesis, metastasis and invasion. Therefore, the inhibition of FAK in endothelial and/or cancer cells is a potential target for anti‑angiogenic therapy. In the present study, a small‑molecule FAK inhibitor, 1,2,4,5-benzenetetramine tetrahydrochloride (Y15), was used to study the effects of FAK inhibition on the adhesion and migration behaviors of vascular endothelial cells (VECs) and human hepatoblastoma cells. Furthermore, the time-dependent differences in proteins associated with the integrin-mediated FAK/Rho GTPases signaling pathway within 2 h were examined. The results indicated that the inhibition of FAK significantly decreased the migration ability of VECs and human hepatoblastoma cells in a dose-dependent manner. Inhibition of FAK promoted cell detachment by decreasing the expression of focal adhesion components, and blocked cell motility by reducing the level of Rho GTPases. However, the expression of crucial proteins involved in integrin-induced signaling in two cell lines exhibited a time-dependent difference with increased duration of FAK inhibitor treatment, suggesting different mechanisms of FAK-mediated cell migration behavior. These results suggest that the mechanism underlying FAK-mediated adhesion and migration behavior differs among various cells, which is expected to provide evidence for future FAK therapy targeted against tumor angiogenesis.

Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells

PubMed Central

Zhang, Yixuan; Li, Qiang; Youn, Ji Youn; Cai, Hua

2017-01-01

The VEGF/VEGFR2/Akt/eNOS/NO pathway is essential to VEGF-induced angiogenesis. We have previously discovered a novel role of calpain in mediating VEGF-induced PI3K/AMPK/Akt/eNOS activation through Ezrin. Here, we sought to identify possible feedback regulation of VEGFR2 by calpain via its substrate protein phosphotyrosine phosphatase 1B (PTP1B), and the relevance of this pathway to VEGF-induced angiogenesis, especially in diabetic wound healing. Overexpression of PTP1B inhibited VEGF-induced VEGFR2 and Akt phosphorylation in bovine aortic endothelial cells, while PTP1B siRNA increased both, implicating negative regulation of VEGFR2 by PTP1B. Calpain inhibitor ALLN induced VEGFR2 activation, which can be completely blocked by PTP1B overexpression. Calpain activation induced by overexpression or Ca/A23187 resulted in PTP1B cleavage, which can be blocked by ALLN. Moreover, calpain activation inhibited VEGF-induced VEGFR2 phosphorylation, which can be restored by PTP1B siRNA. These data implicate calpain/PTP1B negative feedback regulation of VEGFR2, in addition to the primary signaling pathway of VEGF/VEGFR2/calpain/PI3K/AMPK/Akt/eNOS. We next examined a potential role of PTP1B in VEGF-induced angiogenesis. Endothelial cells transfected with PTP1B siRNA showed faster wound closure in response to VEGF. Aortic discs isolated from PTP1B siRNA-transfected mice also had augmented endothelial outgrowth. Importantly, PTP1B inhibition and/or calpain overexpression significantly accelerated wound healing in STZ-induced diabetic mice. In conclusion, our data for the first time demonstrate a calpain/PTP1B/VEGFR2 negative feedback loop in the regulation of VEGF-induced angiogenesis. Modulation of local PTP1B and/or calpain activities may prove beneficial in the treatment of impaired wound healing in diabetes. PMID:27872190

Ankyrin-G Inhibits Endocytosis of Cadherin Dimers.

PubMed

Cadwell, Chantel M; Jenkins, Paul M; Bennett, Vann; Kowalczyk, Andrew P

2016-01-08

Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Therefore, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. However, the mechanisms that control junctional plasticity are not fully understood. Vascular endothelial cadherin (VE-cadherin) is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β- and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of cadherin and stabilizes it at the plasma membrane. Here we report that induced cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Zika virus infection of cellular components of the blood-retinal barriers: implications for viral associated congenital ocular disease.

PubMed

Roach, Tracoyia; Alcendor, Donald J

2017-03-03

Ocular abnormalities present in microcephalic infants with presumed Zika virus (ZIKV) congenital disease includes focal pigment mottling of the retina, chorioretinal atrophy, optic nerve abnormalities, and lens dislocation. Target cells in the ocular compartment for ZIKV infectivity are unknown. The cellular response of ocular cells to ZIKV infection has not been described. Mechanisms for viral dissemination in the ocular compartment of ZIKV-infected infants and adults have not been reported. Here, we identify target cells for ZIKV infectivity in both the inner and outer blood-retinal barriers (IBRB and OBRB), describe the cytokine expression profile in the IBRB after ZIKV exposure, and propose a mechanism for viral dissemination in the retina. We expose primary cellular components of the IBRB including human retinal microvascular endothelial cells, retinal pericytes, and Müller cells as well as retinal pigmented epithelial cells of the OBRB to the PRVABC56 strain of ZIKV. Viral infectivity was analyzed by microscopy, immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR and qRT-PCR). Angiogenic and proinflammatory cytokines were measured by Luminex assays. We find by immunofluorescent staining using the Flavivirus 4G2 monoclonal antibody that retinal endothelial cells and pericytes of the IBRB and retinal pigmented epithelial cells of the OBRB are fully permissive for ZIKV infection but not Müller cells when compared to mock-infected controls. We confirmed ZIKV infectivity in retinal endothelial cells, retinal pericytes, and retinal pigmented epithelial cells by RT-PCR and qRT-PCR using ZIKV-specific oligonucleotide primers. Expression profiles by Luminex assays in retinal endothelial cells infected with ZIKV revealed a marginal increase in levels of beta-2 microglobulin (β2-m), granulocyte macrophage colony-stimulating factor (GMCSF), intercellular adhesion molecule 1 (ICAM-1), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP1), and vascular cell adhesion molecule 1 (VCAM-1) and higher levels of regulated upon activation, normal T cell expressed and presumably secreted (RANTES) but lower levels of interleukin-4 (IL-4) compared to controls. Retinal endothelial cells, retinal pericytes, and retinal pigmented epithelial cells are fully permissive for ZIKV lytic replication and are primary target cells in the retinal barriers for infection. ZIKV infection of retinal endothelial cells and retinal pericytes induces significantly higher levels of RANTES that likely contributes to ocular inflammation.

Combination of PI3K/Akt/mTOR inhibitors and PDT in endothelial and tumor cells

NASA Astrophysics Data System (ADS)

Fateye, Babasola; Chen, Bin

2011-02-01

The PI3/Akt/mTOR kinase signaling pathway is a major signaling pathway in eukaryotic cells, and dysregulation of this signaling pathway has been implicated in tumorigenesis and malignancy in several cancers including prostate cancer. We assessed the effects of combination PI3K pathway inhibition on the efficacy of PDT in human prostate tumor cell line (PC3) and SV40-transformed mouse endothelial cell line (SVEC-40). Combination of PDT and BEZ 235 (BEZ), a pan-PI3/ mTOR kinase inhibitor additively enhanced efficacy of sub-lethal PDT in both cell lines. The combination of the pan-PI3/ mTOR kinase inhibitor LY294002 (LY) with PDT also enhanced efficacy of PDT in PC3 in an additive manner but synergistically in SVEC. In order to determine the mechanism of enhancement of efficacy, we assessed apoptosis and autophagy following PDT. PDT-mediated apoptosis was enhanced in endothelial cells, by both BEZ and LY rapidly after treatment. Compared to SVEC, PC3 cells are apoptosis-deficient and apoptosis was not significantly enhanced by either LY or BEZ. However, lethal PDT of PC3 cells induced a delayed autophagic response which may be enhanced by combination, depending on PI3K inhibitor and dose.

Novel INTeraction of MUC4 and galectin: potential pathobiological implications for metastasis in lethal pancreatic cancer.

PubMed

Senapati, Shantibhusan; Chaturvedi, Pallavi; Chaney, William G; Chakraborty, Subhankar; Gnanapragassam, Vinayaga S; Sasson, Aaron R; Batra, Surinder K

2011-01-15

Several studies have reported aberrant expression of MUC4 in pancreatic cancer (PC), which is associated with tumorigenicity and metastasis. Mechanisms through which MUC4 promote metastasis of PC cells to distant organs are poorly defined. Identification of MUC4-galectin-3 interaction and its effect on the adhesion of cancer cells to endothelial cells were done by immunoprecipitation and cell-cell adhesion assays, respectively. Serum galectin-3 level for normal and PC patients were evaluated through ELISA. In the present study, we have provided clinical evidence that the level of galectin-3 is significantly elevated in the sera of PC patients with metastatic disease compared with patients without metastasis (P = 0.04) and healthy controls (P = 0.00001). Importantly, for the first time, we demonstrate that MUC4 present on the surface of circulating PC cells plays a significant role in the transient and reversible attachment (docking) of circulating tumor cells to the surface of endothelial cells. Further, exogenous galectin-3 at concentrations similar to that found in the sera of PC patients interacts with MUC4 via surface glycans such as T antigens, which results in the clustering of MUC4 on the cell surface and a stronger attachment (locking) of circulating tumor cells to the endothelium. Altogether, these findings suggest that PC cell-associated MUC4 helps in the docking of tumor cells on the endothelial surface. During cancer progression, MUC4-galectin-3 interaction-mediated clustering of MUC4 may expose the surface adhesion molecules, which in turn promotes a stronger attachment (locking) of tumor cells to the endothelial surface. ©2010 AACR.

GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury.

PubMed

Yi, Lei; Huang, Xiaoqin; Guo, Feng; Zhou, Zengding; Chang, Mengling; Huan, Jingning

2017-01-01

The bacterial endotoxin or lipopolysaccharide (LPS) leads to the extensive vascular endothelial cells (EC) injury under septic conditions. Guanine nucleotide exchange factor-H1 (GEF-H1)/ROCK signaling not only involved in LPS-induced overexpression of pro-inflammatory mediator in ECs but also implicated in LPS-induced endothelial hyper-permeability. However, the mechanisms behind LPS-induced GEF-H1/ROCK signaling activation in the progress of EC injury remain incompletely understood. GEF-H1 localized on microtubules (MT) and is suppressed in its MT-bound state. MT disassembly promotes GEF-H1 release from MT and stimulates downstream ROCK-specific GEF activity. Since glycogen synthase kinase (GSK-3beta) participates in regulating MT dynamics under pathologic conditions, we examined the pivotal roles for GSK-3beta in modulating LPS-induced activation of GEF-H1/ROCK, increase of vascular endothelial permeability and severity of acute lung injury (ALI). In this study, we found that LPS induced human pulmonary endothelial cell (HPMEC) monolayers disruption accompanied by increase in GSK-3beta activity, activation of GEF-H1/ROCK signaling and decrease in beta-catenin and ZO-1 expression. Inhibition of GSK-3beta reduced HPMEC monolayers hyper-permeability and GEF-H1/ROCK activity in response to LPS. GSK-3beta/GEF-H1/ROCK signaling is implicated in regulating the expression of beta-catenin and ZO-1. In vivo , GSK-3beta inhibition attenuated LPS-induced activation of GEF-H1/ROCK pathway, lung edema and subsequent ALI. These findings present a new mechanism of GSK-3beta-dependent exacerbation of lung micro-vascular hyper-permeability and escalation of ALI via activation of GEF-H1/ROCK signaling and disruption of intracellular junctional proteins under septic condition.

Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs).

PubMed

DeStefano, Jackson G; Xu, Zinnia S; Williams, Ashley J; Yimam, Nahom; Searson, Peter C

2017-08-04

The endothelial cells that form the lumen of capillaries and microvessels are an important component of the blood-brain barrier. Cell phenotype is regulated by transducing a range of biomechanical and biochemical signals in the local microenvironment. Here we report on the role of shear stress in modulating the morphology, motility, proliferation, apoptosis, and protein and gene expression, of confluent monolayers of human brain microvascular endothelial cells derived from induced pluripotent stem cells. To assess the response of derived human brain microvascular endothelial cells (dhBMECs) to shear stress, confluent monolayers were formed in a microfluidic device. Monolayers were subjected to a shear stress of 4 or 12 dyne cm -2 for 40 h. Static conditions were used as the control. Live cell imaging was used to assess cell morphology, cell speed, persistence, and the rates of proliferation and apoptosis as a function of time. In addition, immunofluorescence imaging and protein and gene expression analysis of key markers of the blood-brain barrier were performed. Human brain microvascular endothelial cells exhibit a unique phenotype in response to shear stress compared to static conditions: (1) they do not elongate and align, (2) the rates of proliferation and apoptosis decrease significantly, (3) the mean displacement of individual cells within the monolayer over time is significantly decreased, (4) there is no cytoskeletal reorganization or formation of stress fibers within the cell, and (5) there is no change in expression levels of key blood-brain barrier markers. The characteristic response of dhBMECs to shear stress is significantly different from human and animal-derived endothelial cells from other tissues, suggesting that this unique phenotype that may be important in maintenance of the blood-brain barrier. The implications of this work are that: (1) in confluent monolayers of dhBMECs, tight junctions are formed under static conditions, (2) the formation of tight junctions decreases cell motility and prevents any morphological transitions, (3) flow serves to increase the contact area between cells, resulting in very low cell displacement in the monolayer, (4) since tight junctions are already formed under static conditions, increasing the contact area between cells does not cause upregulation in protein and gene expression of BBB markers, and (5) the increase in contact area induced by flow makes barrier function more robust.

Obesity and neuroinflammatory phenotype in mice lacking endothelial megalin.

PubMed

Bartolome, Fernando; Antequera, Desiree; Tavares, Eva; Pascual, Consuelo; Maldonado, Rosario; Camins, Antoni; Carro, Eva

2017-01-31

The multiligand receptor megalin controls the brain uptake of a number of ligands, including insulin and leptin. Despite the role of megalin in the transport of these metabolically relevant hormones, the role of megalin at the blood-brain-barrier (BBB) has not yet been explored in the context of metabolic regulation. Here we investigate the role of brain endothelial megalin in energy metabolism and leptin signaling using an endothelial cell-specific megalin deficient (EMD) mouse model. We found megalin is important to protect mice from developing obesity and metabolic syndrome when mice are fed a normal chow diet. EMD mice developed neuroinflammation, by triggering several pro-inflammatory cytokines, displayed reduced neurogenesis and mitochondrial deregulation. These results implicate brain endothelial megalin expression in obesity-related metabolic changes through the leptin signaling pathway proposing a potential link between obesity and neurodegeneration.

Fatty Acid-binding Protein 4, a Point of Convergence for Angiogenic and Metabolic Signaling Pathways in Endothelial Cells*

PubMed Central

Harjes, Ulrike; Bridges, Esther; McIntyre, Alan; Fielding, Barbara A.; Harris, Adrian L.

2014-01-01

Fatty acid-binding protein 4 (FABP4) is an adipogenic protein and is implicated in atherosclerosis, insulin resistance, and cancer. In endothelial cells, FABP4 is induced by VEGFA, and inhibition of FABP4 blocks most of the VEGFA effects. We investigated the DLL4-NOTCH-dependent regulation of FABP4 in human umbilical vein endothelial cells by gene/protein expression and interaction analyses following inhibitor treatment and RNA interference. We found that FABP4 is directly induced by NOTCH. Stimulation of NOTCH signaling with human recombinant DLL4 led to FABP4 induction, independently of VEGFA. FABP4 induction by VEGFA was reduced by blockade of DLL4 binding to NOTCH or inhibition of NOTCH signal transduction. Chromatin immunoprecipitation of the NOTCH intracellular domain showed increased binding to two specific regions in the FABP4 promoter. The induction of FABP4 gene expression was dependent on the transcription factor FOXO1, which was essential for basal expression of FABP4, and FABP4 up-regulation following stimulation of the VEGFA and/or the NOTCH pathway. Thus, we show that the DLL4-NOTCH pathway mediates endothelial FABP4 expression. This indicates that induction of the angiogenesis-restricting DLL4-NOTCH can have pro-angiogenic effects via this pathway. It also provides a link between DLL4-NOTCH and FOXO1-mediated regulation of endothelial gene transcription, and it shows that DLL4-NOTCH is a nodal point in the integration of pro-angiogenic and metabolic signaling in endothelial cells. This may be crucial for angiogenesis in the tumor environment. PMID:24939870

RhoC and ROCKs regulate cancer cell interactions with endothelial cells.

PubMed

Reymond, Nicolas; Im, Jae Hong; Garg, Ritu; Cox, Susan; Soyer, Magali; Riou, Philippe; Colomba, Audrey; Muschel, Ruth J; Ridley, Anne J

2015-06-01

RhoC is a member of the Rho GTPase family that is implicated in cancer progression by stimulating cancer cell invasiveness. Here we report that RhoC regulates the interaction of cancer cells with vascular endothelial cells (ECs), a crucial step in the metastatic process. RhoC depletion by RNAi reduces PC3 prostate cancer cell adhesion to ECs, intercalation between ECs as well as transendothelial migration in vitro. Depletion of the kinases ROCK1 and ROCK2, two known RhoC downstream effectors, similarly decreases cancer interaction with ECs. RhoC also regulates the extension of protrusions made by cancer cells on vascular ECs in vivo. Transient RhoC depletion is sufficient to reduce both early PC3 cell retention in the lungs and experimental metastasis formation in vivo. Our results indicate RhoC plays a central role in cancer cell interaction with vascular ECs, which is a critical event for cancer progression. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Similar NF-κB Gene Signatures in TNF-α Treated Human Endothelial Cells and Breast Tumor Biopsies

PubMed Central

Perrot-Applanat, Martine; Vacher, Sophie; Toullec, Aurore; Pelaez, Irma; Velasco, Guillaume; Cormier, Françoise; Saad, Hanan El Sheikh; Lidereau, Rosette; Baud, Véronique; Bièche, Ivan

2011-01-01

Background Endothelial dysfunction has been implicated in the pathogenesis of diverse pathologies ranging from vascular and immune diseases to cancer. TNF-α is one of the mediators of endothelial dysfunction through the activation of transcription factors, including NF-κB. While HUVEC (macrovascular cells) have been largely used in the past, here, we documented an NF-κB gene signature in TNFα-stimulated microvascular endothelial cells HMEC often used in tumor angiogenesis studies. Methodology/Principal Findings We measured mRNA expression of 55 NF-κB related genes using quantitative RT-PCR in HUVEC and HMEC. Our study identified twenty genes markedly up-regulated in response to TNFα, including adhesion molecules, cytokines, chemokines, and apoptosis regulators, some of them being identified as TNF-α-inducible genes for the first time in endothelial cells (two apoptosis regulators, TNFAIP3 and TNFRSF10B/Trail R2 (DR5), the chemokines GM-CSF/CSF2 and MCF/CSF1, and CD40 and TNF-α itself, as well as NF-κB components (RELB, NFKB1or 50/p105 and NFKB2 or p52/p100). For eight genes, the fold induction was much higher in HMEC, as compared to HUVEC. Most importantly, our study described for the first time a connection between NF-κB activation and the induction of most, if not all, of these genes in HMEC as evaluated by pharmacological inhibition and RelA expression knock-down by RNA interference. Moreover, since TNF-α is highly expressed in tumors, we further applied the NF-κB gene signature documented in TNFα-stimulated endothelial cells to human breast tumors. We found a significant positive correlation between TNF and the majority (85 %) of the identified endothelial TNF-induced genes in a well-defined series of 96 (48 ERα positive and 48 ERα negative) breast tumors. Conclusion/Significance Taken together these data suggest the potential use of this NF-κB gene signature in analyzing the role of TNF-α in the endothelial dysfunction, as well as in breast tumors independently of the presence of ERα. PMID:21754991

Implications Enzymatic Degradation of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red Cell Free Layer of the Rat Cremaster Muscle.

PubMed

Yalcin, Ozlem; Jani, Vivek P; Johnson, Paul C; Cabrales, Pedro

2018-01-01

The endothelial glycocalyx is a complex network of glycoproteins, proteoglycans, and glycosaminoglycans; it lines the vascular endothelial cells facing the lumen of blood vessels forming the endothelial glycocalyx layer (EGL). This study aims to investigate the microvascular hemodynamics implications of the EGL by quantifying changes in blood flow hydrodynamics post-enzymatic degradation of the glycocalyx layer. High-speed intravital microscopy videos of small arteries (around 35 μm) of the rat cremaster muscle were recorded at various time points after enzymatic degradation of the EGL. The thickness of the cell free layer (CFL), blood flow velocity profiles, and volumetric flow rates were quantified. Hydrodynamic effects of the presence of the EGL were observed in the differences between the thickness of CFL in microvessels with an intact EGL and glass tubes of similar diameters. Maximal changes in the thickness of CFL were observed 40 min post-enzymatic degradation of the EGL. Analysis of the frequency distribution of the thickness of CFL allows for estimation of the thickness of the endothelial surface layer (ESL), the plasma layer, and the glycocalyx. Peak flow, maximum velocity, and mean velocity were found to statistically increase by 24, 27, and 25%, respectively, after enzymatic degradation of the glycocalyx. The change in peak-to-peak maximum velocity and mean velocity were found to statistically increase by 39 and 32%, respectively, after 40 min post-enzymatic degradation of the EGL. The bluntness of blood flow velocity profiles was found to be reduced post-degradation of the EGL, as the exclusion volume occupied by the EGL increased the effective volume impermeable to RBCs in microvessels. This study presents the effects of the EGL on microvascular hemodynamics. Enzymatic degradation of the EGL resulted in a decrease in the thickness of CFL, an increase in blood velocity, blood flow, and decrease of the bluntness of the blood flow velocity profile in small arterioles. In summary, the EGL functions as a molecular sieve to solute transport and as a lubrication layer to protect the endothelium from red blood cell (RBC) motion near the vessel wall, determining wall shear stress.

Genetic Analysis Reveals a Longevity-Associated Protein Modulating Endothelial Function and Angiogenesis.

PubMed

Villa, Francesco; Carrizzo, Albino; Spinelli, Chiara C; Ferrario, Anna; Malovini, Alberto; Maciąg, Anna; Damato, Antonio; Auricchio, Alberto; Spinetti, Gaia; Sangalli, Elena; Dang, Zexu; Madonna, Michele; Ambrosio, Mariateresa; Sitia, Leopoldo; Bigini, Paolo; Calì, Gaetano; Schreiber, Stefan; Perls, Thomas; Fucile, Sergio; Mulas, Francesca; Nebel, Almut; Bellazzi, Riccardo; Madeddu, Paolo; Vecchione, Carmine; Puca, Annibale A

2015-07-31

Long living individuals show delay of aging, which is characterized by the progressive loss of cardiovascular homeostasis, along with reduced endothelial nitric oxide synthase activity, endothelial dysfunction, and impairment of tissue repair after ischemic injury. Exploit genetic analysis of long living individuals to reveal master molecular regulators of physiological aging and new targets for treatment of cardiovascular disease. We show that the polymorphic variant rs2070325 (Ile229Val) in bactericidal/permeability-increasing fold-containing-family-B-member-4 (BPIFB4) associates with exceptional longevity, under a recessive genetic model, in 3 independent populations. Moreover, the expression of BPIFB4 is instrumental to maintenance of cellular and vascular homeostasis through regulation of protein synthesis. BPIFB4 phosphorylation/activation by protein-kinase-R-like endoplasmic reticulum kinase induces its complexing with 14-3-3 and heat shock protein 90, which is facilitated by the longevity-associated variant. In isolated vessels, BPIFB4 is upregulated by mechanical stress, and its knock-down inhibits endothelium-dependent vasorelaxation. In hypertensive rats and old mice, gene transfer of longevity-associated variant-BPIFB4 restores endothelial nitric oxide synthase signaling, rescues endothelial dysfunction, and reduces blood pressure levels. Furthermore, BPIFB4 is implicated in vascular repair. BPIFB4 is abundantly expressed in circulating CD34(+) cells of long living individuals, and its knock-down in endothelial progenitor cells precludes their capacity to migrate toward the chemoattractant SDF-1. In a murine model of peripheral ischemia, systemic gene therapy with longevity-associated variant-BPIFB4 promotes the recruitment of hematopoietic stem cells, reparative vascularization, and reperfusion of the ischemic muscle. Longevity-associated variant-BPIFB4 may represent a novel therapeutic tool to fight endothelial dysfunction and promote vascular reparative processes. © 2015 American Heart Association, Inc.

Cellular tropism of human enterovirus D species serotypes EV-94, EV-70, and EV-68 in vitro: implications for pathogenesis.

PubMed

Smura, Teemu; Ylipaasto, Petri; Klemola, Päivi; Kaijalainen, Svetlana; Kyllönen, Lauri; Sordi, Valeria; Piemonti, Lorenzo; Roivainen, Merja

2010-11-01

Enterovirus 94 (EV-94) is an enterovirus serotype described recently which, together with EV-68 and EV-70, forms human enterovirus D species. This study investigates the seroprevalences of these three serotypes and their abilities to infect, replicate, and damage cell types considered to be essential for enterovirus-induced diseases. The cell types studied included human leukocyte cell lines, primary endothelial cells, and pancreatic islets. High prevalence of neutralizing antibodies against EV-68 and EV-94 was found in the Finnish population. The virus strains studied had wide leukocyte tropism. EV-94 and EV-68 were able to produce infectious progeny in leukocyte cell lines with monocytic, granulocytic, T-cell, or B-cell characteristics. EV-94 and EV-70 were capable of infecting primary human umbilical vein endothelial cells, whereas EV-68 had only marginal progeny production and did not induce cytopathic effects in these cells. Intriguingly, EV-94 was able to damage pancreatic islet β-cells, to infect, replicate, and cause necrosis in human pancreatic islets, and to induce proinflammatory and chemoattractive cytokine expression in endothelial cells. These results suggest that HEV-D viruses may be more prevalent than has been thought previously, and they provide in vitro evidence that EV-94 may be a potent pathogen and should be considered a potentially diabetogenic enterovirus type. © 2010 Wiley-Liss, Inc.

Effect of flow on endothelial endocytosis of nanocarriers targeted to ICAM-1

PubMed Central

Bhowmick, Tridib; Berk, Erik; Cui, Xiumin; Muzykantov, Vladimir R.; Muro, Silvia

2011-01-01

Delivery of drugs into the endothelium by nanocarriers targeted to endothelial determinants may improve treatment of vascular maladies. This is the case for intercellular adhesion molecule 1 (ICAM-1), a glycoprotein overexpressed on endothelial cells (ECs) in many pathologies. ICAM-1-targeted nanocarriers bind to and are internalized by ECs via a non-classical pathway, CAM-mediated endocytosis. In this work we studied the effects of endothelial adaptation to physiological flow on the endocytosis of model polymer nanocarriers targeted to ICAM-1 (anti-ICAM/NCs, ~180-nm diameter). Culturing established endothelial-like cells (EAhy926 cells) and primary human umbilical vein ECs (HUVECs) under 4 dyn/cm2 laminar shear stress for 24 h resulted in flow adaptation: cell elongation and formation of actin stress fibers aligned to the flow direction. Fluorescence microscopy showed that flow-adapted cells internalized anti-ICAM/NCs under flow, although at slower rate versus non flow-adapted cells under static incubation (~35% reduction). Uptake was inhibited by amiloride, whereas marginally affected by filipin and cadaverine, implicating that CAM-endocytosis accounts for anti-ICAM/NC uptake under flow. Internalization under flow was more modestly affected by inhibiting protein kinase C, which regulates actin remodeling during CAM-endocytosis. Actin recruitment to stress fibers that maintain the cell shape under flow may delay uptake of anti-ICAM/NCs under this condition by interfering with actin reorganization needed for CAM-endocytosis. Electron microscopy revealed somewhat slow, yet effective endocytosis of anti-ICAM/NCs by pulmonary endothelium after i.v. injection in mice, similar to that of flow-adapted cell cultures: ~40% (30 min) and 80% (3 h) internalization. Similar to cell culture data, uptake was slightly faster in capillaries with lower shear stress. Further, LPS treatment accelerated internalization of anti-ICAM/NCs in mice. Therefore, regulation of endocytosis of ICAM-1-targeted nanocarriers by flow and endothelial status may modulate drug delivery into ECs exposed to different physiological (capillaries vs. arterioles/venules) or pathological (ischemia, inflammation) levels and patterns of blood flow. PMID:21951807

Effect of flow on endothelial endocytosis of nanocarriers targeted to ICAM-1.

PubMed

Bhowmick, Tridib; Berk, Erik; Cui, Xiumin; Muzykantov, Vladimir R; Muro, Silvia

2012-02-10

Delivery of drugs into the endothelium by nanocarriers targeted to endothelial determinants may improve treatment of vascular maladies. This is the case for intercellular adhesion molecule 1 (ICAM-1), a glycoprotein overexpressed on endothelial cells (ECs) in many pathologies. ICAM-1-targeted nanocarriers bind to and are internalized by ECs via a non-classical pathway, CAM-mediated endocytosis. In this work we studied the effects of endothelial adaptation to physiological flow on the endocytosis of model polymer nanocarriers targeted to ICAM-1 (anti-ICAM/NCs, ~180 nm diameter). Culturing established endothelial-like cells (EAhy926 cells) and primary human umbilical vein ECs (HUVECs) under 4 dyn/cm(2) laminar shear stress for 24 h resulted in flow adaptation: cell elongation and formation of actin stress fibers aligned to the flow direction. Fluorescence microscopy showed that flow-adapted cells internalized anti-ICAM/NCs under flow, although at slower rate versus non flow-adapted cells under static incubation (~35% reduction). Uptake was inhibited by amiloride, whereas marginally affected by filipin and cadaverine, implicating that CAM-endocytosis accounts for anti-ICAM/NC uptake under flow. Internalization under flow was more modestly affected by inhibiting protein kinase C, which regulates actin remodeling during CAM-endocytosis. Actin recruitment to stress fibers that maintain the cell shape under flow may delay uptake of anti-ICAM/NCs under this condition by interfering with actin reorganization needed for CAM-endocytosis. Electron microscopy revealed somewhat slow, yet effective endocytosis of anti-ICAM/NCs by pulmonary endothelium after i.v. injection in mice, similar to that of flow-adapted cell cultures: ~40% (30 min) and 80% (3 h) internalization. Similar to cell culture data, uptake was slightly faster in capillaries with lower shear stress. Further, LPS treatment accelerated internalization of anti-ICAM/NCs in mice. Therefore, regulation of endocytosis of ICAM-1-targeted nanocarriers by flow and endothelial status may modulate drug delivery into ECs exposed to different physiological (capillaries vs. arterioles/venules) or pathological (ischemia, inflammation) levels and patterns of blood flow. Copyright © 2011 Elsevier B.V. All rights reserved.

Unique and shared inflammatory profiles of human brain endothelia and pericytes.

PubMed

Smyth, Leon C D; Rustenhoven, Justin; Park, Thomas I-H; Schweder, Patrick; Jansson, Deidre; Heppner, Peter A; O'Carroll, Simon J; Mee, Edward W; Faull, Richard L M; Curtis, Maurice; Dragunow, Mike

2018-05-11

Pericytes and endothelial cells are critical cellular components of the blood-brain barrier (BBB) and play an important role in neuroinflammation. To date, the majority of inflammation-related studies in endothelia and pericytes have been carried out using immortalised cell lines or non-human-derived cells. Whether these are representative of primary human cells is unclear and systematic comparisons of the inflammatory responses of primary human brain-derived pericytes and endothelia has yet to be performed. To study the effects of neuroinflammation at the BBB, primary brain endothelial cells and pericytes were isolated from human biopsy tissue. Culture purity was examined using qPCR and immunocytochemistry. Electrical cell-substrate impedance sensing (ECIS) was used to determine the barrier properties of endothelial and pericyte cultures. Using immunocytochemistry, cytometric bead array, and ECIS, we compared the responses of endothelia and pericytes to a panel of inflammatory stimuli (IL-1β, TNFα, LPS, IFN-γ, TGF-β 1 , IL-6, and IL-4). Secretome analysis was performed to identify unique secretions of endothelia and pericytes in response to IL-1β. Endothelial cells were pure, moderately proliferative, retained the expression of BBB-related junctional proteins and transporters, and generated robust TEER. Both endothelia and pericytes have the same pattern of transcription factor activation in response to inflammatory stimuli but respond differently at the secretion level. Secretome analysis confirmed that endothelia and pericytes have overlapping but distinct secretome profiles in response to IL-1β. We identified several cell-type specific responses, including G-CSF and GM-CSF (endothelial-specific), and IGFBP2 and IGFBP3 (pericyte-specific). Finally, we demonstrated that direct addition of IL-1β, TNFα, LPS, and IL-4 contributed to the loss of endothelial barrier integrity in vitro. Here, we identify important cell-type differences in the inflammatory response of brain pericytes and endothelia and provide, for the first time, a comprehensive profile of the secretions of primary human brain endothelia and pericytes which has implications for understanding how inflammation affects the cerebrovasculature.

Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis

PubMed Central

Retzlaff, Jennifer; Thamm, Kristina; Ghosh, Chandra C.; Ziegler, Wolfgang; Haller, Hermann; Parikh, Samir M.; David, Sascha

2017-01-01

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to an infection leading to systemic inflammation and endothelial barrier breakdown. The vascular-destabilizing factor Angiopoietin-2 (Angpt-2) has been implicated in these processes in humans. Here we screened in an unbiased approach FDA-approved compounds with respect to Angpt-2 suppression in endothelial cells (ECs) in vitro. We identified Flunarizine – a well-known anti-migraine calcium channel (CC) blocker – being able to diminish intracellular Angpt-2 protein in a time- and dose-dependent fashion thereby indirectly reducing the released protein. Moreover, Flunarizine protected ECs from TNFα-induced increase in Angpt-2 transcription and vascular barrier breakdown. Mechanistically, we could exclude canonical Tie2 signalling being responsible but found that three structurally distinct T-type - but not L-type - CC blockers can suppress Angpt-2. Most importantly, experimental increase in intracellular calcium abolished Flunarizine’s effect. Flunarizine was also able to block the injurious increase of Angpt-2 in murine endotoxemia in vivo. This resulted in reduced pulmonary adhesion molecule expression (intercellular adhesion molecule-1) and tissue infiltration of inflammatory cells (Gr-1). Our finding could have therapeutic implications as side effects of Flunarizine are low and specific sepsis therapeutics that target the dysregulated host response are highly desirable. PMID:28276491

Flunarizine suppresses endothelial Angiopoietin-2 in a calcium - dependent fashion in sepsis.

PubMed

Retzlaff, Jennifer; Thamm, Kristina; Ghosh, Chandra C; Ziegler, Wolfgang; Haller, Hermann; Parikh, Samir M; David, Sascha

2017-03-09

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to an infection leading to systemic inflammation and endothelial barrier breakdown. The vascular-destabilizing factor Angiopoietin-2 (Angpt-2) has been implicated in these processes in humans. Here we screened in an unbiased approach FDA-approved compounds with respect to Angpt-2 suppression in endothelial cells (ECs) in vitro. We identified Flunarizine - a well-known anti-migraine calcium channel (CC) blocker - being able to diminish intracellular Angpt-2 protein in a time- and dose-dependent fashion thereby indirectly reducing the released protein. Moreover, Flunarizine protected ECs from TNFα-induced increase in Angpt-2 transcription and vascular barrier breakdown. Mechanistically, we could exclude canonical Tie2 signalling being responsible but found that three structurally distinct T-type - but not L-type - CC blockers can suppress Angpt-2. Most importantly, experimental increase in intracellular calcium abolished Flunarizine's effect. Flunarizine was also able to block the injurious increase of Angpt-2 in murine endotoxemia in vivo. This resulted in reduced pulmonary adhesion molecule expression (intercellular adhesion molecule-1) and tissue infiltration of inflammatory cells (Gr-1). Our finding could have therapeutic implications as side effects of Flunarizine are low and specific sepsis therapeutics that target the dysregulated host response are highly desirable.

Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS.

PubMed

Zhang, Hong-Hai; Lechuga, Thomas J; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

2016-05-01

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-(12)C6 (14)N4-Arg and L-(12)C6 (14)N2-Lys) or "heavy" (L-(13)C6 (15)N4-Arg and L-(13)C6 (15)N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. © 2016 by the Society for the Study of Reproduction, Inc.

Endothelial-regenerating cells: an expanding universe.

PubMed

Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

2010-03-01

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

Genetic framework for GATA factor function in vascular biology.

PubMed

Linnemann, Amelia K; O'Geen, Henriette; Keles, Sunduz; Farnham, Peggy J; Bresnick, Emery H

2011-08-16

Vascular endothelial dysfunction underlies the genesis and progression of numerous diseases. Although the GATA transcription factor GATA-2 is expressed in endothelial cells and is implicated in coronary heart disease, it has been studied predominantly as a master regulator of hematopoiesis. Because many questions regarding GATA-2 function in the vascular biology realm remain unanswered, we used ChIP sequencing and loss-of-function strategies to define the GATA-2-instigated genetic network in human endothelial cells. In contrast to erythroid cells, GATA-2 occupied a unique target gene ensemble consisting of genes encoding key determinants of endothelial cell identity and inflammation. GATA-2-occupied sites characteristically contained motifs that bind activator protein-1 (AP-1), a pivotal regulator of inflammatory genes. GATA-2 frequently occupied the same chromatin sites as c-JUN and c-FOS, heterodimeric components of AP-1. Although all three components were required for maximal AP-1 target gene expression, GATA-2 was not required for AP-1 chromatin occupancy. GATA-2 conferred maximal phosphorylation of chromatin-bound c-JUN at Ser-73, which stimulates AP-1-dependent transactivation, in a chromosomal context-dependent manner. This work establishes a link between a GATA factor and inflammatory genes, mechanistic insights underlying GATA-2-AP-1 cooperativity and a rigorous genetic framework for understanding GATA-2 function in normal and pathophysiological vascular states.

Endogenous developmental endothelial locus-1 limits ischemia-related angiogenesis by blocking inflammation

PubMed Central

Klotzsche - von Ameln, Anne; Cremer, Sebastian; Hoffmann, Jedrzej; Schuster, Peggy; Khedr, Sherif; Korovina, Irina; Troulinaki, Maria; Neuwirth, Ales; Sprott, David; Chatzigeorgiou, Antonios; Economopoulou, Matina; Orlandi, Alessia; Hain, Andreas; Zeiher, Andreas M.; Deussen, Andreas; Hajishengallis, George; Dimmeler, Stefanie; Chavakis, Triantafyllos; Chavakis, Emmanouil

2017-01-01

We have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of β2-integrin–dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischemia-related angiogenesis. Intriguingly, Del-1–deficient mice displayed increased neovascularization in two independent ischemic models (retinopathy of prematurity and hind-limb ischemia), as compared to Del-1–proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischemic neovascularization in Del-1-deficiency was linked to higher infiltration of the ischemic tissue by CD45+ hematopoietic and immune cells. Moreover, Del-1-deficiency promoted β2-integrin–dependent adhesion of hematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischemic muscles in vivo. Consistently, the increased hind limb ischemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the β2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularization in Del-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognized function of endogenous Del-1 as a local inhibitor of ischemia-induced angiogenesis by restraining LFA-1–dependent homing of pro-angiogenic hematopoietic cells to ischemic tissues. Our findings are relevant for the optimization of therapeutic approaches in the context of ischemic diseases. PMID:28447099

Zinc regulates iNOS-derived nitric oxide formation in endothelial cells.

PubMed

Cortese-Krott, Miriam M; Kulakov, Larissa; Opländer, Christian; Kolb-Bachofen, Victoria; Kröncke, Klaus-D; Suschek, Christoph V

2014-01-01

Aberrant production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of endothelial dysfunction and vascular disease. Mechanisms responsible for the fine-tuning of iNOS activity in inflammation are still not fully understood. Zinc is an important structural element of NOS enzymes and is known to inhibit its catalytical activity. In this study we aimed to investigate the effects of zinc on iNOS activity and expression in endothelial cells. We found that zinc down-regulated the expression of iNOS (mRNA+protein) and decreased cytokine-mediated activation of the iNOS promoter. Zinc-mediated regulation of iNOS expression was due to inhibition of NF-κB transactivation activity, as determined by a decrease in both NF-κB-driven luciferase reporter activity and expression of NF-κB target genes, including cyclooxygenase 2 and IL-1β. However, zinc did not affect NF-κB translocation into the nucleus, as assessed by Western blot analysis of nuclear and cytoplasmic fractions. Taken together our results demonstrate that zinc limits iNOS-derived high output NO production in endothelial cells by inhibiting NF-κB-dependent iNOS expression, pointing to a role of zinc as a regulator of iNOS activity in inflammation.

Zinc regulates iNOS-derived nitric oxide formation in endothelial cells

PubMed Central

Cortese-Krott, Miriam M.; Kulakov, Larissa; Opländer, Christian; Kolb-Bachofen, Victoria; Kröncke, Klaus-D.; Suschek, Christoph V.

2014-01-01

Aberrant production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of endothelial dysfunction and vascular disease. Mechanisms responsible for the fine-tuning of iNOS activity in inflammation are still not fully understood. Zinc is an important structural element of NOS enzymes and is known to inhibit its catalytical activity. In this study we aimed to investigate the effects of zinc on iNOS activity and expression in endothelial cells. We found that zinc down-regulated the expression of iNOS (mRNA+protein) and decreased cytokine-mediated activation of the iNOS promoter. Zinc-mediated regulation of iNOS expression was due to inhibition of NF-κB transactivation activity, as determined by a decrease in both NF-κB-driven luciferase reporter activity and expression of NF-κB target genes, including cyclooxygenase 2 and IL-1β. However, zinc did not affect NF-κB translocation into the nucleus, as assessed by Western blot analysis of nuclear and cytoplasmic fractions. Taken together our results demonstrate that zinc limits iNOS-derived high output NO production in endothelial cells by inhibiting NF-κB-dependent iNOS expression, pointing to a role of zinc as a regulator of iNOS activity in inflammation. PMID:25180171

Endocardial Hippo signaling regulates myocardial growth and cardiogenesis.

PubMed

Artap, Stanley; Manderfield, Lauren J; Smith, Cheryl L; Poleshko, Andrey; Aghajanian, Haig; See, Kelvin; Li, Li; Jain, Rajan; Epstein, Jonathan A

2018-08-01

The Hippo signaling pathway has been implicated in control of cell and organ size, proliferation, and endothelial-mesenchymal transformation. This pathway impacts upon two partially redundant transcription cofactors, Yap and Taz, that interact with other factors, including members of the Tead family, to affect expression of downstream genes. Yap and Taz have been shown to regulate, in a cell-autonomous manner, myocardial proliferation, myocardial hypertrophy, regenerative potential, and overall size of the heart. Here, we show that Yap and Taz also play an instructive, non-cell-autonomous role in the endocardium of the developing heart to regulate myocardial growth through release of the paracrine factor, neuregulin. Without endocardial Yap and Taz, myocardial growth is impaired causing early post-natal lethality. Thus, the Hippo signaling pathway regulates cell size via both cell-autonomous and non-cell-autonomous mechanisms. Furthermore, these data suggest that Hippo may regulate organ size via a sensing and paracrine function in endothelial cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Anti-angiogenic and vascular disrupting effects of C9, a new microtubule-depolymerizing agent

PubMed Central

Ren, Xuan; Dai, Mei; Lin, Li-Ping; Li, Pui-Kai; Ding, Jian

2009-01-01

Background and purpose: The critical role of blood supply in the growth of solid tumours makes blood vessels an ideal target for anti-tumour drug discovery. The anti-angiogenic and vascular disrupting activities of C9, a newly synthesized microtubule-depolymerizing agent, were investigated with several in vitro and in vivo models. Possible mechanisms involved in its activity were also assessed. Experimental approach: Microtubule-depolymerizing actions were assessed by surface plasmon resonance binding, competitive inhibition and cytoskeleton immunofluorescence. Anti-angiogenic and vascular disrupting activities were tested on proliferation, migration, tube formation with human umbilical vein endothelial cells, and in rat aortic ring, chick chorioallantoic membrane and Matrigel plug assays. Western blots and Rho activation assays were employed to examine the role of Raf-MEK-ERK (mitogen-activated ERK kinase, extracellular signal-regulated kinase) and Rho/Rho kinase signalling. Key results: C9 inhibited proliferation, migration and tube formation of endothelial cells and inhibited angiogenesis in aortic ring and chick chorioallantoic membrane assays. C9 induced disassembly of microtubules in endothelial cells and down-regulated Raf-MEK-ERK signalling activated by pro-angiogenic factors. In addition, C9 disrupted capillary-like networks and newly formed vessels in vitro and rapidly decreased perfusion of neovasculature in vivo. Endothelial cell contraction and membrane blebbing induced by C9 in neovasculature was dependent on the Rho/Rho kinase pathway. Conclusions and implications: Anti-angiogenic and vascular disruption by C9 was associated with changes in morphology and function of endothelial cells, involving the Raf-MEK-ERK and Rho/Rho kinase signalling pathways. These findings strongly suggest that C9 is a new microtubule-binding agent that could effectively target tumour vasculature. PMID:19302593

Abacavir induces platelet-endothelium interactions by interfering with purinergic signalling: A step from inflammation to thrombosis.

PubMed

Alvarez, Angeles; Rios-Navarro, Cesar; Blanch-Ruiz, Maria Amparo; Collado-Diaz, Victor; Andujar, Isabel; Martinez-Cuesta, Maria Angeles; Orden, Samuel; Esplugues, Juan V

2017-05-01

The controversy connecting Abacavir (ABC) with cardiovascular disease has been fuelled by the lack of a credible mechanism of action. ABC shares structural similarities with endogenous purines, signalling molecules capable of triggering prothrombotic/proinflammatory programmes. Platelets are leading actors in the process of thrombosis. Our study addresses the effects of ABC on interactions between platelets and other vascular cells, while exploring the adhesion molecules implicated and the potential interference with the purinergic signalling pathway. The effects of ABC on platelet aggregation and platelet-endothelium interactions were evaluated, respectively, with an aggregometer and a flow chamber system that reproduced conditions in vivo. The role of adhesion molecules and purinergic receptors in endothelial and platelet populations was assessed by selective pre-incubation with specific antagonists and antibodies. ABC and carbovir triphosphate (CBT) levels were evaluated by HPLC. The results showed that ABC promoted the adherence of platelets to endothelial cells, a crucial step for the formation of thrombi. This was not a consequence of a direct effect of ABC on platelets, but resulted from activation of the endothelium via purinergic ATP-P2X 7 receptors, which subsequently triggered an interplay between P-selectin and ICAM-1 on endothelial cells with constitutively expressed GPIIb/IIIa and GPIbα on platelets. ABC did not induce platelet activation (P-selectin expression or Ca 2+ mobilization) or aggregation, even at high concentrations. CBT levels in endothelial cells were lower than those required to induce platelet-endothelium interactions. Thus, ABC interference with endothelial purinergic signalling leads to platelet recruitment. This highlights the endothelium as the main cell target of ABC in this interaction, which is in line with previous experimental evidence that ABC induces manifestations of vascular inflammation. Copyright © 2017 Elsevier B.V. All rights reserved.

Importance of mitochondrial calcium uniporter in high glucose-induced endothelial cell dysfunction.

PubMed

Chen, Wei; Yang, Jie; Chen, Shuhua; Xiang, Hong; Liu, Hengdao; Lin, Dan; Zhao, Shaoli; Peng, Hui; Chen, Pan; Chen, Alex F; Lu, Hongwei

2017-11-01

Mitochondrial Ca 2+ overload is implicated in hyperglycaemia-induced endothelial cell dysfunction, but the key molecular events responsible remain unclear. We examined the involvement of mitochondrial calcium uniporter, which mediates mitochondrial Ca 2+ uptake, in endothelial cell dysfunction resulting from high-glucose treatment. Human umbilical vein endothelial cells were exposed to various glucose concentrations and to high glucose (30 mM) following mitochondrial calcium uniporter inhibition or activation with ruthenium red and spermine, respectively. Subsequently, mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA and protein expression was measured by real-time polymerase chain reaction and western blotting. Ca 2+ concentrations were analysed by laser confocal microscopy, and cytoplasmic and mitochondrial oxidative stress was detected using 2',7'-dichlorofluorescein diacetate and MitoSOX Red, respectively. Apoptosis was assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and a wound-healing assay was performed using an in vitro model. High glucose markedly upregulated mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA expression, as well as protein production, in a dose- and time-dependent manner with a maximum effect demonstrated at 72 h and 30 mM glucose concentration. Moreover, high-glucose treatment significantly raised both mitochondrial and cytoplasmic Ca 2+ and reactive oxygen species levels, increased apoptosis and compromised wound healing (all p < 0.05). These effects were enhanced by spermine and completely negated by ruthenium red, which are known to activate and inhibit mitochondrial calcium uniporter, respectively. Mitochondrial calcium uniporter plays an important role in hyperglycaemia-induced endothelial cell dysfunction and may constitute a therapeutic target to reduce vascular complications in diabetes.

Initial investigation of SU-8 photopolymer as a material for noninvasive endothelial cell research platforms

NASA Astrophysics Data System (ADS)

Westwood, S.; Gojova, A.; Kuo, B.; Barakat, A. I.; Gray, B. L.

2007-01-01

This paper presents a preliminary investigation in the usage of the micromachining polymer material SU-8 for the noninvasive shape control and functional study of vascular endothelial cells (ECs). We previously demonstrated a silicon and glass modular microinstrument platform that allowed for a wide range of EC functional response studies. However, we expect SU-8 to provide a more versatile fabrication technology and material for microchannel fabrication and instrumentation, since it is capable of achieving high aspect ratio sensor-compatible structures through simple photopatterning. In this paper, SU-8 microchannels were fabricated on glass slides for straightforward optical observation and biological sampling. Channel widths ranged from 50 to 210 µm, length varied from 100 to 2100 µm, with depth fixed at 100 µm. We plated bovine aortic endothelial cells (BAECs) in the microchannels and used image analysis to determine cellular elongation and orientation. Similar to silicon-on-glass microchannels, the cells become more elongated and oriented along the microchannel axis as the width of the microchannel decreases. Initial results indicate cells plate in the microchannels and on the SU-8 surfaces, whereas in a previous silicon microchannel study, cells plated exclusively on the glass bottom surfaces. This finding has implications for SU-8 as a structural material for microchannel instrumentation.

Activation of c-Jun N-terminal kinase and apoptosis in endothelial cells mediated by endogenous generation of hydrogen peroxide

NASA Technical Reports Server (NTRS)

Ramachandran, Anup; Moellering, Douglas; Go, Young-Mi; Shiva, Sruti; Levonen, Anna-Liisa; Jo, Hanjoong; Patel, Rakesh P.; Parthasarathy, Sampath; Darley-Usmar, Victor M.

2002-01-01

Reactive oxygen species have been implicated in the activation of signal transduction pathways. However, extracellular addition of oxidants such as hydrogen peroxide (H2O2) often requires concentrations that cannot be readily achieved under physiological conditions to activate biological responses such as apoptosis. Explanations for this discrepancy have included increased metabolism of H2O2 in the extracellular environment and compartmentalization within the cell. We have addressed this issue experimentally by examining the induction of apoptosis of endothelial cells induced by exogenous addition of H2O2 and by a redox cycling agent, 2,3-dimethoxy-1,4-naphthoquinone, that generates H2O2 in cells. Here we show that low nanomolar steady-state concentrations (0.1-0.5 nmol x min(-1) x 10(6) cells) of H2O2 generated intracellularly activate c-Jun N terminal kinase and initiate apoptosis in endothelial cells. A comparison with bolus hydrogen peroxide suggests that the low rate of intracellular formation of this reactive oxygen species results in a similar profile of activation for both c-Jun N terminal kinase and the initiation of apoptosis. However, a detailed analysis reveals important differences in both the duration and profile for activation of these signaling pathways.

Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

PubMed Central

Roth Flach, Rachel J.; Skoura, Athanasia; Matevossian, Anouch; Danai, Laura V.; Zheng, Wei; Cortes, Christian; Bhattacharya, Samit K.; Aouadi, Myriam; Hagan, Nana; Yawe, Joseph C.; Vangala, Pranitha; Menendez, Lorena Garcia; Cooper, Marcus P.; Fitzgibbons, Timothy P.; Buckbinder, Leonard; Czech, Michael P.

2015-01-01

Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe−/− mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe−/− and Ldlr−/− mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFκB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis. PMID:26688060

Nitrosonifedipine Ameliorates the Progression of Type 2 Diabetic Nephropathy by Exerting Antioxidative Effects

PubMed Central

Ishizawa, Keisuke; Izawa-Ishizawa, Yuki; Yamano, Noriko; Urushihara, Maki; Sakurada, Takumi; Imanishi, Masaki; Fujii, Shoko; Nuno, Asami; Miyamoto, Licht; Kihira, Yoshitaka; Ikeda, Yasumasa; Kagami, Shoji; Kobori, Hiroyuki; Tsuchiya, Koichiro; Tamaki, Toshiaki

2014-01-01

Diabetic nephropathy (DN) is the major cause of end-stage renal failure. Oxidative stress is implicated in the pathogenesis of DN. Nitrosonifedipine (NO-NIF) is a weak calcium channel blocker that is converted from nifedipine under light exposure. Recently, we reported that NO-NIF has potential as a novel antioxidant with radical scavenging abilities and has the capacity to treat vascular dysfunction by exerting an endothelial protective effect. In the present study, we extended these findings by evaluating the efficacy of NO-NIF against DN and by clarifying the mechanisms of its antioxidative effect. In a model of type 2 DN (established in KKAy mice), NO-NIF administration reduced albuminuria and proteinuria as well as glomerular expansion without affecting glucose metabolism or systolic blood pressure. NO-NIF also suppressed renal and systemic oxidative stress and decreased the expression of intercellular adhesion molecule (ICAM)-1, a marker of endothelial cell injury, in the glomeruli of the KKAy mice. Similarly, NO-NIF reduced albuminuria, oxidative stress, and ICAM-1 expression in endothelial nitric oxide synthase (eNOS) knockout mice. Moreover, NO-NIF suppressed urinary angiotensinogen (AGT) excretion and intrarenal AGT protein expression in proximal tubular cells in the KKAy mice. On the other hand, hyperglycemia-induced mitochondrial superoxide production was not attenuated by NO-NIF in cultured endothelial cells. These findings suggest that NO-NIF prevents the progression of type 2 DN associated with endothelial dysfunction through selective antioxidative effects. PMID:24489716

Bidirectional Signaling of Mammary Epithelium and Stroma: Implications for Breast Cancer—Preventive Actions of Dietary Factors

USDA-ARS?s Scientific Manuscript database

The mammary gland is composed of two major cellular compartments: a highly dynamic epithelium that undergoes cycles of proliferation, differentiation, and apoptosis in response to local and endocrine signals and the underlying stroma comprised of fibroblasts, endothelial cells, and adipocytes that c...

Substrate stiffness-dependent exacerbation of endothelial permeability and inflammation: mechanisms and potential implications in ALI and PH (2017 Grover Conference Series)

PubMed Central

Karki, Pratap; Birukova, Anna A.

2018-01-01

The maintenance of endothelial barrier integrity is absolutely essential to prevent the vascular leak associated with pneumonia, pulmonary edema resulting from inhalation of toxins, acute elevation to high altitude, traumatic and septic lung injury, acute lung injury (ALI), and its life-threatening complication, acute respiratory distress syndrome (ARDS). In addition to the long-known edemagenic and inflammatory agonists, emerging evidences suggest that factors of endothelial cell (EC) mechanical microenvironment such as blood flow, mechanical strain of the vessel, or extracellular matrix stiffness also play an essential role in the control of endothelial permeability and inflammation. Recent studies from our group and others have demonstrated that substrate stiffening causes endothelial barrier disruption and renders EC more susceptible to agonist-induced cytoskeletal rearrangement and inflammation. Further in vivo studies have provided direct evidence that proinflammatory stimuli increase lung microvascular stiffness which in turn exacerbates endothelial permeability and inflammation and perpetuates a vicious circle of lung inflammation. Accumulating evidence suggests a key role for RhoA GTPases signaling in stiffness-dependent mechanotransduction mechanisms defining EC permeability and inflammatory responses. Vascular stiffening is also known to be a key contributor to other cardiovascular diseases such as arterial pulmonary hypertension (PH), although the precise role of stiffness in the development and progression of PH remains to be elucidated. This review summarizes the current understanding of stiffness-dependent regulation of pulmonary EC permeability and inflammation, and discusses potential implication of pulmonary vascular stiffness alterations at macro- and microscale in development and modulation of ALI and PH. PMID:29714090

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. These results indicate that following TBI, the cerebral endothelium undergoes vascular remodeling through shedding of eMVs containing TJPs and endothelial markers. The detection of this shedding potentially allows for a novel methodology for real-time monitoring of cerebral vascular health (remodeling), BBB status and neuroinflammation following a TBI event.

Reduced Ang2 expression in aging endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at; Ebenbauer, B.; Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna

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

Cell Permeability, Migration, and Reactive Oxygen Species Induced by Multi-Walled Carbon Nanotubes in Human Microvascular Endothelial Cells

PubMed Central

Pacurari, M; Qian, Y; Fu, W; Schwegler-Berry, D; Ding, M; Castranova, V; Guo, NL

2011-01-01

Multi-walled carbon nanotubes (MWCNT) have elicited great interest in biomedical applications due to their extraordinary physical, chemical, and optical properties. Intravenous administration of MWCNT-based medical imaging agents and drugs in animal models was utilized. However, the potential harmful health effects of MWCNT administration in humans have not yet been elucidated. Furthermore, to date, there are no apparent reports regarding the precise mechanisms of translocation of MWCNT into target tissues and organs from blood circulation. This study demonstrates that exposure to MWCNT leads to an increase in cell permeability in human microvascular endothelial cells (HMVEC). The results obtained from this study also showed that the MWCNT-induced rise in endothelial permeability is mediated by reactive oxygen species (ROS) production and actin filament remodeling. In addition, it was found that MWCNT promoted cell migration in HMVEC. Mechanistically, MWCNT exposure elevated the levels of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) in HMVEC. Taken together, these results provide new insights into the bioreactivity of MWCNT, which may have implications in the biomedical application of MWCNT in vascular targeting, imaging, and drug delivery. The results generated from this study also elucidate the potential adverse effects of MWCNT exposure on humans at the cellular level. PMID:22129238

Scleroderma Related Lung Disease: Is There a Pathogenic Role for Adipokines?

PubMed Central

Haley, Shannon; Shah, Dilip; Romero, Freddy; Summer, Ross

2013-01-01

Scleroderma is a systemic autoimmune disease of unknown etiology whose hallmark features include endothelial cell dysfunction, fibroblast proliferation and immune dysregulation. Although virtually any organ can be pathologically involved in scleroderma, lung complications including interstitial lung disease (ILD) and pulmonary arterial hypertension (PAH) are the leading cause of death in patients with this condition. Currently, the molecular mechanisms leading to development of scleroderma-related lung disease are poorly understood; however, the systemic nature of this condition has led many to implicate circulating factors in the pathogenesis of some of its organ impairment. In this article, we focus on a new class of circulating factors derived from adipose-tissue called adipokines, which are known to be altered in scleroderma. Recently, the adipokines adiponectin and leptin have been found to regulate biological activities in endothelial, fibroblast and immune cell types in lung and in many other tissues. The pleiotropic nature of these circulating factors and their functional activity on many cell types implicated in the pathogenesis of ILD and PAH suggest these hormones may play a mechanistic role in the onset and/or progression of scleroderma-related lung diseases. PMID:24173692

Essential roles of angiotensin II in vascular endothelial growth factor expression in sleep apnea syndrome.

PubMed

Takahashi, Susumu; Nakamura, Yutaka; Nishijima, Tsuguo; Sakurai, Shigeru; Inoue, Hiroshi

2005-09-01

Hypoxia-induced endothelial cell dysfunction has been implicated in increased cardiovascular disease associated with obstructive sleep apnea syndrome (OSAS). OSAS mediates hypertension by stimulating angiotensin II (Ang II) production. Hypoxia and Ang II are the major stimuli of vascular endothelial growth factor (VEGF), which is a potent angiogenic cytokine and also contributes to the atherogenic process itself. We observed serum Ang II and VEGF levels and peripheral blood mononuclear cell (PBMC) and neutrophil VEGF expression. Compared to controls, subjects with OSAS had significantly increased levels of serum Ang II and VEGF and VEGF mRNA expression in their leukocytes. To examine whether Ang II stimulates VEGF expression in OSAS, we treated PBMCs obtained from control subjects with Ang II and with an Ang II receptor type 1 (AT(1)) blocker, olmesartan. We observed an increased expression of VEGF in the Ang II-stimulated PBMCs and decreased in VEGF mRNA and protein expression in the PBMCs treated with olmesartan. These findings suggest that the Ang II-AT(1) receptors pathway potentially are involved in OSAS and VEGF-induced vascularity and that endothelial dysfunction might be linked to this change in Ang II activity within leukocytes of OSAS patients.

Adipokines, Biomarkers of Endothelial Activation, and Metabolic Syndrome in Patients with Ankylosing Spondylitis

PubMed Central

López-Mejías, Raquel; Miranda-Filloy, José A.; Ubilla, Begoña; Carnero-López, Beatriz; Blanco, Ricardo; Pina, Trinitario; González-Juanatey, Carlos; Llorca, Javier; González-Gay, Miguel A.

2014-01-01

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease associated with accelerated atherosclerosis and increased risk of cardiovascular (CV) disease. AS patients also display a high prevalence of features clustered under the name of metabolic syndrome (MeS). Anti-TNF-α therapy was found to be effective to treat AS patients by suppressing inflammation and also improving endothelial function. Previously, it was demonstrated that a short infusion of anti-TNF-α monoclonal antibodyinfliximab induced a rapid and dramatic reduction in serum insulin levels and insulin resistance along with a rapid improvement of insulin sensitivity in nondiabetic AS patients. The role of adipokines, MeS-related biomarkers and biomarkers of endothelial cell activation and inflammation seem to be relevant in different chronic inflammatory diseases. However, its implication in AS has not been fully established. Therefore, in this review we summarize the recent advances in the study of the involvement of these molecules in CV disease or MeS in AS. The assessment of adipokines and biomarkers of endothelial cell activation and MeS may be of potential relevance in the stratification of the CV risk of patients with AS. PMID:24757680

Endothelial cell repopulation after stenting determines in-stent neointima formation: effects of bare-metal vs. drug-eluting stents and genetic endothelial cell modification.

PubMed

Douglas, Gillian; Van Kampen, Erik; Hale, Ashley B; McNeill, Eileen; Patel, Jyoti; Crabtree, Mark J; Ali, Ziad; Hoerr, Robert A; Alp, Nicholas J; Channon, Keith M

2013-11-01

Understanding endothelial cell repopulation post-stenting and how this modulates in-stent restenosis is critical to improving arterial healing post-stenting. We used a novel murine stent model to investigate endothelial cell repopulation post-stenting, comparing the response of drug-eluting stents with a primary genetic modification to improve endothelial cell function. Endothelial cell repopulation was assessed en face in stented arteries in ApoE(-/-) mice with endothelial-specific LacZ expression. Stent deployment resulted in near-complete denudation of endothelium, but was followed by endothelial cell repopulation, by cells originating from both bone marrow-derived endothelial progenitor cells and from the adjacent vasculature. Paclitaxel-eluting stents reduced neointima formation (0.423 ± 0.065 vs. 0.240 ± 0.040 mm(2), P = 0.038), but decreased endothelial cell repopulation (238 ± 17 vs. 154 ± 22 nuclei/mm(2), P = 0.018), despite complete strut coverage. To test the effects of selectively improving endothelial cell function, we used transgenic mice with endothelial-specific overexpression of GTP-cyclohydrolase 1 (GCH-Tg) as a model of enhanced endothelial cell function and increased NO production. GCH-Tg ApoE(-/-) mice had less neointima formation compared with ApoE(-/-) littermates (0.52 ± 0.08 vs. 0.26 ± 0.09 mm(2), P = 0.039). In contrast to paclitaxel-eluting stents, reduced neointima formation in GCH-Tg mice was accompanied by increased endothelial cell coverage (156 ± 17 vs. 209 ± 23 nuclei/mm(2), P = 0.043). Drug-eluting stents reduce not only neointima formation but also endothelial cell repopulation, independent of strut coverage. In contrast, selective targeting of endothelial cell function is sufficient to improve endothelial cell repopulation and reduce neointima formation. Targeting endothelial cell function is a rational therapeutic strategy to improve vascular healing and decrease neointima formation after stenting.

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

PubMed

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

2017-09-15

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

Substrate effects on endothelial cell adherence rates.

PubMed

Scott, W J; Mann, P

1990-01-01

Endothelial cell attachment to a synthetic substrate was studied using an in vitro model system. Attachment rate was defined as the number of tritium-labeled endothelial cells attached to a synthetic substrate after 30 minutes. The surface of the synthetic substrate was chemically modified with either laminin or fibronectin. Labeled endothelial cells attached more rapidly to synthetic substrate, chemically modified with biomolecules, as compared with the untreated substrate controls. Unlabeled endothelial cells were grown to confluency on a second set of modified and untreated substrates. The cells were removed with 1% Triton, and the rate of re-endothelialization with tritium-labeled endothelial cells was determined. The rate was 11-13 times that of the same cells on untreated substrate. These data confirm that biomolecules increase the attachment rate of endothelial cells to synthetic substrate, and also suggest that endothelial cells may secrete a Triton-insoluble product (Sigma, St. Louis, MO) into subendothelial matrix that increases re-endothelialization.

An evaluation of image quality and accuracy of eye bank measurement of donor cornea endothelial cell density in the Specular Microscopy Ancillary Study.

PubMed

Lass, Jonathan H; Gal, Robin L; Ruedy, Katrina J; Benetz, Beth Ann; Beck, Roy W; Baratz, Keith H; Holland, Edward J; Kalajian, Andrea; Kollman, Craig; Manning, Francis J; Mannis, Mark J; McCoy, Kristen; Montoya, Monty; Stulting, Doyle; Xing, Dongyuan

2005-03-01

The Specular Microscopy Ancillary Study was designed to examine donor corneal endothelial specular image quality, compare the central endothelial cell density determined by eye banks with the endothelial cell density determined by a central specular microscopy reading center, and evaluate donor factors that may have an impact on specular image quality and endothelial cell density accuracy. Nonrandomized comparative trial. Endothelial specular images of donor corneas assigned in the Cornea Donor Study. Certified readers assessed donor image quality (analyzable from fair to excellent vs. unanalyzable) and determined the central endothelial cell density. Independent adjudication was performed if there was a difference in the quality of grading or if the endothelial cell density varied by > or =5.0% between readers. Average reading center-determined endothelial cell density was compared with the endothelial cell density determined by each eye bank. Evaluation of image quality and accuracy of endothelial cell density. Of 688 donor endothelial images submitted by 23 eye banks, 663 (96%) were analyzable (excellent, 40 [6%]; good, 302 [44%]; fair, 321 [47%]), and 25 (4%) were unanalyzable by reading center standards. In situ retrieval and greater epithelial exposure correlated with a higher image quality grading. The eye bank-determined endothelial cell density of 434 of the 663 (65%) analyzable images were within 10% of the endothelial cell density determined by the reading center, whereas 185 (28%) were more than 10% higher and 44 (7%) were more than 10% lower. Greater variation in endothelial cell density between the eye banks and the reading center was observed with shorter time of death to preservation, presence of an epithelial defect, folds in Descemet's membrane, lower image quality, and the use of fixed-frame or center method endothelial cell density analysis. Overall, donor endothelial specular image quality and accuracy of endothelial cell density determination were good. However, the data suggest that factors that may affect image quality and contribute to variation in interpretation of the endothelial cell density should be addressed, because the donor endothelial cell density is an important parameter for assessing long-term corneal graft survival.

C-reactive protein induces matrix metalloproteinase-1 and -10 in human endothelial cells: implications for clinical and subclinical atherosclerosis.

PubMed

Montero, Ines; Orbe, Josune; Varo, Nerea; Beloqui, Oscar; Monreal, José I; Rodríguez, José A; Díez, Javier; Libby, Peter; Páramo, José A

2006-04-04

We examined the effect of C-reactive protein (CRP) on matrix metalloproteinase (MMP) and inhibitor expression in endothelial cells and in patients with clinical and subclinical atherosclerosis. In addition to predicting atherosclerotic vascular disease, CRP may directly promote a proinflammatory/proatherosclerotic phenotype. Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were incubated in the presence or absence of CRP (50 mug/ml). Microarray analysis, real-time polymerase chain reaction, immunological and activity assays for MMPs were performed. Specific inhibitors of mitogen-activated protein kinase pathway were used. The MMP-1 and -10 plasma levels were measured in apparently healthy subjects (n = 70). Immunolocalization of CRP, MMP-1, and MMP-10 was performed in human mammary arteries and carotid endarterectomy specimens. C-reactive protein augmented MMP-1 and -10 messenger ribonucleic acid expression in HUVEC (p < 0.05) and HAEC (p < 0.01). C-reactive protein stimulation also increased MMP-1 and -10 protein in conditioned culture medium (p < 0.001), as well as MMP activity (p = 0.001). Specific inhibition of p38 or MEK abolished the CRP induction of the MMP-1, whereas MMP-10 induction blockade required the simultaneous inhibition of p38 and Jun N-terminal kinase pathways. Subjects with CRP values >3 mg/l (n = 37) had increased plasma MMP-1 and -10 (p < 0.05), the association being significant after adjustment for confounding variables (p = 0.04 and p = 0.008, respectively). The MMP-10 levels were elevated in subjects with higher carotid intima-media thickness (p = 0.009). Increased CRP and MMP-10 colocalized in endothelial layer and macrophage-rich areas in advanced atherosclerotic plaques. Increased local and systemic CRP-related MMP activation might provide a link between inflammation and plaque vulnerability.

Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eum, Sung Yong, E-mail: seum@miami.edu; Jaraki, Dima; András, Ibolya E.

Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2)more » after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft-associated PP2A/MMP-2 induced PCB153-induced dysfunction of occludin. • Disrupted lipid rafts modulated PCB153-induced increase of permeability. • Lipid rafts act as a signaling platform for PCB153-induced dysfunction of occludin.« less

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 pathophysiology associated with diabetic microangiopathy.

A selective antagonist reveals a potential role of G protein-coupled receptor 55 in platelet and endothelial cell function.

PubMed

Kargl, Julia; Brown, Andrew J; Andersen, Liisa; Dorn, Georg; Schicho, Rudolf; Waldhoer, Maria; Heinemann, Akos

2013-07-01

The G protein-coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiologic functions, its role remains enigmatic owing mainly to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca²⁺ release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB₁ or CB₂). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.

Endothelial Targeting of Semi-permeable Polymer Nanocarriers for Enzyme Therapies

PubMed Central

Dziubla, Thomas D; Shuvaev, Vladimir V.; Hong, Nan Kang; Hawkins, Brian; Muniswamy, Madesh; Takano, Hajime; Simone, Eric; Nakada, Marian T.; Fisher, Aron; Albelda, Steven M.; Muzykantov, Vladimir R.

2007-01-01

The medical utility of proteins, e.g. therapeutic enzymes, is greatly restricted by their liable nature and inadequate delivery. Most therapeutic enzymes do not accumulate in their targets and are inactivated by proteases. Targeting of enzymes encapsulated into substrate-permeable Polymeric Nano-Carriers (PNC) impermeable for proteases might overcome these limitations. To test this hypothesis, we designed endothelial targeted PNC loaded with catalase, the H2O2-detoxifying enzyme, and tested if this approach protects against vascular oxidative stress, a pathological process implicated in ischemia-reperfusion and other disease conditions. Encapsulation of catalase (MW 240KD), peroxidase (MW 42kD) and xanthine oxidase (XO, MW 300 kD) into ~300nm diameter PNC composed of co-polymers of PEG-PLGA (polyethylene glycol and poly-lactic/poly-glycolic acid) was in the range ~10% for all enzymes. PNC/catalase and PNC/peroxidase were protected from external proteolysis and exerted the enzymatic activity on their PNC diffusible substrates, H2O2 and ortho-phenylendiamine, whereas activity of encapsulated XO was negligible due to polymer impermeability to the substrate. PNC targeted to platelet-endothelial cell adhesion molecule-1 delivered active encapsulated catalase to endothelial cells and protected the endothelium against oxidative stress in cell culture and animal studies. Vascular targeting of PNC-loaded detoxifying enzymes may find wide medical applications including management of oxidative stress and other toxicities. PMID:17950837

G-protein-coupled receptor kinase 2 and endothelial dysfunction: molecular insights and pathophysiological mechanisms

PubMed Central

Taguchi, Kumiko; Matsumoto, Takayuki; Kobayashi, Tsuneo

2015-01-01

Smooth muscle cells (SMC) and endothelial cells are the major cell types in blood vessels. The principal function of vascular SMC in the body is to regulate blood flow and pressure through contraction and relaxation. The endothelium performs a crucial role in maintaining vascular integrity by achieving whole-organ metabolic homeostasis via the production of factors associated with vasoconstriction or vasorelaxation. In this review, we have focused on the production of nitric oxide (NO), a vasorelaxation factor. The extent of NO production represents a key marker in vascular health. A decrease in NO is capable of inducing pathological conditions associated with endothelial dysfunction, such as obesity, diabetes, cardiovascular disease, and atherosclerosis. Recent studies have strongly implicated the involvement of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cardiovascular disease. Vasculature which is affected by insulin resistance and type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction by reducing intracellular NO. GRK2 activation also induces changes in the subcellular localization of GRK2 itself and also of β-arrestin 2, a downstream protein. In this review, we describe the pathophysiological mechanisms of insulin resistance and diabetes, focusing on the signal transduction for NO production via GRK2 and β-arrestin 2, providing novel insights into the potential field of translational investigation in the treatment of diabetic complications. PMID:26447102

G-protein-coupled receptor kinase 2 and endothelial dysfunction: molecular insights and pathophysiological mechanisms.

PubMed

Taguchi, Kumiko; Matsumoto, Takayuki; Kobayashi, Tsuneo

2015-01-01

Smooth muscle cells (SMC) and endothelial cells are the major cell types in blood vessels. The principal function of vascular SMC in the body is to regulate blood flow and pressure through contraction and relaxation. The endothelium performs a crucial role in maintaining vascular integrity by achieving whole-organ metabolic homeostasis via the production of factors associated with vasoconstriction or vasorelaxation. In this review, we have focused on the production of nitric oxide (NO), a vasorelaxation factor. The extent of NO production represents a key marker in vascular health. A decrease in NO is capable of inducing pathological conditions associated with endothelial dysfunction, such as obesity, diabetes, cardiovascular disease, and atherosclerosis. Recent studies have strongly implicated the involvement of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cardiovascular disease. Vasculature which is affected by insulin resistance and type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction by reducing intracellular NO. GRK2 activation also induces changes in the subcellular localization of GRK2 itself and also of β-arrestin 2, a downstream protein. In this review, we describe the pathophysiological mechanisms of insulin resistance and diabetes, focusing on the signal transduction for NO production via GRK2 and β-arrestin 2, providing novel insights into the potential field of translational investigation in the treatment of diabetic complications.

Brain vascular heterogeneity: implications for disease pathogenesis and design of in vitro blood-brain barrier models.

PubMed

Noumbissi, Midrelle E; Galasso, Bianca; Stins, Monique F

2018-04-23

The vertebrate blood-brain barrier (BBB) is composed of cerebral microvascular endothelial cells (CEC). The BBB acts as a semi-permeable cellular interface that tightly regulates bidirectional molecular transport between blood and the brain parenchyma in order to maintain cerebral homeostasis. The CEC phenotype is regulated by a variety of factors, including cells in its immediate environment and within functional neurovascular units. The cellular composition of the brain parenchyma surrounding the CEC varies between different brain regions; this difference is clearly visible in grey versus white matter. In this review, we discuss evidence for the existence of brain vascular heterogeneity, focusing on differences between the vessels of the grey and white matter. The region-specific differences in the vasculature of the brain are reflective of specific functions of those particular brain areas. This BBB-endothelial heterogeneity may have implications for the course of pathogenesis of cerebrovascular diseases and neurological disorders involving vascular activation and dysfunction. This heterogeneity should be taken into account when developing BBB-neuro-disease models representative of specific brain areas.

Cancer Cells Regulate Biomechanical Properties of Human Microvascular Endothelial Cells*

PubMed Central

Mierke, Claudia Tanja

2011-01-01

Metastasis is a key event of malignant tumor progression. The capability to metastasize depends on the ability of the cancer cell to migrate into connective tissue, adhere, and possibly transmigrate through the endothelium. Previously we reported that the endothelium does not generally act as barrier for cancer cells to migrate in three-dimensional extracellular matrices (3D-ECMs). Instead, the endothelium acts as an enhancer or a promoter for the invasiveness of certain cancer cells. How invasive cancer cells diminish the endothelial barrier function still remains elusive. Therefore, this study investigates whether invasive cancer cells can decrease the endothelial barrier function through alterations of endothelial biomechanical properties. To address this, MDA-MB-231 breast cancer cells were used that invade deeper and more numerous into 3D-ECMs when co-cultured with microvascular endothelial cells. Using magnetic tweezer measurements, MDA-MB-231 cells were found to alter the mechanical properties of endothelial cells by reducing endothelial cell stiffness. Using spontaneous bead diffusion, actin cytoskeletal remodeling dynamics were shown to be increased in endothelial cells co-cultured with MDA-MB-231 cells compared with mono-cultured endothelial cells. In addition, knockdown of the α5 integrin subunit in highly transmigrating α5β1high cells derived from breast, bladder, and kidney cancer cells abolished the endothelial invasion-enhancing effect comparable with the inhibition of myosin light chain kinase. These results indicate that the endothelial invasion-enhancing effect is α5β1 integrin-dependent. Moreover, inhibition of Rac-1, Rho kinase, MEK kinase, and PI3K reduced the endothelial invasion-enhancing effect, indicating that signaling via small GTPases may play a role in the endothelial facilitated increased invasiveness of cancer cells. In conclusion, decreased stiffness and increased cytoskeletal remodeling dynamics of endothelial cells may account for the breakdown of endothelial barrier function, suggesting that biomechanical alterations are sufficient to facilitate the transmigration and invasion of invasive cancer cells into 3D-ECMs. PMID:21940631

A branched chain amino acid metabolite drives vascular transport of fat and causes insulin resistance

PubMed Central

Jang, Cholsoon; Oh, Sungwhan F; Wada, Shogo; Rowe, Glenn C; Liu, Laura; Chan, Mun Chun; Rhee, James; Hoshino, Atsushi; Kim, Boa; Ibrahim, Ayon; Baca, Luisa G; Kim, Esl; Ghosh, Chandra C; Parikh, Samir M; Jiang, Aihua; Chu, Qingwei; Forman, Daniel E.; Lecker, Stewart H.; Krishnaiah, Saikumari; Rabinowitz, Joshua D; Weljie, Aalim M; Baur, Joseph A; Kasper, Dennis L; Arany, Zoltan

2016-01-01

Epidemiological and experimental data implicate branched chain amino acids (BCAAs) in the development of insulin resistance, but the mechanisms underlying this link remain unclear.1–3 Insulin resistance in skeletal muscle stems from excess accumulation of lipid species4, a process that requires blood-borne lipids to first traverse the blood vessel wall. Little is known, however, of how this trans-endothelial transport occurs or is regulated. Here, we leverage PGC-1α, a transcriptional coactivator that regulates broad programs of FA consumption, to identify 3-hydroxy-isobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, as a novel paracrine regulator of trans-endothelial fatty acids (FA) transport. 3-HIB is secreted from muscle cells, activates endothelial FA transport, stimulates muscle FA uptake in vivo, and promotes muscle lipid accumulation and insulin resistance in animals. Conversely, inhibiting the synthesis of 3-HIB in muscle cells blocks the promotion of endothelial FA uptake. 3-HIB levels are elevated in muscle from db/db mice and from subjects with diabetes. These data thus unveil a novel mechanism that regulates trans-endothelial flux of FAs, revealing 3-HIB as a new bioactive signaling metabolite that links the regulation of FA flux to BCAA catabolism and provides a mechanistic explanation for how increased BCAA catabolic flux can cause diabetes. PMID:26950361

Inhibition of the proliferation and acceleration of migration of vascular endothelial cells by increased cysteine-rich motor neuron 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakashima, Yukiko; Morimoto, Mayuka; Toda, Ken-ichi

2015-07-03

Cysteine-rich motor neuron 1 (CRIM1) is upregulated only in extracellular matrix gels by angiogenic factors such as vascular endothelial growth factor (VEGF). It then plays a critical role in the tube formation of endothelial cells. In the present study, we investigated the effects of increased CRIM1 on other endothelial functions such as proliferation and migration. Knock down of CRIM1 had no effect on VEGF-induced proliferation or migration of human umbilical vein endothelial cells (HUVECs), indicating that basal CRIM1 is not involved in the proliferation or migration of endothelial cells. Stable CRIM1-overexpressing endothelial F-2 cells, termed CR1 and CR2, were constructed,more » because it was difficult to prepare monolayer HUVECs that expressed high levels of CRIM1. Proliferation was reduced and migration was accelerated in both CR1 and CR2 cells, compared with normal F-2 cells. Furthermore, the transient overexpression of CRIM1 resulted in decreased proliferation and increased migration of bovine aortic endothelial cells. In contrast, neither proliferation nor migration of COS-7 cells were changed by the overexpression of CRIM1. These results demonstrate that increased CRIM1 reduces the proliferation and accelerates the migration of endothelial cells. These CRIM1 effects might contribute to tube formation of endothelial cells. CRIM1 induced by angiogenic factors may serve as a regulator in endothelial cells to switch from proliferating cells to morphological differentiation. - Highlights: • CRIM1 was upregulated only in tubular endothelial cells, but not in monolayers. • Increased CRIM1 reduced the proliferation of endothelial cells. • Increased CRIM1 accelerated the migration of endothelial cells. • Increased CRIM1 had no effect on the proliferation or migration of COS-7 cells.« less

Isolation and Characterization of Rat Pituitary Endothelial Cells

PubMed Central

Chaturvedi, Kirti; Sarkar, Dipak K.

2010-01-01

Most previous studies that determined the effect of estradiol on angiogenesis used endothelial cells from nonpituitary sources. Because pituitary tumor tissue receives its blood supply via portal and arterial circulation, it is important to use pituitary-derived endothelial cells in studying pituitary angiogenesis. We have developed a magnetic separation technique to isolate endothelial cells from pituitary tissues and have characterized these cells in primary cultures. Endothelial cells of the pituitary showed the existence of endothelial cell marker, CD31, and of von Willebrand factor protein. These cells in cultures also showed immunore-activity of estrogen receptors alpha and beta. The angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor, significantly increased proliferation and migration of the pituitary-derived endothelial cells in primary cultures. These results suggest that a magnetic separation technique can be used for enrichment of pituitary-derived endothelial cells for determination of cellular mechanisms governing the vascularization in the pituitary. PMID:17028416

Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells

PubMed Central

Ding, Bi-Sen; Gomi, Kazunori; Rafii, Shahin; Crystal, Ronald G.; Walters, Matthew S.

2015-01-01

ABSTRACT Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell–endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal ‘crosstalk’ between human airway basal cells and endothelial cells that regulates proliferation of basal cells. PMID:26116571

Activation of PPARbeta/delta induces endothelial cell proliferation and angiogenesis.

PubMed

Piqueras, Laura; Reynolds, Andrew R; Hodivala-Dilke, Kairbaan M; Alfranca, Arántzazu; Redondo, Juan M; Hatae, Toshihisa; Tanabe, Tadashi; Warner, Timothy D; Bishop-Bailey, David

2007-01-01

The role of the nuclear receptor peroxisome-proliferator activated receptor (PPAR)-beta/delta in endothelial cells remains unclear. Interestingly, the selective PPARbeta/delta ligand GW501516 is in phase II clinical trials for dyslipidemia. Here, using GW501516, we have assessed the involvement of PPARbeta/delta in endothelial cell proliferation and angiogenesis. Western blot analysis indicated PPARbeta/delta was expressed in primary human umbilical and aortic endothelial cells, and in the endothelial cell line, EAHy926. Treatment with GW501516 increased human endothelial cell proliferation and morphogenesis in cultures in vitro, endothelial cell outgrowth from murine aortic vessels in vitro, and angiogenesis in a murine matrigel plug assay in vivo. GW501516 induced vascular endothelial cell growth factor mRNA and peptide release, as well as adipose differentiation-related protein (ADRP), a PPARbeta/delta target gene. GW501516-induced proliferation, morphogenesis, vascular endothelial growth factor (VEGF), and ADRP were absent in endothelial cells transfected with dominant-negative PPARbeta/delta. Furthermore, treatment of cells with cyclo-VEGFI, a VEGF receptor1/2 antagonist, abolished GW501516-induced endothelial cell proliferation and tube formation. PPARbeta/delta is a novel regulator of endothelial cell proliferation and angiogenesis through VEGF. The use of GW501516 to treat dyslipidemia may need to be carefully monitored in patients susceptible to angiogenic disorders.

Angiotensin II receptor one (AT1) mediates dextrose induced endoplasmic reticulum stress and superoxide production in human coronary artery endothelial cells.

PubMed

Haas, Michael J; Onstead-Haas, Luisa; Lee, Tracey; Torfah, Maisoon; Mooradian, Arshag D

2016-10-01

Renin-angiotensin-aldosterone system (RAAS) has been implicated in diabetes-related vascular complications partly through oxidative stress. To determine the role of angiotensin II receptor subtype one (AT1) in dextrose induced endoplasmic reticulum (ER) stress, another cellular stress implicated in vascular disease. Human coronary artery endothelial cells with or without AT1 receptor knock down were treated with 27.5mM dextrose for 24h in the presence of various pharmacologic blockers of RAAS and ER stress and superoxide (SO) production were measured. Transfection of cells with AT1 antisense RNA knocked down cellular AT1 by approximately 80%. The ER stress was measured using the placental alkaline phosphatase (ES-TRAP) assay and western blot analysis of glucose regulated protein 78 (GRP78), c-jun-N-terminal kinase 1 (JNK1), phospho-JNK1, eukaryotic translation initiation factor 2α (eIF2α) and phospho-eIF2α measurements. Superoxide (SO) generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride (MCLA) chemiluminescence. In cells with AT1 knock down, dextrose induced ER stress was significantly blunted and treatment with 27.5mM dextrose resulted in significantly smaller increase in SO production compared to 27.5mM dextrose treated and sham transfected cells. Dextrose induced ER stress was reduced with pharmacologic blockers of AT1 (losartan and candesartan) and mineralocorticoid receptor blocker (spironolactone) but not with angiotensin converting enzyme inhibitors (captopril and lisinopril). The dextrose induced SO generation was inhibited by all pharmacologic blockers of RAAS tested. The results indicate that dextrose induced ER stress and SO production in endothelial cells are mediated at least partly through AT1 receptor activation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Production of soluble Neprilysin by endothelial cells.

PubMed

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

2014-04-04

A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35±0.70 and 6.54±0.41 μmols of substrate cleaved over 3h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC50 values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37±1.43 and 38.30±4.70, respectively as a % of control; P<0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17. Copyright © 2014 Elsevier Inc. All rights reserved.

The contribution of CXCL12-expressing radial glia cells to neuro-vascular patterning during human cerebral cortex development

PubMed Central

Errede, Mariella; Girolamo, Francesco; Rizzi, Marco; Bertossi, Mirella; Roncali, Luisa; Virgintino, Daniela

2014-01-01

This study was conducted on human developing brain by laser confocal and transmission electron microscopy (TEM) to make a detailed analysis of important features of blood-brain barrier (BBB) microvessels and possible control mechanisms of vessel growth and differentiation during cerebral cortex vascularization. The BBB status of cortex microvessels was examined at a defined stage of cortex development, at the end of neuroblast waves of migration, and before cortex lamination, with BBB-endothelial cell markers, namely tight junction (TJ) proteins (occludin and claudin-5) and influx and efflux transporters (Glut-1 and P-glycoprotein), the latter supporting evidence for functional effectiveness of the fetal BBB. According to the well-known roles of astroglia cells on microvessel growth and differentiation, the early composition of astroglia/endothelial cell relationships was analyzed by detecting the appropriate astroglia, endothelial, and pericyte markers. GFAP, chemokine CXCL12, and connexin 43 (Cx43) were utilized as markers of radial glia cells, CD105 (endoglin) as a marker of angiogenically activated endothelial cells (ECs), and proteoglycan NG2 as a marker of immature pericytes. Immunolabeling for CXCL12 showed the highest level of the ligand in radial glial (RG) fibers in contact with the growing cortex microvessels. These specialized contacts, recognizable on both perforating radial vessels and growing collaterals, appeared as CXCL12-reactive en passant, symmetrical and asymmetrical, vessel-specific RG fiber swellings. At the highest confocal resolution, these RG varicosities showed a CXCL12-reactive dot-like content whose microvesicular nature was confirmed by ultrastructural observations. A further analysis of RG varicosities reveals colocalization of CXCL12 with Cx43, which is possibly implicated in vessel-specific chemokine signaling. PMID:25360079

Liposome-based glioma targeted drug delivery enabled by stable peptide ligands.

PubMed

Wei, Xiaoli; Gao, Jie; Zhan, Changyou; Xie, Cao; Chai, Zhilan; Ran, Danni; Ying, Man; Zheng, Ping; Lu, Weiyue

2015-11-28

The treatment of glioma is one of the most challenging tasks in clinic. As an intracranial tumor, glioma exhibits many distinctive characteristics from other tumors. In particular, various barriers including enzymatic barriers in the blood and brain capillary endothelial cells, blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB) rigorously prevent drug and drug delivery systems from reaching the tumor site. To tackle this dilemma, we developed a liposomal formulation to circumvent multiple-barriers by modifying the liposome surface with proteolytically stable peptides, (D)CDX and c(RGDyK). (D)CDX is a D-peptide ligand of nicotine acetylcholine receptors (nAChRs) on the BBB, and c(RGDyK) is a ligand of integrin highly expressed on the BBTB and glioma cells. Lysosomal compartments of brain capillary endothelial cells are implicated in the transcytosis of those liposomes. However, both peptide ligands displayed exceptional stability in lysosomal homogenate, ensuring that intact ligands could exert subsequent exocytosis from brain capillary endothelial cells and glioma targeting. In the cellular uptake studies, dually labeled liposomes could target both brain capillary endothelial cells and tumor cells, effectively traversing the BBB and BBTB monolayers, overcoming enzymatic barrier and targeting three-dimensional tumor spheroids. Its targeting ability to intracranial glioma was further verified in vivo by ex vivo imaging and histological studies. As a result, doxorubicin liposomes modified with both (D)CDX and c(RGDyK) presented better anti-glioma effect with prolonged median survival of nude mice bearing glioma than did unmodified liposomes and liposomes modified with individual peptide ligand. In conclusion, the liposome suggested in the present study could effectively overcome multi-barriers and accomplish glioma targeted drug delivery, validating its potential value in improving the therapeutic efficacy of doxorubicin for glioma. Copyright © 2015 Elsevier B.V. All rights reserved.

Using Magnets and Magnetic Beads to Dissect Signaling Pathways Activated by Mechanical Tension Applied to Cells

PubMed Central

Marjoram, R.J.; Guilluy, C; Burridge, K.

2015-01-01

Cellular tension has implications in normal biology and pathology. Membrane adhesion receptors serve as conduits for mechanotransduction that lead to cellular responses. Ligand-conjugated magnetic beads are a useful tool in the study of how cells sense and respond to tension. Here we detail methods for their use in applying tension to cells and strategies for analyzing the results. We demonstrate the methods by analyzing mechanotransduction through VE-cadherin on endothelial cells using both permanent magnets and magnetic tweezers. PMID:26427549

Intracellular L-arginine concentration does not determine NO production in endothelial cells: Implications on the 'L-arginine paradox'

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Soyoung; Mohan, Srinidi; Fung, Ho-Leung, E-mail: hlfung@buffalo.edu

2011-11-04

Highlights: Black-Right-Pointing-Pointer Our findings provide a possible solution to the 'L-arginine paradox'. Black-Right-Pointing-Pointer Extracellular L-arginine concentration is the major determinant of NO production. Black-Right-Pointing-Pointer Cellular L-arginine action is limited by cellular ARG transport, not the K{sub m} of NOS. Black-Right-Pointing-Pointer We explain how L-arginine supplementation can work to increase endothelial function. -- Abstract: We examined the relative contributory roles of extracellular vs. intracellular L-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of {sup 15}N{sub 4}-ARG, ARG, or L-arginine ethyl ester (ARG-EE) for 2 h.more » To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, {sup 15}N{sub 4}-ARG, dimethylarginines, and L-citrulline by an LC-MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by{sup 15}N-nitrite or estimated {sup 15}N{sub 3}-citrulline concentrations when {sup 15}N{sub 4}-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced {sup 15}N{sub 4}-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by {sup 15}N-nitrite, total nitrite and {sup 15}N{sub 3}-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the 'L-arginine paradox' should not consider intracellular ARG concentration as a reference point.« less

Strategies to reverse endothelial progenitor cell dysfunction in diabetes.

PubMed

Petrelli, Alessandra; Di Fenza, Raffaele; Carvello, Michele; Gatti, Francesca; Secchi, Antonio; Fiorina, Paolo

2012-01-01

Bone-marrow-derived cells-mediated postnatal vasculogenesis has been reported as the main responsible for the regulation of vascular homeostasis in adults. Since their discovery, endothelial progenitor cells have been depicted as mediators of postnatal vasculogenesis for their peculiar phenotype (partially staminal and partially endothelial), their ability to differentiate in endothelial cell line and to be incorporated into the vessels wall during ischemia/damage. Diabetes mellitus, a condition characterized by cardiovascular disease, nephropathy, and micro- and macroangiopathy, showed a dysfunction of endothelial progenitor cells. Herein, we review the mechanisms involved in diabetes-related dysfunction of endothelial progenitor cells, highlighting how hyperglycemia affects the different steps of endothelial progenitor cells lifetime (i.e., bone marrow mobilization, trafficking into the bloodstream, differentiation in endothelial cells, and homing in damaged tissues/organs). Finally, we review preclinical and clinical strategies that aim to revert diabetes-induced dysfunction of endothelial progenitor cells as a means of finding new strategies to prevent diabetic complications.

Characterisation of Weibel-Palade body fusion by amperometry in endothelial cells reveals fusion pore dynamics and the effect of cholesterol on exocytosis.

PubMed

Cookson, Emma A; Conte, Ianina L; Dempster, John; Hannah, Matthew J; Carter, Tom

2013-12-01

Regulated secretion from endothelial cells is mediated by Weibel-Palade body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs, and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, whereas cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, whereas its supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics and highlight an important role for cholesterol in the regulation of WPB exocytosis.

Molecular Mechanism for Cellular Response to β-Escin and Its Therapeutic Implications.

PubMed

Domanski, Dominik; Zegrocka-Stendel, Oliwia; Perzanowska, Anna; Dutkiewicz, Malgorzata; Kowalewska, Magdalena; Grabowska, Iwona; Maciejko, Dorota; Fogtman, Anna; Dadlez, Michal; Koziak, Katarzyna

2016-01-01

β-escin is a mixture of triterpene saponins isolated from the horse chestnut seeds (Aesculus hippocastanum L.). The anti-edematous, anti-inflammatory and venotonic properties of β-escin have been the most extensively clinically investigated effects of this plant-based drug and randomized controlled trials have proved the efficacy of β-escin for the treatment of chronic venous insufficiency. However, despite the clinical recognition of the drug its pharmacological mechanism of action still remains largely elusive. To determine the cellular and molecular basis for the therapeutic effectiveness of β-escin we performed discovery and targeted proteomic analyses and in vitro evaluation of cellular and molecular responses in human endothelial cells under inflammatory conditions. Our results demonstrate that in endothelial cells β-escin potently induces cholesterol synthesis which is rapidly followed with marked fall in actin cytoskeleton integrity. The concomitant changes in cell functioning result in a significantly diminished responses to TNF-α stimulation. These include reduced migration, alleviated endothelial monolayer permeability, and inhibition of NFκB signal transduction leading to down-expression of TNF-α-induced effector proteins. Moreover, the study provides evidence for novel therapeutic potential of β-escin beyond the current vascular indications.

Molecular Mechanism for Cellular Response to β-Escin and Its Therapeutic Implications

PubMed Central

Perzanowska, Anna; Dutkiewicz, Malgorzata; Kowalewska, Magdalena; Grabowska, Iwona; Maciejko, Dorota; Fogtman, Anna; Dadlez, Michal; Koziak, Katarzyna

2016-01-01

β-escin is a mixture of triterpene saponins isolated from the horse chestnut seeds (Aesculus hippocastanum L.). The anti-edematous, anti-inflammatory and venotonic properties of β-escin have been the most extensively clinically investigated effects of this plant-based drug and randomized controlled trials have proved the efficacy of β-escin for the treatment of chronic venous insufficiency. However, despite the clinical recognition of the drug its pharmacological mechanism of action still remains largely elusive. To determine the cellular and molecular basis for the therapeutic effectiveness of β-escin we performed discovery and targeted proteomic analyses and in vitro evaluation of cellular and molecular responses in human endothelial cells under inflammatory conditions. Our results demonstrate that in endothelial cells β-escin potently induces cholesterol synthesis which is rapidly followed with marked fall in actin cytoskeleton integrity. The concomitant changes in cell functioning result in a significantly diminished responses to TNF-α stimulation. These include reduced migration, alleviated endothelial monolayer permeability, and inhibition of NFκB signal transduction leading to down-expression of TNF-α—induced effector proteins. Moreover, the study provides evidence for novel therapeutic potential of β-escin beyond the current vascular indications. PMID:27727329

Signaling hierarchy regulating human endothelial cell development.

PubMed

Kelly, Melissa A; Hirschi, Karen K

2009-05-01

Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

BCL2 expression in CD105 positive neoangiogenic cells and tumor progression in angioimmunoblastic T-cell lymphoma.

PubMed

Ratajczak, Philippe; Leboeuf, Christophe; Wang, Li; Brière, Josette; Loisel-Ferreira, Irmine; Thiéblemont, Catherine; Zhao, Wei-Li; Janin, Anne

2012-06-01

The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a crosstalk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein coexpression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells. In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were coexpressed. BCL2 was expressed only in neoangiogenic CD34(+)CD105(+) endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34(+) endothelial cells, but not in CD3(+)CD10(+)lymphoma cells, or in CD34(+) endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34(+)CD105(+) neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH. BCL2 expression by CD105(+) neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.

Rapid isolation of choriocapillary endothelial cells by Lycopersicon esculentum-coated Dynabeads.

PubMed

Hoffmann, S; Spee, C; Murata, T; Cui, J Z; Ryan, S J; Hinton, D R

1998-10-01

In vitro studies of choroidal endothelial cells may be critical for understanding the pathogenesis of neovascularization in age-related macular degeneration, since endothelial cells from different sites are highly heterogeneous in their morphology and behavior. Isolation of choroidal endothelial cells is complicated and labor intensive because of the small size of the choroid and the difficulty of excluding contaminating cells. We describe a rapid, simplified method for the isolation of bovine choroidal endothelial cells using microdissection followed by the use of superparamagnetic beads (Dynabeads) coated with the endothelial cell-specific lectin Lycopersicon esculentum, which selectively binds to fucose residues on the endothelial cell surface. Cells bound to beads are isolated using a magnetic particle concentrator. Isolated cells grew to confluence in a monolayer with a cobblestone morphology and were shown to be endothelial cells by their greater than 95% immunoreactivity to von Willebrand factor and phagocytosis of dil-acetylated LDL. Isolated cells grew as tubes in three-dimensional cultures. This method markedly reduces the time needed for pure culture of cells and makes the in vitro study of choroidal endothelial cells practical and reproducible.

The controversial origin of pericytes during angiogenesis - Implications for cell-based therapeutic angiogenesis and cell-based therapies.

PubMed

Blocki, Anna; Beyer, Sebastian; Jung, Friedrich; Raghunath, Michael

2018-01-01

Pericytes reside within the basement membrane of small vessels and are often in direct cellular contact with endothelial cells, fulfilling important functions during blood vessel formation and homeostasis. Recently, these pericytes have been also identified as mesenchymal stem cells. Mesenchymal stem cells, and especially their specialized subpopulation of pericytes, represent promising candidates for therapeutic angiogenesis applications, and have already been widely applied in pre-clinical and clinical trials. However, cell-based therapies of ischemic diseases (especially of myocardial infarction) have not resulted in significant long-term improvement. Interestingly, pericytes from a hematopoietic origin were observed in embryonic skin and a pericyte sub-population expressing leukocyte and monocyte markers was described during adult angiogenesis in vivo. Since mesenchymal stem cells do not express hematopoietic markers, the latter cell type might represent an alternative pericyte population relevant to angiogenesis. Therefore, we sourced blood-derived angiogenic cells (BDACs) from monocytes that closely resembled hematopoietic pericytes, which had only been observed in vivo thus far. BDACs displayed many pericytic features and exhibited enhanced revascularization and functional tissue regeneration in a pre-clinical model of critical limb ischemia. Comparison between BDACs and mesenchymal pericytes indicated that BDACs (while resembling hematopoietic pericytes) enhanced early stages of angiogenesis, such as endothelial cell sprouting. In contrast, mesenchymal pericytes were responsible for blood vessel maturation and homeostasis, while reducing endothelial sprouting.Since the formation of new blood vessels is crucial during therapeutic angiogenesis or during integration of implants into the host tissue, hematopoietic pericytes (and therefore BDACs) might offer an advantageous addition or even an alternative for cell-based therapies.

Apolipoprotein CIII-induced THP-1 cell adhesion to endothelial cells involves pertussis toxin-sensitive G protein- and protein kinase C alpha-mediated nuclear factor-kappaB activation.

PubMed

Kawakami, Akio; Aikawa, Masanori; Nitta, Noriko; Yoshida, Masayuki; Libby, Peter; Sacks, Frank M

2007-01-01

Plasma apolipoprotein CIII (apoCIII) independently predicts risk for coronary heart disease (CHD). We recently reported that apoCIII directly enhances adhesion of human monocytes to endothelial cells (ECs), and identified the activation of PKC alpha as a necessary upstream event of enhanced monocyte adhesion. This study tested the hypothesis that apoCIII activates PKC alpha in human monocytic THP-1 cells, leading to NF-kappaB activation. Among inhibitors specific to PKC activators, phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 limited apoCIII-induced PKC alpha activation and THP-1 cell adhesion. ApoCIII increased PC-PLC activity in THP-1 cells, resulting in PKC alpha activation. Pertussis toxin (PTX) inhibited apoCIII-induced PC-PLC activation and subsequent PKC alpha activation, implicating PTX-sensitive G protein pathway. ApoCIII further activated nuclear factor-kappaB (NF-kappaB) through PKC alpha in THP-1 cells and augmented beta1-integrin expression. The NF-kappaB inhibitor peptide SN50 partially inhibited apoCIII-induced beta1-integrin expression and THP-1 cell adhesion. ApoCIII-rich VLDL had similar effects to apoCIII alone. PTX-sensitive G protein pathway participates critically in PKC alpha stimulation in THP-1 cells exposed to apoCIII, activating NF-kappaB, and increasing beta1-integrin. This action causes monocytic cells to adhere to endothelial cells. Furthermore, because leukocyte NF-kappaB activation contributes to inflammatory aspects of atherogenesis, apoCIII may stimulate diverse inflammatory responses through monocyte activation.

Disruption of astrocyte-vascular coupling and the blood-brain barrier by invading glioma cells

PubMed Central

Watkins, Stacey; Robel, Stefanie; Kimbrough, Ian F.; Robert, Stephanie M.; Ellis-Davies, Graham; Sontheimer, Harald

2014-01-01

Astrocytic endfeet cover the entire cerebral vasculature and serve as exchange sites for ions, metabolites, and energy substrates from the blood to the brain. They maintain endothelial tight junctions that form the blood-brain barrier (BBB) and release vasoactive molecules that regulate vascular tone. Malignant gliomas are highly invasive tumors that use the perivascular space for invasion and co-opt existing vessels as satellite tumors form. Here we use a clinically relevant mouse model of glioma and find that glioma cells, as they populate the perivascular space of pre-existing vessels, displace astrocytic endfeet from endothelial or vascular smooth muscle cells. This causes a focal breach in the BBB. Furthermore, astrocyte-mediated gliovascular coupling is lost, and glioma cells seize control over regulation of vascular tone through Ca2+-dependent release of K+. These findings have important clinical implications regarding blood flow in the tumor-associated brain and the ability to locally deliver chemotherapeutic drugs in disease. PMID:24943270

Reduced survival in patients with early-stage non-small-cell lung cancer is associated with high pleural endothelial progenitor cell levels.

PubMed

Pirro, Matteo; Cagini, Lucio; Mannarino, Massimo R; Andolfi, Marco; Potenza, Rossella; Paciullo, Francesco; Bianconi, Vanessa; Frangione, Maria Rosaria; Bagaglia, Francesco; Puma, Francesco; Mannarino, Elmo

2016-12-01

Endothelial progenitor cells are capable of contributing to neovascularization in tumours. In patients with either malignant or transudative pleural effusion, we tested the presence of pleural endothelial progenitor cells. We also measured the number of endothelial progenitor cells in post-surgery pleural drainage of either patients with early non-small-cell lung cancer or control patients with benign lung disease undergoing pulmonary resection. The prospective influence of post-surgery pleural-drainage endothelial progenitor cells on cancer recurrence/survival was investigated. Pleural endothelial progenitor cell levels were quantified by fluorescence-activated cell sorting analysis in pleural effusion of 15 patients with late-stage non-small-cell lung cancer with pleural involvement and in 15 control patients with congestive heart failure. Also, pleural-drainage endothelial progenitor cells were measured in pleural-drainage fluid 48 h after surgery in 64 patients with early-stage non-small-cell lung cancer and 20 benign lung disease patients undergoing pulmonary resection. Cancer recurrence and survival was evaluated in patients with high pleural-drainage endothelial progenitor cell levels. The number of pleural endothelial progenitor cells was higher in non-small-cell lung cancer pleural effusion than in transudative pleural effusion. Also, pleural-drainage endothelial progenitor cell levels were higher in patients with non-small-cell lung cancer than in patients with benign lung disease undergoing pulmonary resection (P < 0.05). Non-small-cell lung cancer patients with high pleural-drainage endothelial progenitor cell levels had a significantly 4.9 higher rate of cancer recurrence/death than patients with lower pleural-drainage endothelial progenitor cell levels, irrespective of confounders. Endothelial progenitor cells are present in the pleural effusion and are higher in patients with late-stage non-small-cell lung cancer with pleural involvement than in congestive heart failure patients. Endothelial progenitor cell levels are higher in the post-surgery pleural drainage of patients with non-small-cell lung cancer than in non-neoplastic pleural-drainage fluid. High pleural-drainage endothelial progenitor cell levels in patients undergoing pulmonary resection for early non-small-cell lung cancer predict an increased risk of cancer recurrence and death. © The Author 2016. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Magnetic beads (Dynabead) toxicity to endothelial cells at high bead concentration: implication for tissue engineering of vascular prosthesis.

PubMed

Tiwari, A; Punshon, G; Kidane, A; Hamilton, G; Seifalian, A M

2003-10-01

Magnetic beads (Dynabeads) have been used for the purification of endothelial cells. One application for this procedure may be for single-stage seeding of bypass grafts. The number of endothelial cells (EC) isolated is crucial and therefore to increase the number of cells extracted, a higher number of Dynabeads per cell may need to be used. The effect of large numbers of CD31 Dynabeads on cell proliferation/metabolism is unknown. We undertook this study using CD31-coated Dynabeads and EC from human umbilical vein. EC were coated at concentrations of 4, 10, or 50 beads per cell. The cells were cultured for 6 days with control being normal EC. Cellular proliferation was assessed by trypsinization of cells and metabolism assessed with an Alamar blue viability assay. In a further experiment a compliant polyurethane graft was single-stage seeded with both coated Dynabeads and normal EC. The results showed that using a higher number of beads per cell resulted in a reduction in cell proliferation and a reduction in cell metabolism. The total number of Dynabeads-coated cells in culture compared to controls (%) by day 6 were 30.7 +/- 2.56, 41.3 +/- 9.8 and 59.2 +/- 7.3 for 50, 10, and 4 beads per cell, respectively. The corresponding results for Alamar blue were 43.7 +/- 1.2, 61.8 +/- 1.4, and 72.1 +/- 4.3. The seeded grafts showed reduced metabolism with the Dynabeads-coated EC. In conclusion, high numbers of beads per cell have a late detrimental effect on cell proliferation and metabolism. Therefore for single-stage seeding lower numbers of Dynabeads will need to be used with resultant reduction in the number of available EC.

Heme oxygenase-1 protects INF-gamma primed endothelial cells from Jurkat T-cell adhesion.

PubMed

Du, D; Chang, S; Chen, B; Zhou, H; Chen, Z K

2007-12-01

The heme oxygenase-1 (HO-1) system is associated with the rate-limiting step of conversion of heme, one of the most critical roles in cytoprotective mechanisms. Our study investigated its potential role in protection of endothelial cells from T cells. The recombinant plasmid pcDNA3-HO-1 was transfected into endothelial cells. Indirect fluorescent staining was used to examine the expression of HO-1 protein. Then endothelial cells primed by INF-gamma were mixed in culture with Jurkat T cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). The number of adhesive Jurkat T cells was determined using FACS to evaluate the adhesion effect. After being cultured with endothelial cells, the cell cycle of Jurkat T cells was detected using FACS. Expression of HO-1 on endothelial cells conferred significant protection against Jurkat T-cell-mediated adhesion. The rate of Jurkat T-cell adhesions was reduced to 19.06%, in contrast with 31.42% in the control group (P<.05). After using ZnPP, an inhibitor of HO-1, the rate of Jurkat T-cell adhesion recovered to 29.08%. The binding activities between endothelial cells and Jurkat T cells was blocked by HO-1 expression. The proliferation of Jurkat T cells was inhibited after culture with endothelial cells, which had been transfected with HO-1, which blocked cell cycle entry of T cells. More than 60% of Jurkat T cells remained in G0/G1 compared with 40% among the control group. HO-1 directly protected endothelial cells primed by INF-gamma from Jurkat T cells and down-regulated the expression of HLA-DR on the surface of endothelial cells. These results indicated that transgenic expression of HO-1 may be useful to prevent lymphocytes from responding to endothelial cells.

Protein Kinase-C Beta Contributes to Impaired Endothelial Insulin Signaling in Humans with Diabetes Mellitus

PubMed Central

Tabit, Corey E; Shenouda, Sherene M; Holbrook, Monica; Fetterman, Jessica L; Kiani, Soroosh; Frame, Alissa A; Kluge, Matthew A; Held, Aaron; Dohadwala, Mustali; Gokce, Noyan; Farb, Melissa; Rosenzweig, James; Ruderman, Neil; Vita, Joseph A; Hamburg, Naomi M

2013-01-01

Background Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling through the activity of protein kinase C-β (PKCβ) and nuclear factor κB (NFκB) reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus. Methods and Results We measured protein expression and insulin response in freshly isolated endothelial cells from patients with Type 2 diabetes mellitus (n=40) and non-diabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes (P=0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in non-diabetic subjects but not in diabetic patients (P=0.003) consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=−0.541, P=0.02) Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes. Endothelial NFκB activation was higher in diabetes and was reduced with PKCβ inhibition. Conclusions We provide evidence for the presence of altered eNOS activation, reduced insulin action and inflammatory activation in the endothelium of patients with diabetes. Our findings implicate PKCβ activity in endothelial insulin resistance. PMID:23204109

LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

PubMed Central

Figarola, James L.; Singhal, Jyotsana; Rahbar, Samuel; Awasthi, Sanjay

2014-01-01

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis. PMID:24615331

In chronic spontaneous urticaria, high numbers of dermal endothelial cells, but not mast cells, are linked to recurrent angio-oedema.

PubMed

Terhorst, D; Koti, I; Krause, K; Metz, M; Maurer, M

2018-03-01

Chronic spontaneous urticaria (CSU) is an inflammatory skin disorder characterized by recurrent weals, angio-oedema or both. Recent studies have shown that the number of endothelial cells is increased in the skin of patients with CSU, but the underlying mechanisms and clinical implications of this are unclear. To evaluate whether mast cell (MC) or endothelial cell (EC) numbers correlate with CSU and whether they are relevant for disease duration, disease activity or the presence of clinical features. We determined the numbers of CD31+ ECs and MCs in nonlesional skin of 30 patients with CSU using quantitative histomorphometry, and assessed their correlation with each other and with clinical features such as disease duration, disease activity and occurrence of angio-oedema. The numbers of MCs and ECs were high in the nonlesional skin of patients with CSU, but did not correlate with each other. Neither MC number nor EC number correlated with disease duration or disease activity. Interestingly, patients with high numbers of cutaneous CD31+ ECs had higher rates of recurrent angio-oedema and vice versa. Based on these findings, we speculate that vascular remodelling and MC hyperplasia in patients with CSU occurs independently and via different mechanisms. Targeting of the mechanisms that drive neoangiogenesis in CSU may result in novel therapeutic strategies for the management of patients with angio-oedema. © 2017 British Association of Dermatologists.

Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin*

PubMed Central

Parker, William H.; Qu, Zhi-chao; May, James M.

2015-01-01

Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. PMID:26152729

Endothelial microparticle uptake in target cells is annexin I/phosphatidylserine receptor dependent and prevents apoptosis.

PubMed

Jansen, Felix; Yang, Xiaoyan; Hoyer, Friedrich Felix; Paul, Kathrin; Heiermann, Nadine; Becher, Marc Ulrich; Abu Hussein, Nebal; Kebschull, Moritz; Bedorf, Jörg; Franklin, Bernardo S; Latz, Eicke; Nickenig, Georg; Werner, Nikos

2012-08-01

Endothelial microparticles (EMP) are released from activated or apoptotic cells, but their effect on target cells and the exact way of incorporation are largely unknown. We sought to determine the uptake mechanism and the biological effect of EMP on endothelial and endothelial-regenerating cells. EMP were generated from starved endothelial cells and isolated by ultracentrifugation. Caspase 3 activity assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that EMP protect target endothelial cells against apoptosis in a dose-dependent manner. Proteomic analysis was performed to identify molecules contained in EMP, which might be involved in EMP uptake. Expression of annexin I in EMP was found and confirmed by Western blot, whereas the corresponding receptor phosphatidylserine receptor was present on endothelial target cells. Silencing either annexin I on EMP or phosphatidylserine receptor on target cells using small interfering RNA showed that the uptake of EMP by human coronary artery endothelial cells is annexin I/phosphatidylserine receptor dependent. Annexin I-downregulated EMP abrogated the EMP-mediated protection against apoptosis of endothelial target cells. p38 activation was found to mediate camptothecin-induced apoptosis. Finally, human coronary artery endothelial cells pretreated with EMP inhibited camptothecin-induced p38 activation. EMP are incorporated by endothelial cells in an annexin I/phosphatidylserine receptor-dependent manner and protect target cells against apoptosis. Inhibition of p38 activity is involved in EMP-mediated protection against apoptosis.

Fibroblast TGF-beta Signaling in Breast Development and Cancer

DTIC Science & Technology

2010-09-01

hypermetylation. Among the genes implicated are GSTP1 , APC, RASSF1A, H1N-1 and RAR2 [18,19]. More research is needed to clarify the contribution of any...KG, Wallis BS, Hanson JC, Chuaqui RF, et al. Tumor-associated endothelial cells display GSTP1 and RARbeta2 promoter methylation in human prostate

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.

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 cellsmore » (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.« less

Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

PubMed

Merna, Nick; Wong, Andrew K; Barahona, Victor; Llanos, Pierre; Kunar, Balvir; Palikuqi, Brisa; Ginsberg, Michael; Rafii, Shahin; Rabbany, Sina Y

2018-04-17

Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm 2 for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease. © 2018 John Wiley & Sons Ltd.

Are there Race-Dependent Endothelial Cell Responses to Exercise?

PubMed Central

Brown, Michael D.; Feairheller, Deborah L.

2013-01-01

African Americans have endothelial dysfunction which likely contributes to their high prevalence of hypertension. Endothelial cell responses to stimuli could play a role in the development of endothelial dysfunction and hypertension. High physiological levels of vascular laminar shear stress can profoundly alter endothelial cell phenotype. It is not known whether there are race-dependent endothelial cell responses to laminar shear stress. PMID:23262464

MicroRNA-10b downregulation mediates acute rejection of renal allografts by derepressing BCL2L11

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xiaoyou; Dong, Changgui; Jiang, Zhengyao

Kidney transplantation is the major therapeutic option for end-stage kidney diseases. However, acute rejection could cause allograft loss in some of these patients. Emerging evidence supports that microRNA (miRNA) dysregulation is implicated in acute allograft rejection. In this study, we used next-generation sequencing to profile miRNA expression in normal and acutely rejected kidney allografts. Among 75 identified dysregulated miRNAs, miR-10b was the most significantly downregulated miRNAs in rejected allografts. Transfecting miR-10b inhibitor into human renal glomerular endothelial cells recapitulated key features of acute allograft rejection, including endothelial cell apoptosis, release of pro-inflammatory cytokines (interleukin-6, tumor necrosis factor α, interferon-γ, andmore » chemokine (C–C motif) ligand 2) and chemotaxis of macrophages whereas transfection of miR-10b mimics had opposite effects. Downregulation of miR-10b directly derepressed the expression of BCL2L11 (an apoptosis inducer) as revealed by luciferase reporter assay. Taken together, miR-10b downregulation mediates many aspects of disease pathogenicity of acute kidney allograft rejection. Restoring miR-10b expression in glomerular endothelial cells could be a novel therapeutic approach to reduce acute renal allograft loss. - Highlights: • miR-10b was the most downregulated microRNAs in acutely rejected renal allografts. • miR-10b downregulation triggered glomerular endothelial cell apoptosis. • miR-10b downregulation induced release of pro-inflammatory cytokines. • miR-10b downregulation derepressed its pro-apoptotic target BCL2L11.« less

2-Chlorohexadecanoic acid induces ER stress and mitochondrial dysfunction in brain microvascular endothelial cells.

PubMed

Bernhart, Eva; Kogelnik, Nora; Prasch, Jürgen; Gottschalk, Benjamin; Goeritzer, Madeleine; Depaoli, Maria Rosa; Reicher, Helga; Nusshold, Christoph; Plastira, Ioanna; Hammer, Astrid; Fauler, Günter; Malli, Roland; Graier, Wolfgang F; Malle, Ernst; Sattler, Wolfgang

2018-05-01

Peripheral leukocytes induce blood-brain barrier (BBB) dysfunction through the release of cytotoxic mediators. These include hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H 2 O 2 -chloride system of activated phagocytes. HOCl targets the endogenous pool of ether phospholipids (plasmalogens) generating chlorinated inflammatory mediators like e.g. 2-chlorohexadecanal and its conversion product 2-chlorohexadecanoic acid (2-ClHA). In the cerebrovasculature these compounds inflict damage to brain microvascular endothelial cells (BMVEC) that form the morphological basis of the BBB. To follow subcellular trafficking of 2-ClHA we synthesized a 'clickable' alkyne derivative (2-ClHyA) that phenocopied the biological activity of the parent compound. Confocal and superresolution structured illumination microscopy revealed accumulation of 2-ClHyA in the endoplasmic reticulum (ER) and mitochondria of human BMVEC (hCMEC/D3 cell line). 2-ClHA and its alkyne analogue interfered with protein palmitoylation, induced ER-stress markers, reduced the ER ATP content, and activated transcription and secretion of interleukin (IL)-6 as well as IL-8. 2-ClHA disrupted the mitochondrial membrane potential and induced procaspase-3 and PARP cleavage. The protein kinase R-like ER kinase (PERK) inhibitor GSK2606414 suppressed 2-ClHA-mediated activating transcription factor 4 synthesis and IL-6/8 secretion, but showed no effect on endothelial barrier dysfunction and cleavage of procaspase-3. Our data indicate that 2-ClHA induces potent lipotoxic responses in brain endothelial cells and could have implications in inflammation-induced BBB dysfunction. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

The emerging role of the endocannabinoid system in cardiovascular disease

PubMed Central

2009-01-01

Endocannabinoids are endogenous bioactive lipid mediators present both in the brain and various peripheral tissues, which exert their biological effects via interaction with specific G-protein-coupled cannabinoid receptors, the CB1 and CB2. Pathological overactivation of the endocannabinoid system (ECS) in various forms of shock and heart failure may contribute to the underlying pathology and cardiodepressive state by the activation of the cardiovascular CB1 receptors. Furthermore, tonic activation of CB1 receptors by endocannabinoids has also been implicated in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes, such as plasma lipid alterations, abdominal obesity, hepatic steatosis, inflammation, and insulin and leptin resistance. In contrast, activation of CB2 receptors in immune cells exerts various immunomodulatory effects, and the CB2 receptors in endothelial and inflammatory cells appear to limit the endothelial inflammatory response, chemotaxis, and inflammatory cell adhesion and activation in atherosclerosis and reperfusion injury. Here, we will overview the cardiovascular actions of endocannabinoids and the growing body of evidence implicating the dysregulation of the ECS in a variety of cardiovascular diseases. We will also discuss the therapeutic potential of the modulation of the ECS by selective agonists/antagonists in various cardiovascular disorders associated with inflammation and tissue injury, ranging from myocardial infarction and heart failure to atherosclerosis and cardiometabolic disorders. PMID:19357846

Coculture with endothelial cells reduces the population of cycling LeX neural precursors but increases that of quiescent cells with a side population phenotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathieu, Celine; Fouchet, Pierre; Gauthier, Laurent R.

2006-04-01

Neural stem cell proliferation and differentiation are regulated by external cues from their microenvironment. As endothelial cells are closely associated with neural stem cell in brain germinal zones, we investigated whether endothelial cells may interfere with neurogenesis. Neural precursor cells (NPC) from telencephalon of EGFP mouse embryos were cocultured in direct contact with endothelial cells. Endothelial cells did not modify the overall proliferation and apoptosis of neural cells, albeit they transiently delayed spontaneous apoptosis. These effects appeared to be specific to endothelial cells since a decrease in proliferation and a raise in apoptosis were observed in cocultures with fibroblasts. Endothelialmore » cells stimulated the differentiation of NPC into astrocytes and into neurons, whereas they reduced differentiation into oligodendrocytes in comparison to adherent cultures on polyornithine. Determination of NPC clonogenicity and quantification of LeX expression, a marker for NPC, showed that endothelial cells decreased the number of cycling NPC. On the other hand, the presence of endothelial cells increased the number of neural cells having 'side population' phenotype, another marker reported on NPC, which we have shown to contain quiescent cells. Thus, we show that endothelial cells may regulate neurogenesis by acting at different level of NPC differentiation, proliferation and quiescence.« less

Clinicopathologic and prognostic implications of progranulin in breast carcinoma.

PubMed

Li, Li-qin; Huang, Hui-lian; Ping, Jin-liang; Wang, Xiao-hong; Zhong, Jing; Dai, Li-cheng

2011-07-05

Progranulin is a newly discovered 88-kDa glycoprotein originally purified from the highly tumorigenic mouse teratoma-derived cell line PC. Its expression is closely correlated with the development and metastasis of several cancers. However, no immunohistochemical evidence currently exists to correlate progranulin expression with clinicopathologic features in breast carcinoma biopsies, and the role of progranulin as a new marker of metastatic risk and prognosis in breast cancer has not yet been studied. The aim of this study was to investigate the clinicopathologic and prognostic implications of progranulin expression in breast carcinoma and its correlation with tumor angiogenesis. Progranulin expression was determined immunohistochemically in 183 surgical specimens from patients with breast cancer and 20 tissue samples from breast fibroadenomas. The tumor angiogenesis-related biomarker, vascular endothelial growth factor was assayed and microvessel density was assessed by counting vascular endothelial cells in tumor tissues labeled with endoglin antibody. The relationship between progranulin expression and the clinicopathologic data were analyzed. Progranulin proteins were overexpressed in breast cancer. The level of progranulin expression was significantly correlated with tumor size (P = 0.004), lymph node metastasis (P < 0.001) and TNM staging (P < 0.001). High progranulin expression was associated with higher tumor angiogenesis, reflected by increased vascular endothelial growth factor expression (P < 0.001) and higher microvessel density (P = 0.002). Progranulin may be a valuable marker for assessing the metastasis and prognosis of breast cancer, and could provide the basis for new combination regimens with antiangiogenic activity.

Vascular Endothelial Growth Factor and Angiogenesis in the Regulation of Cutaneous Wound Repair

PubMed Central

Johnson, Kelly E.; Wilgus, Traci A.

2014-01-01

Significance: Angiogenesis, the growth of new blood vessels from existing vessels, is an important aspect of the repair process. Restoration of blood flow to damaged tissues provides oxygen and nutrients required to support the growth and function of reparative cells. Vascular endothelial growth factor (VEGF) is one of the most potent proangiogenic growth factors in the skin, and the amount of VEGF present in a wound can significantly impact healing. Recent Advances: The activity of VEGF was once considered to be specific for endothelial cells lining the inside of blood vessels, partly because VEGF receptor (VEGFR) expression was believed to be restricted to endothelial cells. It is now known, however, that VEGFRs can be expressed by a variety of other cell types involved in wound repair. For example, keratinocytes and macrophages, which both carry out important functions during wound healing, express VEGFRs and are capable of responding directly to VEGF. Critical Issues: The mechanisms by which VEGF promotes angiogenesis are well established. Recent studies, however, indicate that VEGF can directly affect the activity of several nonendothelial cell types present in the skin. The implications of these extra-angiogenic effects of VEGF on wound repair are not yet known, but they suggest that this growth factor may play a more complex role during wound healing than previously believed. Future Directions: Despite the large number of studies focusing on VEGF and wound healing, it is clear that the current knowledge of how VEGF contributes to the repair of skin wounds is incomplete. Further research is needed to obtain a more comprehensive understanding of VEGF activities during the wound healing process. PMID:25302139

Negative regulatory role of PI3-kinase in TNF-induced tumor necrosis.

PubMed

Matschurat, Susanne; Blum, Sabine; Mitnacht-Kraus, Rita; Dijkman, Henry B P M; Kanal, Levent; De Waal, Robert M W; Clauss, Matthias

2003-10-20

Tissue factor is the prime initiator of blood coagulation. Expression of tissue factor in tumor endothelial cells leads to thrombus formation, occlusion of vessels and development of hemorrhagic infarctions in the tumor tissue, often followed by regression of the tumor. Tumor cells produce endogenous vascular endothelial growth factor (VEGF), which sensitizes endothelial cells for systemically administered tumor necrosis factor alpha (TNF alpha) and synergistically enhances the TNF-induced expression of tissue factor. We have analyzed the pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells (HUVECs) after combined stimulation with TNF and VEGF. By using specific low molecular weight inhibitors, we demonstrated that protein kinase C (PKC), p44/42 and p38 mitogen-activated protein (MAP) kinases, and stress-activated protein kinase (JNK) are essentially involved in the induction of tissue factor. In contrast, the application of wortmannin, an inhibitor of phosphatidylinositol 3 (PI3)-kinase, led to strongly enhanced expression of tissue factor in TNF- and VEGF-treated cells, implicating a negative regulatory role for PI3-kinase. In vivo, the application of wortmannin promoted the formation of TNF-induced hemorrhages and intratumoral necroses in murine meth A tumors. The co-injection of wortmannin lowered the effective dose of applied TNF. Therefore, it is conceivable that the treatment of TNF-sensitive tumors with a combination of TNF and wortmannin will ensure the selective damage of the tumor endothelium and minimize the risk of systemic toxicity of TNF. TNF-treatment in combination with specific inhibition of PI3-kinase is a novel concept in anti-cancer therapy. Copyright 2003 Wiley-Liss, Inc.

[Interaction of FABP4 with plasma membrane proteins of endothelial cells].

PubMed

Saavedra, Paula; Girona, Josefa; Aragonès, Gemma; Cabré, Anna; Guaita, Sandra; Heras, Mercedes; Masana, Lluís

2015-01-01

Fatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells. HUVEC cells were incubated with and without FABP4 (100 ng/ml) for 5 minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker. A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100 ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs. The results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33 kDa containing FAPB4 and its putative receptor. The existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular. Copyright © 2014 Sociedad Española de Arteriosclerosis. Published by Elsevier España. All rights reserved.

Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo.

PubMed

Massberg, Steffen; Konrad, Ildiko; Schürzinger, Katrin; Lorenz, Michael; Schneider, Simon; Zohlnhoefer, Dietlind; Hoppe, Katharina; Schiemann, Matthias; Kennerknecht, Elisabeth; Sauer, Susanne; Schulz, Christian; Kerstan, Sandra; Rudelius, Martina; Seidl, Stefan; Sorge, Falko; Langer, Harald; Peluso, Mario; Goyal, Pankaj; Vestweber, Dietmar; Emambokus, Nikla R; Busch, Dirk H; Frampton, Jon; Gawaz, Meinrad

2006-05-15

The accumulation of smooth muscle and endothelial cells is essential for remodeling and repair of injured blood vessel walls. Bone marrow-derived progenitor cells have been implicated in vascular repair and remodeling; however, the mechanisms underlying their recruitment to the site of injury remain elusive. Here, using real-time in vivo fluorescence microscopy, we show that platelets provide the critical signal that recruits CD34+ bone marrow cells and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells to sites of vascular injury. Correspondingly, specific inhibition of platelet adhesion virtually abrogated the accumulation of both CD34+ and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells at sites of endothelial disruption. Binding of bone marrow cells to platelets involves both P-selectin and GPIIb integrin on platelets. Unexpectedly, we found that activated platelets secrete the chemokine SDF-1alpha, thereby supporting further primary adhesion and migration of progenitor cells. These findings establish the platelet as a major player in the initiation of vascular remodeling, a process of fundamental importance for vascular repair and pathological remodeling after vascular injury.

Production of soluble Neprilysin by endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@monash.edu; Rajapakse, Niwanthi W.; Minond, Dmitriy

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

MicroRNA-34a regulation of endothelial senescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ito, Takashi; Yagi, Shusuke; Yamakuchi, Munekazu, E-mail: munekazu_yamakuchi@urmc.rochester.edu

2010-08-06

Research highlights: {yields} MicroRNA-34a (miR-34a) regulates senescence and cell cycle progression in endothelial cells. {yields} MiR-34a expression increases during endothelial cell senescence and in older mice. {yields} SIRT1 is a miR-34a target gene in endothelial cells. {yields} SIRT1 mediates the effects of miR-34a upon cell senescence in endothelial cells. -- Abstract: Endothelial senescence is thought to play a role in cardiovascular diseases such as atherosclerosis. We hypothesized that endothelial microRNAs (miRNAs) regulate endothelial survival and senescence. We found that miR-34a is highly expressed in primary endothelial cells. We observed that miR-34a expression increases in senescent human umbilical cord vein endothelialmore » cells (HUVEC) and in heart and spleen of older mice. MiR-34a over-expression induces endothelial cell senescence and also suppresses cell proliferation by inhibiting cell cycle progression. Searching for how miR-34a affects senescence, we discovered that SIRT1 is a target of miR-34a. Over-expressing miR-34a inhibits SIRT1 protein expression, and knocking down miR-34a enhances SIRT1 expression. MiR-34a triggers endothelial senescence in part through SIRT1, since forced expression of SIRT1 blocks the ability of miR-34a to induce senescence. Our data suggest that miR-34a contributes to endothelial senescence through suppression of SIRT1.« less

Brain-derived neurotrophic factor secreted by the cerebral endothelium: A new actor of brain function?

PubMed

Marie, Christine; Pedard, Martin; Quirié, Aurore; Tessier, Anne; Garnier, Philippe; Totoson, Perle; Demougeot, Céline

2018-06-01

Low cerebral levels of brain-derived neurotrophic factor (BDNF), which plays a critical role in many brain functions, have been implicated in neurodegenerative, neurological and psychiatric diseases. Thus, increasing BDNF levels in the brain is considered an attractive possibility for the prevention/treatment of various brain diseases. To date, BDNF-based therapies have largely focused on neurons. However, given the cross-talk between endothelial cells and neurons and recent evidence that BDNF expressed by the cerebral endothelium largely accounts for BDNF levels present in the brain, it is likely that BDNF-based therapies would be most effective if they also targeted the cerebral endothelium. In this review, we summarize the available knowledge about the biology and actions of BDNF derived from endothelial cells of the cerebral microvasculature and we emphasize the remaining gaps and shortcomings.

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells.

PubMed

Zhang, Jue; Chu, Li-Fang; Hou, Zhonggang; Schwartz, Michael P; Hacker, Timothy; Vickerman, Vernella; Swanson, Scott; Leng, Ning; Nguyen, Bao Kim; Elwell, Angela; Bolin, Jennifer; Brown, Matthew E; Stewart, Ron; Burlingham, William J; Murphy, William L; Thomson, James A

2017-07-25

Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice – Implications for Superficial Erosion

PubMed Central

Franck, Grégory; Mawson, Thomas; Sausen, Grasiele; Salinas, Manuel; Masson, Gustavo Santos; Cole, Andrew; Beltrami-Moreira, Marina; Chatzizisis, Yiannis; Quillard, Thibault; Tesmenitsky, Yevgenia; Shvartz, Eugenia; Sukhova, Galina K.; Swirski, Filip K.; Nahrendorf, Matthias; Aikawa, Elena; Croce, Kevin J.; Libby, Peter

2017-01-01

Rationale Superficial erosion currently causes up to a third of acute coronary syndromes (ACS), yet we lack understanding of its mechanisms. Thrombi due to superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. Objective This study tested in vivo the involvement of disturbed flow, and of neutrophils, hyaluronan, and TLR2 ligation in superficial intimal injury, a process implicated in superficial erosion. Methods and Results : In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell (EC) activation, neutrophil accumulation, EC death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. TLR2 agonism activated luminal ECs, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing EC injury and local thrombosis (p<0.05). Conclusions These results implicate flow disturbance, neutrophils, and TLR2 signaling as mechanisms that contribute to superficial erosion, a cause of ACS of likely growing importance in the statin era. PMID:28428204

Quantification of Malignant Breast Cancer Cell MDA-MB-231 Transmigration across Brain and Lung Microvascular Endothelium

PubMed Central

Fan, Jie; Fu, Bingmei M.

2015-01-01

Tumor cell extravasation through the endothelial barrier forming the microvessel wall is a crucial step during tumor metastasis. However, where, how and how fast tumor cells transmigrate through endothelial barriers remain unclear. Using an in vitro transwell model, we performed a transmigration assay of malignant breast tumor cells (MDA-MB-231) through brain and lung microvascular endothelial monolayers under control and pathological conditions. The locations and rates of tumor cell transmigration as well as the changes in the structural components (integrity) of endothelial monolayers were quantified by confocal microscopy. Endothelial monolayer permeability to albumin Palbumin was also quantified under the same conditions. We found that about 98% of transmigration occurred at the joints of endothelial cells instead of cell bodies; tumor cell adhesion and transmigration degraded endothelial surface glycocalyx and disrupted endothelial junction proteins, consequently increased Palbumin; more tumor cells adhered to and transmigrated through the endothelial monolayer with higher Palbumin; Palbumin and tumor transmigration were increased by vascular endothelial growth factor (VEGF), a representative of cytokines, and lipopolysaccharides (LPS), a typical systemic inflammatory factor, but reduced by adenosine 3′, 5′-cyclic monophosphate (cAMP). These results suggest that reinforcing endothelial structural integrity is an effective approach for inhibiting tumor extravasation. PMID:26603751

Spirulina platensis and phycocyanobilin activate atheroprotective heme oxygenase-1: a possible implication for atherogenesis.

PubMed

Strasky, Zbynek; Zemankova, Lenka; Nemeckova, Ivana; Rathouska, Jana; Wong, Ronald J; Muchova, Lucie; Subhanova, Iva; Vanikova, Jana; Vanova, Katerina; Vitek, Libor; Nachtigal, Petr

2013-11-01

Spirulina platensis, a water blue-green alga, has been associated with potent biological effects, which might have important relevance in atheroprotection. We investigated whether S. platensis or phycocyanobilin (PCB), its tetrapyrrolic chromophore, can activate atheroprotective heme oxygenase-1 (Hmox1), a key enzyme in the heme catabolic pathway responsible for generation of a potent antioxidant bilirubin, in endothelial cells and in a mouse model of atherosclerosis. In vitro experiments were performed on EA.hy926 endothelial cells exposed to extracts of S. platensis or PCB. In vivo studies were performed on ApoE-deficient mice fed a cholesterol diet and S. platensis. The effect of these treatments on Hmox1, as well as other markers of oxidative stress and endothelial dysfunction, was then investigated. Both S. platensis and PCB markedly upregulated Hmox1 in vitro, and a substantial overexpression of Hmox1 was found in aortic atherosclerotic lesions of ApoE-deficient mice fed S. platensis. In addition, S. platensis treatment led to a significant increase in Hmox1 promoter activity in the spleens of Hmox-luc transgenic mice. Furthermore, both S. platensis and PCB were able to modulate important markers of oxidative stress and endothelial dysfunction, such as eNOS, p22 NADPH oxidase subunit, and/or VCAM-1. Both S. platensis and PCB activate atheroprotective HMOX1 in endothelial cells and S. platensis increased the expression of Hmox1 in aortic atherosclerotic lesions in ApoE-deficient mice, and also in Hmox-luc transgenic mice beyond the lipid lowering effect. Therefore, activation of HMOX1 and the heme catabolic pathway may represent an important mechanism of this food supplement for the reduction of atherosclerotic disease.

Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling

PubMed Central

Holopainen, Tanja; Räsänen, Markus; Anisimov, Andrey; Tuomainen, Tomi; Zheng, Wei; Tvorogov, Denis; Hulmi, Juha J.; Andersson, Leif C.; Cenni, Bruno; Tavi, Pasi; Mervaala, Eero; Kivelä, Riikka; Alitalo, Kari

2015-01-01

Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy. PMID:26430242

Preconditioning with Endoplasmic Reticulum Stress Ameliorates Endothelial Cell Inflammation

PubMed Central

Leonard, Antony; Paton, Adrienne W.; El-Quadi, Monaliza; Paton, James C.; Fazal, Fabeha

2014-01-01

Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability. PMID:25356743

Preconditioning with endoplasmic reticulum stress ameliorates endothelial cell inflammation.

PubMed

Leonard, Antony; Paton, Adrienne W; El-Quadi, Monaliza; Paton, James C; Fazal, Fabeha

2014-01-01

Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability.

Endothelial Inflammatory Transcriptional Responses Induced by Plasma Following Inhalation of Diesel Emissions

PubMed Central

Schisler, Jonathan C.; Ronnebaum, Sarah M.; Madden, Michael; Channell, Meghan M.; Campen, Matthew J.; Willis, Monte S.

2016-01-01

Background Air pollution, especially emissions derived from traffic sources, is associated with adverse cardiovascular outcomes. However, it remains unclear how inhaled factors drive extrapulmonary pathology. Objectives Previously, we found that canonical inflammatory response transcripts were elevated in cultured endothelial cells treated with plasma obtained after exposure compared with pre-exposure samples or filtered air (sham) exposures. While the findings confirmed the presence of bioactive factor(s) in the plasma after diesel inhalation, we wanted to better examine the complete genomic response to investigate 1) major responsive transcripts and 2) collected response pathways and ontogeny that may help to refine this method and inform the pathogenesis. Methods We assayed endothelial RNA with gene expression microarrays, examining the responses of cultured endothelial cells to plasma obtained from 6 healthy human subjects exposed to 100 μg/m3 diesel exhaust or filtered air for 2 h on separate occasions. In addition to pre-exposure baseline samples, we investigated samples obtained immediately-post and 24h-post exposure. Results Microarray analysis of the coronary artery endothelial cells challenged with plasma identified 855 probes that changed over time following diesel exhaust exposure. Over-representation analysis identified inflammatory cytokine pathways were upregulated both at the 2 and 24 h condition. Novel pathways related to FOX transcription factors and secreted extracellular factors were also identified in the microarray analysis. Conclusions These outcomes are consistent with our recent findings that plasma contains bioactive and inflammatory factors following pollutant inhalation. The specific study design implicates a novel pathway related to inflammatory blood borne components that may drive the extrapulmonary toxicity of ambient air pollutants. PMID:25942053

Rac regulates vascular endothelial growth factor stimulated motility.

PubMed

Soga, N; Connolly, J O; Chellaiah, M; Kawamura, J; Hruska, K A

2001-01-01

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood. Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF. These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.

Morphology and vasoactive hormone profiles from endothelial cells derived from stem cells of different sources.

PubMed

Reed, Daniel M; Foldes, Gabor; Kirkby, Nicholas S; Ahmetaj-Shala, Blerina; Mataragka, Stefania; Mohamed, Nura A; Francis, Catherine; Gara, Edit; Harding, Sian E; Mitchell, Jane A

2014-12-12

Endothelial cells form a highly specialised lining of all blood vessels where they provide an anti-thrombotic surface on the luminal side and protect the underlying vascular smooth muscle on the abluminal side. Specialised functions of endothelial cells include their unique ability to release vasoactive hormones and to morphologically adapt to complex shear stress. Stem cell derived-endothelial cells have a growing number of applications and will be critical in any organ regeneration programme. Generally endothelial cells are identified in stem cell studies by well-recognised markers such as CD31. However, the ability of stem cell-derived endothelial cells to release vasoactive hormones and align with shear stress has not been studied extensively. With this in mind, we have compared directly the ability of endothelial cells derived from a range of stem cell sources, including embryonic stem cells (hESC-EC) and adult progenitors in blood (blood out growth endothelial cells, BOEC) with those cultured from mature vessels, to release the vasoconstrictor peptide endothelin (ET)-1, the cardioprotective hormone prostacyclin, and to respond morphologically to conditions of complex shear stress. All endothelial cell types, except hESC-EC, released high and comparable levels of ET-1 and prostacyclin. Under static culture conditions all endothelial cell types, except for hESC-EC, had the typical cobblestone morphology whilst hESC-EC had an elongated phenotype. When cells were grown under shear stress endothelial cells from vessels (human aorta) or BOEC elongated and aligned in the direction of shear. By contrast hESC-EC did not align in the direction of shear stress. These observations show key differences in endothelial cells derived from embryonic stem cells versus those from blood progenitor cells, and that BOEC are more similar than hESC-EC to endothelial cells from vessels. This may be advantageous in some settings particularly where an in vitro test bed is required. However, for other applications, because of low ET-1 release hESC-EC may prove to be protected from vascular inflammation. Copyright © 2014 Elsevier Inc. All rights reserved.

NO Signaling in the Cardiovascular System and Exercise.

PubMed

Fernandes, Tiago; Gomes-Gatto, Camila V; Pereira, Noemy P; Alayafi, Yahya R; das Neves, Vander J; Oliveira, Edilamar M

2017-01-01

Nitric oxide (NO) is a small molecule implicated in multiple signal transduction pathways thus contributing to the regulation of many cellular functions. The identification of NO synthase (NOS) isoforms and the subsequent characterization of the mechanisms of cell activation of the enzymes permitted the partial understanding of both the physiological and pathological processes. NO bioavailability plays an important role in the pathophysiology of cardiovascular disease and its reduction in endothelial cells is strictly associated to endothelial dysfunction which, in turn, correlates with cardiovascular mortality. Indeed, endothelial NO synthase (eNOS) has a key role in limiting cardiac dysfunction and remodeling in heart diseases, in part by decreasing myocyte hypertrophy. Conversely, exercise training is recommended to prevent and treat cardiovascular diseases-associated disorders at least by enhanced NO synthase activity and expression, and increased production of antioxidants, which prevents premature breakdown of NO. Exercise training may cause an improvement in endothelial function for both experimental animals and humans; Studies in both healthy subjects and patients with impaired NO-related vasorelaxation remarked exercise training ability to improve vascular structure and function and endothelial homeostasis. This chapter will briefly consider the importance of NO signaling in the maintenance of cardiovascular physiology, and discuss recent insights into the effect of exercise training on the signaling pathways that modulate NO synthesis and degradation in health and cardiovascular disease. In addition, we will highlight the molecular mechanisms via which microRNAs (miRs) target NO signaling in the cardiovascular system, and NO as a candidate molecule for development of new therapies.

A branched-chain amino acid metabolite drives vascular fatty acid transport and causes insulin resistance.

PubMed

Jang, Cholsoon; Oh, Sungwhan F; Wada, Shogo; Rowe, Glenn C; Liu, Laura; Chan, Mun Chun; Rhee, James; Hoshino, Atsushi; Kim, Boa; Ibrahim, Ayon; Baca, Luisa G; Kim, Esl; Ghosh, Chandra C; Parikh, Samir M; Jiang, Aihua; Chu, Qingwei; Forman, Daniel E; Lecker, Stewart H; Krishnaiah, Saikumari; Rabinowitz, Joshua D; Weljie, Aalim M; Baur, Joseph A; Kasper, Dennis L; Arany, Zoltan

2016-04-01

Epidemiological and experimental data implicate branched-chain amino acids (BCAAs) in the development of insulin resistance, but the mechanisms that underlie this link remain unclear. Insulin resistance in skeletal muscle stems from the excess accumulation of lipid species, a process that requires blood-borne lipids to initially traverse the blood vessel wall. How this trans-endothelial transport occurs and how it is regulated are not well understood. Here we leveraged PPARGC1a (also known as PGC-1α; encoded by Ppargc1a), a transcriptional coactivator that regulates broad programs of fatty acid consumption, to identify 3-hydroxyisobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, as a new paracrine regulator of trans-endothelial fatty acid transport. We found that 3-HIB is secreted from muscle cells, activates endothelial fatty acid transport, stimulates muscle fatty acid uptake in vivo and promotes lipid accumulation in muscle, leading to insulin resistance in mice. Conversely, inhibiting the synthesis of 3-HIB in muscle cells blocks the ability of PGC-1α to promote endothelial fatty acid uptake. 3-HIB levels are elevated in muscle from db/db mice with diabetes and from human subjects with diabetes, as compared to those without diabetes. These data unveil a mechanism in which the metabolite 3-HIB, by regulating the trans-endothelial flux of fatty acids, links the regulation of fatty acid flux to BCAA catabolism, providing a mechanistic explanation for how increased BCAA catabolic flux can cause diabetes.

Biglycan potentially regulates angiogenesis during fracture repair by altering expression and function of endostatin

PubMed Central

Myren, Maja; Kirby, David J.; Noonan, Megan L.; Maeda, Azusa; Owens, Rick T.; Ricard-Blum, Sylvie; Kram, Vardit; Kilts, Tina M.; Young, Marian F.

2016-01-01

The small proteoglycan biglycan (Bgn) is highly expressed in the organic matrix of bone and plays a role in bone formation. Previous work implicated Bgn in vessel growth during bone healing (1). By infusing barium sulfate (BaSO4) into WT and Bgn-deficient mice we discovered the positive effect of Bgn in modulating angiogenesis during fracture healing. Using micro-computed tomography angiography we found significant differences in the vessel size and volume among other parameters. To further understand the mechanistic basis for this, we explored the relationship between Bgn and the anti-angiogenic protein endostatin. Immunohistochemistry (IHC) showed co-localization of Bgn and endostatin in regions of bone formation, with increased endostatin staining in Bgn-KO compared to WT at 14 days post-fracture. To further elucidate the relationship between Bgn and endostatin, an endothelial cell tube formation assay was used. This study showed that endothelial cells treated with endostatin had significantly decreased vessel length and vessel branches compared to untreated cells, while cells treated with endostatin and Bgn at a 1:1 molar ratio had vessel length and vessel branches comparable to untreated cells. This indicated that Bgn was able to mitigate the inhibitory effect of endostatin on endothelial cell growth. In summary, these results suggest that Bgn is needed for proper blood vessel formation during fracture healing, and one mechanism by which Bgn impacts angiogenesis is through inhibition of endostatin. PMID:27072616

Inhibition of c-Met reduces lymphatic metastasis in RIP-Tag2 transgenic mice

PubMed Central

Sennino, Barbara; Ishiguro-Oonuma, Toshina; Schriver, Brian J.; Christensen, James G.; McDonald, Donald M.

2013-01-01

Inhibition of vascular endothelial growth factor (VEGF) signaling can promote lymph node metastasis in preclinical models, but the mechanism is not fully understood, and successful methods of prevention have not been found. Signaling of hepatocyte growth factor (HGF) and its receptor c-Met can promote the growth of lymphatics and metastasis of some tumors. We sought to explore the contributions of c-Met signaling to lymph node metastasis after inhibition of VEGF signaling. In particular, we examined whether c-Met is upregulated in lymphatics in or near pancreatic neuroendocrine tumors in RIP-Tag2 transgenic mice and whether lymph node metastasis can be reduced by concurrent inhibition of VEGF and c-Met signaling. Inhibition of VEGF signaling by anti-VEGF antibody or sunitinib in mice from age 14 to 17 weeks was accompanied by more intratumoral lymphatics, more tumor cells inside lymphatics, and more lymph node metastases. Under these conditions, lymphatic endothelial cells - like tumor cells - had strong immunoreactivity for c-Met and phospho-c-Met. c-Met blockade by the selective inhibitor PF-04217903 significantly reduced metastasis to local lymph nodes. Together, these results indicate that inhibition of VEGF signaling in RIP-Tag2 mice upregulates c-Met expression in lymphatic endothelial cells, increases the number of intratumoral lymphatics and number of tumor cells within lymphatics, and promotes metastasis to local lymph nodes. Prevention of lymph node metastasis by PF-04217903 in this setting implicates c-Met signaling in tumor cell spread to lymph nodes. PMID:23576559

Molecular expression in transfected corneal endothelial cells

NASA Astrophysics Data System (ADS)

Wang, Fan; Miao, Zhuang; Lu, Chengwei; Hao, Jilong

2017-10-01

To investigate the capability of human corneal endothelial cells serving as immunological cells. Expression of HLA-DP, -DQ, -DR, CD40, CD80, and CD86 was determined by immunohistochemical methods. Meanwhile, purified peripheral blood mononuclear cells were cocultured with human corneal endothelial cells which were pre-treated with and without -IFN respectively, activation of lymphocytes was determined by FACS analysis. In coculture system, T lymphocyte was activated by corneal endothelial cells, HLA-DP, -DQ, -DR and CD40 expression were increased by - IFN induction. Costimulatory molecular CD80 was shown on the endothelial cells. Human corneal endothelial cells were assumed to be involved in the corneal transplantation rejection process as potential antigen presenting cells.

Endothelial Cell Implantation and Survival within Experimental Gliomas

NASA Astrophysics Data System (ADS)

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

1994-10-01

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

Endothelial cell regulation of leukocyte infiltration in inflammatory tissues

PubMed Central

Mantovani, A.; Introna, M.; Dejana, E.

1995-01-01

Endothelial cells play an important, active role in the onset and regulation of inflammatory and immune reactions. Through the production of chemokines they attract leukocytes and activate their adhesive receptors. This leads to the anchorage of leukocytes to the adhesive molecules expressed on the endothelial surface. Leukocyte adhesion to endothelial cells is frequently followed by their extravasation. The mechanisms which regulate the passage of leukocytes through endothelial clefts remain to be clarified. Many indirect data suggest that leukocytes might transfer signals to endothelial cells both through the release of active agents and adhesion to the endothelial cell surface. Adhesive molecules (such as PECAM) on the endothelial cell surface might also ‘direct’ leukocytes through the intercellular junction by haptotaxis. The information available on the molecular structure and functional properties of endothelial chemokines, adhesive molecules or junction organization is still fragmentary. Further work is needed to clarify how they interplay in regulating leukocyte infiltration into tissues. PMID:18475659

Geometric control of capillary architecture via cell-matrix mechanical interactions.

PubMed

Sun, Jian; Jamilpour, Nima; Wang, Fei-Yue; Wong, Pak Kin

2014-03-01

Capillary morphogenesis is a multistage, multicellular activity that plays a pivotal role in various developmental and pathological situations. In-depth understanding of the regulatory mechanism along with the capability of controlling the morphogenic process will have direct implications on tissue engineering and therapeutic angiogenesis. Extensive research has been devoted to elucidate the biochemical factors that regulate capillary morphogenesis. The roles of geometric confinement and cell-matrix mechanical interactions on the capillary architecture, nevertheless, remain largely unknown. Here, we show geometric control of endothelial network topology by creating physical confinements with microfabricated fences and wells. Decreasing the thickness of the matrix also results in comparable modulation of the network architecture, supporting the boundary effect is mediated mechanically. The regulatory role of cell-matrix mechanical interaction on the network topology is further supported by alternating the matrix stiffness by a cell-inert PEG-dextran hydrogel. Furthermore, reducing the cell traction force with a Rho-associated protein kinase inhibitor diminishes the boundary effect. Computational biomechanical analysis delineates the relationship between geometric confinement and cell-matrix mechanical interaction. Collectively, these results reveal a mechanoregulation scheme of endothelial cells to regulate the capillary network architecture via cell-matrix mechanical interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Platelets secrete stromal cell–derived factor 1α and recruit bone marrow–derived progenitor cells to arterial thrombi in vivo

PubMed Central

Massberg, Steffen; Konrad, Ildiko; Schürzinger, Katrin; Lorenz, Michael; Schneider, Simon; Zohlnhoefer, Dietlind; Hoppe, Katharina; Schiemann, Matthias; Kennerknecht, Elisabeth; Sauer, Susanne; Schulz, Christian; Kerstan, Sandra; Rudelius, Martina; Seidl, Stefan; Sorge, Falko; Langer, Harald; Peluso, Mario; Goyal, Pankaj; Vestweber, Dietmar; Emambokus, Nikla R.; Busch, Dirk H.; Frampton, Jon; Gawaz, Meinrad

2006-01-01

The accumulation of smooth muscle and endothelial cells is essential for remodeling and repair of injured blood vessel walls. Bone marrow–derived progenitor cells have been implicated in vascular repair and remodeling; however, the mechanisms underlying their recruitment to the site of injury remain elusive. Here, using real-time in vivo fluorescence microscopy, we show that platelets provide the critical signal that recruits CD34+ bone marrow cells and c-Kit+ Sca-1+ Lin− bone marrow–derived progenitor cells to sites of vascular injury. Correspondingly, specific inhibition of platelet adhesion virtually abrogated the accumulation of both CD34+ and c-Kit+ Sca-1+ Lin− bone marrow–derived progenitor cells at sites of endothelial disruption. Binding of bone marrow cells to platelets involves both P-selectin and GPIIb integrin on platelets. Unexpectedly, we found that activated platelets secrete the chemokine SDF-1α, thereby supporting further primary adhesion and migration of progenitor cells. These findings establish the platelet as a major player in the initiation of vascular remodeling, a process of fundamental importance for vascular repair and pathological remodeling after vascular injury. PMID:16618794

[The role of endothelial cells and endothelial precursor cells in angiogenesis].

PubMed

Poreba, Małgorzata; Usnarska-Zubkiewicz, Lidia; Kuliczkowski, Kazimierz

2006-01-01

Endothelium plays a key role in maintenance of vascular homeostasis in human organism. According to new data endothelial cells and hematopoietic cells have a common precursor in prenatal life--a hemangioblast, which explains the fact of sharing the same determinants on the surface of both type of cells. Circulating endothelial precursors were identified in adults and this suggests that hemangioblasts may be present not only during embriogenesis. In some clinical situations the increased numbers of endothelial cells and endothelial precursors were noted, and especially in patients with neoplastic diseases, which is probably the result of increased angiogenesis. Endothelial precursors are thought to be the promice for therapeutic purposes in future--to increase local angiogenesis.

Mesenchymal-endothelial-transition contributes to cardiac neovascularization

PubMed Central

Ubil, Eric; Duan, Jinzhu; Pillai, Indulekha C.L.; Rosa-Garrido, Manuel; Wu, Yong; Bargiacchi, Francesca; Lu, Yan; Stanbouly, Seta; Huang, Jie; Rojas, Mauricio; Vondriska, Thomas M.; Stefani, Enrico; Deb, Arjun

2014-01-01

Endothelial cells contribute to a subset of cardiac fibroblasts by undergoing endothelial-to-mesenchymal-transition, but whether cardiac fibroblasts can adopt an endothelial cell fate and directly contribute to neovascularization after cardiac injury is not known. Here, using genetic fate map techniques, we demonstrate that cardiac fibroblasts rapidly adopt an endothelial cell like phenotype after acute ischemic cardiac injury. Fibroblast derived endothelial cells exhibit anatomical and functional characteristics of native endothelial cells. We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate. Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast derived endothelial cells, reduces post infarct vascular density and worsens cardiac function. Conversely, stimulation of the p53 pathway in cardiac fibroblasts augments mesenchymal to endothelial transition, enhances vascularity and improves cardiac function. These observations demonstrate that mesenchymal-to-endothelial-transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair. PMID:25317562

Comparison of apoptosis in human primary pulmonary endothelial cells and a brain microvascular endothelial cell line co-cultured with Plasmodium falciparum field isolates.

PubMed

Essone, Jean Claude Biteghe Bi; N'Dilimabaka, Nadine; Ondzaga, Julien; Lekana-Douki, Jean Bernard; Mba, Dieudonné Nkoghe; Deloron, Philippe; Mazier, Dominique; Gay, Frédrérick; Touré Ndouo, Fousseyni S

2017-06-27

Plasmodium falciparum infection can progress unpredictably to severe forms including respiratory distress and cerebral malaria. The mechanisms underlying the variable natural course of malaria remain elusive. The cerebral microvascular endothelial cells-D3 and lung endothelial cells both from human were cultured separately and challenged with P. falciparum field isolates taken directly from malaria patients or 3D7 strain (in vitro maintained culture). The capacity of these P. falciparum isolates to induce endothelial cell apoptosis via cytoadherence or not was then assessed. Overall, 27 P. falciparum isolates were collected from patients with uncomplicated malaria (n = 25) or severe malaria (n = 2). About half the isolates (n = 17) were able to bind brain endothelial cells (12 isolates, 44%) or lung endothelial cells (17 isolates, 63%) or both (12 isolates, 44%). Sixteen (59%) of the 27 isolates were apoptogenic for brain and/or lung endothelial cells. The apoptosis stimulus could be cytoadherence, direct cell-cell contact without cytoadherence, or diffusible soluble factors. While some of the apoptogenic isolates used two stimuli (direct contact with or without cytoadherence, plus soluble factors) to induce apoptosis, others used only one. Among the 16 apoptogenic isolates, eight specifically targeted brain endothelial cells, one lung endothelial cells, and seven both. These results indicate that the brain microvascular cell line was more susceptible to apoptosis triggered by P. falciparum than the primary pulmonary endothelial cells and may have relevance to host-parasite interaction.

Cell-SELEX-Based Identification of a Human and Mouse Cross-Reactive Endothelial Cell-Internalizing Aptamer.

PubMed

Dua, Pooja; Kang, Sinae; Shin, Hye-Soo; Kim, Soyoun; Lee, Dong-Ki

2018-04-02

Increased interest and insights gained by researchers on the roles of endothelial cells in the pathophysiology of cancer, inflammatory, and cardiovascular diseases have led to the design of pharmacological interventions aimed at the endothelium lining in the diseased sites. Toward this end, we used established brain microvascular endothelial cell lines mouse (bEND3), human (hCMEC/D3), and Toggle Cell-SELEX to identify a species cross-reactive, endothelial cell-internalizing aptamer R11-3. This 2'F-modified RNA aptamer is specific for endothelial cells as no internalization was seen with cells of nonendothelial origin. R11-3 was truncated in size, and its potential in endothelial targeted therapeutics was established using VEGFR2 targeting long interfering RNA (liRNA) aptamer chimera. Due to its specificity for both mouse and human endothelial cells, we believe that this aptamer not only fits for development of endothelial targeted drug development for human diseases but is also suitable for preclinical evaluation in mice.

Hydrogen-Rich Medium Attenuated Lipopolysaccharide-Induced Monocyte-Endothelial Cell Adhesion and Vascular Endothelial Permeability via Rho-Associated Coiled-Coil Protein Kinase.

PubMed

Xie, Keliang; Wang, Weina; Chen, Hongguang; Han, Huanzhi; Liu, Daquan; Wang, Guolin; Yu, Yonghao

2015-07-01

Sepsis is the leading cause of death in critically ill patients. In recent years, molecular hydrogen, as an effective free radical scavenger, has been shown a selective antioxidant and anti-inflammatory effect, and it is beneficial in the treatment of sepsis. Rho-associated coiled-coil protein kinase (ROCK) participates in junction between normal cells, and regulates vascular endothelial permeability. In this study, we used lipopolysaccharide to stimulate vascular endothelial cells and explored the effects of hydrogen-rich medium on the regulation of adhesion of monocytes to endothelial cells and vascular endothelial permeability. We found that hydrogen-rich medium could inhibit adhesion of monocytes to endothelial cells and decrease levels of adhesion molecules, whereas the levels of transepithelial/endothelial electrical resistance values and the expression of vascular endothelial cadherin were increased after hydrogen-rich medium treatment. Moreover, hydrogen-rich medium could lessen the expression of ROCK, as a similar effect of its inhibitor Y-27632. In addition, hydrogen-rich medium could also inhibit adhesion of polymorphonuclear neutrophils to endothelial cells. In conclusion, hydrogen-rich medium could regulate adhesion of monocytes/polymorphonuclear neutrophils to endothelial cells and vascular endothelial permeability, and this effect might be related to the decreased expression of ROCK protein.

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. © 2014 International Federation for Cell Biology.

Connexin43high prostate cancer cells induce endothelial connexin43 up-regulation through the activation of intercellular ERK1/2-dependent signaling axis.

PubMed

Piwowarczyk, Katarzyna; Paw, Milena; Ryszawy, Damian; Rutkowska-Zapała, Magdalena; Madeja, Zbigniew; Siedlar, Maciej; Czyż, Jarosław

2017-06-01

Connexin(Cx)43 regulates the invasive potential of prostate cancer cells and participates in their extravasation. To address the role of endothelial Cx43 in this process, we analyzed Cx43 regulation in human umbilical vein endothelial cells in the proximity of Cx43 high (DU-145 and MAT-LyLu) and Cx43 low prostate cancer cells (PC-3 and AT-2). Endothelial Cx43 up-regulation was observed during the diapedesis of DU-145 and MAT-LyLu cells. This process was attenuated by transient Cx43 silencing in cancer cells and by chemical inhibition of ERK1/2-dependent signaling in endothelial cells. Cx43 expression in endothelial cells was insensitive to the inhibition of gap junctional intercellular coupling between Cx43 high prostate cancer and endothelial cells by 18α-glycyrrhetinic acid. Instead, endothelial Cx43 up-regulation was correlated with the local contraction of endothelial cells and with their activation in the proximity of Cx43 high DU-145 and MAT-LyLu cells. It was also sensitive to pro-inflammatory factors secreted by peripheral blood monocytes, such as TNFα. In contrast to Cx43 low AT-2 cells, Cx43 low PC-3 cells produced angioactive factors that locally activated the endothelial cells in the absence of endothelial Cx43 up-regulation. Collectively, these data show that Cx43 low and Cx43 high prostate cancer cells can adapt discrete, Cx43-independent and Cx43-dependent strategies of diapedesis. Our observations identify a novel strategy of prostate cancer cell diapedesis, which depends on the activation of intercellular Cx43/ERK1/2/Cx43 signaling axis at the interfaces between Cx43 high prostate cancer and endothelial cells. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Interleukin-34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment.

PubMed

Ségaliny, Aude I; Mohamadi, Amel; Dizier, Blandine; Lokajczyk, Anna; Brion, Régis; Lanel, Rachel; Amiaud, Jérôme; Charrier, Céline; Boisson-Vidal, Catherine; Heymann, Dominique

2015-07-01

Interleukin-34 (IL-34) was recently characterized as the M-CSF "twin" cytokine, regulating the proliferation/differentiation/survival of myeloid cells. The implication of M-CSF in oncology was initially suspected by the reduced metastatic dissemination in knock-out mice, due to angiogenesis impairment. Based on this observation, our work studied the involvement of IL-34 in the pathogenesis of osteosarcoma. The in vivo effects of IL-34 were assessed on tissue vasculature and macrophage infiltration in a murine preclinical model based on a paratibial inoculation of human osteosarcoma cells overexpressing or not IL-34 or M-CSF. In vitro investigations using endothelial cell precursors and mature HUVEC cells were performed to analyse the involvement of IL-34 in angiogenesis and myeloid cell adhesion. The data revealed that IL-34 overexpression was associated with the progression of osteosarcoma (tumor growth, lung metastases) and an increase of neo-angiogenesis. In vitro analyses demonstrated that IL-34 stimulated endothelial cell proliferation and vascular cord formation. Pre-treatment of endothelial cells by chondroitinases/heparinases reduced the formation of vascular tubes and abolished the associated cell signalling. In addition, IL-34 increased the in vivo recruitment of M2 tumor-associated macrophages into the tumor tissue. IL-34 increased in vitro monocyte/CD34(+) cell adhesion to activated HUVEC monolayers under physiological shear stress conditions. This work also demonstrates that IL-34 is expressed by osteosarcoma cells, is regulated by TNF-α, IL-1β, and contributes to osteosarcoma growth by increasing the neo-angiogenesis and the recruitment of M2 macrophages. By promoting new vessel formation and extravasation of immune cells, IL-34 may play a key role in tumor development and inflammatory diseases. © 2014 UICC.

West Nile virus-induced cell adhesion molecules on human brain microvascular endothelial cells regulate leukocyte adhesion and modulate permeability of the in vitro blood-brain barrier model.

PubMed

Roe, Kelsey; Orillo, Beverly; Verma, Saguna

2014-01-01

Characterizing the mechanisms by which West Nile virus (WNV) causes blood-brain barrier (BBB) disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs) in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE). Infection with WNV (NY99 strain) significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1) did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101) strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via 'Trojan horse' route, and improve WNV disease outcome.

The Volatile Anesthetic Isoflurane Increases Endothelial Adenosine Generation via Microparticle Ecto-5′-Nucleotidase (CD73) Release

PubMed Central

Kim, Mihwa; Ham, Ahrom; Kim, Katelyn Yu-Mi; Brown, Kevin M.; Lee, H. Thomas

2014-01-01

Endothelial dysfunction is common in acute and chronic organ injury. Isoflurane is a widely used halogenated volatile anesthetic during the perioperative period and protects against endothelial cell death and inflammation. In this study, we tested whether isoflurane induces endothelial ecto-5′-nucleotidase (CD73) and cytoprotective adenosine generation to protect against endothelial cell injury. Clinically relevant concentrations of isoflurane induced CD73 activity and increased adenosine generation in cultured human umbilical vein or mouse glomerular endothelial cells. Surprisingly, isoflurane-mediated induction of endothelial CD73 activity occurred within 1 hr and without synthesizing new CD73. We determined that isoflurane rapidly increased CD73 containing endothelial microparticles into the cell culture media. Indeed, microparticles isolated from isoflurane-treated endothelial cells had significantly higher CD73 activity as well as increased CD73 protein. In vivo, plasma from mice anesthetized with isoflurane had significantly higher endothelial cell-derived CD144+ CD73+ microparticles and had increased microparticle CD73 activity compared to plasma from pentobarbital-anesthetized mice. Supporting a critical role of CD73 in isoflurane-mediated endothelial protection, a selective CD73 inhibitor (APCP) prevented isoflurane-induced protection against human endothelial cell inflammation and apoptosis. In addition, isoflurane activated endothelial cells Rho kinase evidenced by myosin phosphatase target subunit-1 and myosin light chain phosphorylation. Furthermore, isoflurane-induced release of CD73 containing microparticles was significantly attenuated by a selective Rho kinase inhibitor (Y27632). Taken together, we conclude that the volatile anesthetic isoflurane causes Rho kinase-mediated release of endothelial microparticles containing preformed CD73 and increase adenosine generation to protect against endothelial apoptosis and inflammation. PMID:24945528

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.

microRNAs as Pharmacological Targets in Endothelial Cell Function and Dysfunction

PubMed Central

Chamorro-Jorganes, Aránzazu; Araldi, Elisa; Suárez, Yajaira

2013-01-01

Endothelial cell dysfunction is a term which implies the dysregulation of normal endothelial cell functions, including impairment of the barrier functions, control of vascular tone, disturbance of proliferative, migratory and morphogenic capacities of endothelial cells, as well as control of leukocyte trafficking. MicroRNAs (miRNAs) are short non-coding RNAs that have emerged as critical regulators of gene expression acting predominantly at the post-transcriptional level. This review summarizes the latest insights in the identification of endothelial-specific miRNAs and their targets, as well as their roles in controlling endothelial cell functions in both autocrine and paracrine manner. In addition, we discuss the therapeutic potential for the treatment of endothelial cell dysfunction and associated vascular pathophysiological conditions. PMID:23603154

Decellularized Human Kidney Cortex Hydrogels Enhance Kidney Microvascular Endothelial Cell Maturation and Quiescence.

PubMed

Nagao, Ryan J; Xu, Jin; Luo, Ping; Xue, Jun; Wang, Yi; Kotha, Surya; Zeng, Wen; Fu, Xiaoyun; Himmelfarb, Jonathan; Zheng, Ying

2016-10-01

The kidney peritubular microvasculature is highly susceptible to injury from drugs and toxins, often resulting in acute kidney injury and progressive chronic kidney disease. Little is known about the process of injury and regeneration of human kidney microvasculature, resulting from the lack of appropriate kidney microvascular models that can incorporate the proper cells, extracellular matrices (ECMs), and architectures needed to understand the response and contribution of individual vascular components in these processes. In this study, we present methods to recreate the human kidney ECM (kECM) microenvironment by fabricating kECM hydrogels derived from decellularized human kidney cortex. The majority of native matrix proteins, such as collagen-IV, laminin, and heparan sulfate proteoglycan, and their isoforms were preserved in similar proportions as found in normal kidneys. Human kidney peritubular microvascular endothelial cells (HKMECs) became more quiescent when cultured on this kECM gel compared with culture on collagen-I-assessed using phenotypic, genotypic, and functional assays; whereas human umbilical vein endothelial cells became stimulated on kECM gels. We demonstrate for the first time that human kidney cortex can form a hydrogel suitable for use in flow-directed microphysiological systems. Our findings strongly suggest that selecting the proper ECM is a critical consideration in the development of vascularized organs on a chip and carries important implications for tissue engineering of all vascularized organs.

On the Normal Force Mechanotransduction of Human Umbilical Vein Endothelial Cells

NASA Astrophysics Data System (ADS)

Vahabikashi, Amir; Wang, Qiuyun; Wilson, James; Wu, Qianhong; Vucbmss Team

2016-11-01

In this paper, we report a cellular biomechanics study to examine the normal force mechanotransduction of Human Umbilical Vein Endothelial Cells (HUVECs) with their implications on hypertension. Endothelial cells sense mechanical forces and adjust their structure and function accordingly. The mechanotransduction of normal forces plays a vital role in hypertension due to the higher pressure buildup inside blood vessels. Herein, HUVECs were cultured to full confluency and then exposed to different mechanical loadings using a novel microfluidic flow chamber. One various pressure levels while keeps the shear stress constant inside the flow chamber. Three groups of cells were examined, the control group (neither shear nor normal stresses), the normal pressure group (10 dyne/cm2 of shear stress and 95 mmHg of pressure), and the hypertensive group (10 dyne/cm2 of shear stress and 142 mmHg of pressure). Cellular response characterized by RT-PCR method indicates that, COX-2 expressed under normal pressure but not high pressure; Mn-SOD expressed under both normal and high pressure while this response was stronger for normal pressure; FOS and e-NOS did not respond under any condition. The differential behavior of COX-2 and Mn-SOD in response to changes in pressure, is instrumental for better understanding the pathogenesis of hypertensive cardiovascular diseases. This research was supported by the National Science Foundation under Award #1511096.

Everolimus Inhibits Anti-HLA I Antibody-Mediated Endothelial Cell Signaling, Migration and Proliferation More Potently than Sirolimus

PubMed Central

Jin, Yi-Ping; Valenzuela, Nicole M.; Ziegler, Mary E.; Rozengurt, Enrique; Reed, Elaine F.

2017-01-01

Antibody (Ab) crosslinking of HLA I molecules on the surface of endothelial cells triggers proliferative and pro-survival intracellular signaling, which is implicated in the process of chronic allograft rejection, also known as transplant vasculopathy. The purpose of this study was to investigate the role of mammalian target of rapamycin (mTOR) in HLA I antibody-induced signaling cascades. Everolimus provides a tool to establish how the mTOR signal network regulates HLA I-mediated migration, proliferation, and survival. We found that everolimus inhibits mTORC1 by disassociating Raptor from mTOR, thereby preventing class I-induced phosphorylation of mTOR, p70S6K, S6RP, and 4E-BP1, and resultant class I-stimulated cell migration and proliferation. Furthermore, we found that everolimus inhibits class I-mediated mTORC2 activation (1) by disassociating Rictor and Sin1 from mTOR; (2) by preventing class I-stimulated Akt phosphorylation; and (3) by preventing class I-mediated ERK phosphorylation. These results suggest that everolimus is more effective than sirolimus at antagonizing both mTORC1 and mTORC2, the latter of which is critical in endothelial cell functional changes leading to transplant vasculopathy in solid organ transplantation after HLA I crosslinking. Our findings point to a potential therapeutic effect of everolimus in prevention of chronic antibody-mediated rejection. PMID:24580843

Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

PubMed

Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

2001-04-01

The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

Multi-ligand nanoparticles for targeted drug delivery to the injured vascular wall

NASA Astrophysics Data System (ADS)

Kona, Soujanya

Pathological conditions like coronary artery disease, acute myocardial infarction, stroke, and peripheral artery diseases as well as cardiovascular interventions used in the treatment of coronary artery diseases such as angioplasty and stenting damage/injure the blood vessel wall, leading to inflamed or activated endothelial cells that have been implicated in events leading to thrombosis, inflammation, and restenosis. Oral administration of anti-coagulant and anti-inflammatory drugs causes systemic toxicity, bleeding, patient incompliance, and inadequate amounts of drugs at the injured area. Though drug-eluting stents have shown therapeutic benefits, complications such as in-stent restenosis and late thrombosis still remain and are a cause for concern. Rapid growth in the field of nanotechnology and nanoscience in recent years has paved the way for new targeted and controlled drug delivery strategies. In this perspective, the development of biodegradable nanoparticles for targeted intracellular drug delivery to the inflamed endothelial cells may offer an improved avenue for treatment of cardiovascular diseases. The major objective of this research was to develop "novel multi-ligand nanoparticles," as drug carriers that can efficiently target and deliver therapeutic agents to the injured/inflamed vascular cells under dynamic flow conditions. Our approach mimics the natural binding ability of platelets to injured/activated endothelial cells through glycoprotein Ib (GPIb) bound to P-selectin expressed on inflamed endothelial cells and to the subendothelium through GPIb binding to von Willebrand factor (vWF) deposited onto the injured vascular wall. Our design also exploits the natural cell membrane translocation ability of the internalizing cell peptide - trans-activating transcriptor (TAT) to enhance the nanoparticle uptake by the targeted cells. Our hypothesis is that these multi-ligand nanoparticles would show an increased accumulation at the injury site since GPIb specially binds to both P-selectin expressed on damaged endothelial cells and vWF deposited on injured subendothelium while the cell penetrating peptide -- TAT would facilitate enhanced uptake of these nanoparticles by the damaged vascular cells. To test this hypothesis, fluorescent drug loaded poly (D, L-lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG) nanoparticles (PLGA-PEG NPs) were formulated using a standard double emulsion method. We further conjugated GPIb and TAT via carbodiimide and avidin-biotin chemistry to the PLGA-PEG nanoparticles. Characterization of these nanoparticles indicated the average size to be about 200nm. Endothelial cell uptake studies indicated an optimal nanoparticle incubation time of one hour and optimal dose of 400 mug/ml. Biocompatibility results showed these particles to be non-toxic to endothelial cells. Moreover, dexamethasone release profiles from the nanoparticles demonstrated their ability to provide a sustained drug release over four weeks. Static and dynamic uptake studies of control, GPIb-conjugated, and GPIb-TAT-conjugated PLGA-PEG nanoparticles on activated endothelial cells exhibited an increased adhesion and uptake of GPIb-TAT conjugated PLGA-PEG nanoparticles compared to control nanoparticles. A similar trend of significantly higher adhesion of GPIb-TAT conjugated PLGA-PEG nanoparticles to the injured vessel wall was also observed in preliminary ex-vivo studies using the rat carotid injury model. These results suggest that "our novel multi-ligand NPs" would provide a unique active targeting strategy. This system would rapidly target and deliver therapeutic agents to the injured vascular wall under flow conditions. It could also serve as an effective therapeutic delivery system to treat the complications associated with cardiovascular diseases.

Formation of a PKCζ/β-catenin complex in endothelial cells promotes angiopoietin-1–induced collective directional migration and angiogenic sprouting

PubMed Central

Oubaha, Malika; Lin, Michelle I.; Margaron, Yoran; Filion, Dominic; Price, Emily N.; Zon, Leonard I.; Côté, Jean-François

2012-01-01

Angiogenic sprouting requires that cell-cell contacts be maintained during migration of endothelial cells. Angiopoietin-1 (Ang-1) and vascular endothelial growth factor act oppositely on endothelial cell junctions. We found that Ang-1 promotes collective and directional migration and, in contrast to VEGF, induces the formation of a complex formed of atypical protein kinase C (PKC)-ζ and β-catenin at cell-cell junctions and at the leading edge of migrating endothelial cells. This complex brings Par3, Par6, and adherens junction proteins at the front of migrating cells to locally activate Rac1 in response to Ang-1. The colocalization of PKCζ and β-catenin at leading edge along with PKCζ-dependent stabilization of cell-cell contacts promotes directed and collective endothelial cell migration. Consistent with these results, down-regulation of PKCζ in endothelial cells alters Ang-1–induced sprouting in vitro and knockdown in developing zebrafish results in intersegmental vessel defects caused by a perturbed directionality of tip cells and by loss of cell contacts between tip and stalk cells. These results reveal that PKCζ and β-catenin function in a complex at adherens junctions and at the leading edge of migrating endothelial cells to modulate collective and directional migration during angiogenesis. PMID:22936663

NUP160-SLC43A3 is a novel recurrent fusion oncogene in angiosarcoma.

PubMed

Shimozono, Naoki; Jinnin, Masatoshi; Masuzawa, Mamiko; Masuzawa, Mikio; Wang, Zhongzhi; Hirano, Ayaka; Tomizawa, Yukiko; Etoh-Kira, Tomomi; Kajihara, Ikko; Harada, Miho; Fukushima, Satoshi; Ihn, Hironobu

2015-11-01

Angiosarcoma is a malignant vascular tumor originating from endothelial cells of blood vessels or lymphatic vessels. The specific driver mutations in angiosarcoma remain unknown. In this study, we investigated this issue by transcriptome sequencing of patient-derived angiosarcoma cells (ISO-HAS), identifying a novel fusion gene NUP160-SLC43A3 found to be expressed in 9 of 25 human angiosarcoma specimens that were examined. In tumors harboring the fusion gene, the duration between the onset of symptoms and the first hospital visit was significantly shorter, suggesting more rapid tumor progression. Stable expression of the fusion gene in nontransformed human dermal microvascular endothelial cells elicited a gene-expression pattern mimicking ISO-HAS cells and increased cell proliferation, an effect traced in part to NUP160 truncation. Conversely, RNAi-mediated attenuation of NUP160 in ISO-HAS cells decreased cell number. Confirming the oncogenic effects of the fusion protein, subcutaneous implantation of NUP160-SLC43A3-expressing fibroblasts induced tumors resembling human angiosarcoma. Collectively, our findings advance knowledge concerning the genetic causes of angiosarcoma, with potential implications for new diagnostic and therapeutic approaches. ©2015 American Association for Cancer Research.

Effect of microbubble ligation to cells on ultrasound signal enhancement: implications for targeted imaging.

PubMed

Lankford, Miles; Behm, Carolyn Z; Yeh, James; Klibanov, Alexander L; Robinson, Peter; Lindner, Jonathan R

2006-10-01

Molecular imaging with contrast-enhanced ultrasound (CEU) relies on the detection of microbubbles retained in regions of disease. The aim of this study was to determine whether microbubble attachment to cells influences their acoustic signal generation and stability. Biotinylated microbubbles were attached to streptavidin-coated plates to derive density versus intensity relations during low- and high-power imaging. To assess damping from microbubble attachment to solid or cell surfaces, in vitro imaging was performed for microbubbles charge-coupled to methacrylate spheres and for vascular cell adhesion molecule-1-targeted microbubbles attached to endothelial cells. Signal enhancement on plates increased according to acoustic power and microbubble site density up to 300 mm. Microbubble signal was reduced by attachment to solid spheres during high- and low-power imaging but was minimally reduced by attachment to endothelial cells and only at low power. Attachment of targeted microbubbles to rigid surfaces results in damping and a reduction of their acoustic signal, which is not seen when microbubbles are attached to cells. A reliable concentration versus intensity relationship can be expected from microbubble attachment to 2-dimensional surfaces until a very high site density is reached.

Inhibition of angiogenesis in vitro by Arg-Gly-Asp-containing synthetic peptide.

PubMed Central

Nicosia, R. F.; Bonanno, E.

1991-01-01

This study was designed to evaluate the effect of the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) on angiogenesis in serum-free collagen gel culture of rat aorta. The GRGDS peptide contains the amino acid sequence Arg-Gly-Asp (RGD), which has been implicated as a recognition site in interactions between extracellular matrix (ECM) molecules and cell membrane receptors. RGD-containing synthetic peptides are known to inhibit attachment of endothelial cells to substrates, but their effect on angiogenesis has not been fully characterized. Aortic explants embedded in collagen gel in the absence of GRGDS generated branching microvessels through a process of endothelial migration and proliferation. Addition of GRGDS to the culture medium caused a marked inhibition of angiogenesis. In contrast, GRGES, a control peptide lacking the RGD sequence, failed to inhibit angiogenesis. The inhibitory effect of GRGDS was nontoxic and reversible. The angiogenic activity of aortic explants previously inhibited with GRGDS could be restored by incubating the cultures in GRGDS-free medium. These findings suggest that angiogenesis is an anchorage-dependent process that can be inhibited by interfering with the attachment of endothelial cells to the ECM. It also indicates that synthetic peptides can be used as probes to study the mechanisms by which the ECM regulates angiogenesis. Images Figure 1 PMID:1707235

Angiogenic dysfunction in bone marrow-derived early outgrowth cells from diabetic animals is attenuated by SIRT1 activation.

PubMed

Yuen, Darren A; Zhang, Yanling; Thai, Kerri; Spring, Christopher; Chan, Lauren; Guo, Xiaoxin; Advani, Andrew; Sivak, Jeremy M; Gilbert, Richard E

2012-12-01

Impaired endothelial repair is a key contributor to microvascular rarefaction and consequent end-organ dysfunction in diabetes. Recent studies suggest an important role for bone marrow-derived early outgrowth cells (EOCs) in mediating endothelial repair, but the function of these cells is impaired in diabetes, as in advanced age. We sought to determine whether diabetes-associated EOC dysfunction might be attenuated by pharmacological activation of silent information regulator protein 1 (SIRT1), a lysine deacetylase implicated in nutrient-dependent life span extension in mammals. Despite being cultured in normal (5.5 mM) glucose for 7 days, EOCs from diabetic rats expressed less SIRT1 mRNA, induced less endothelial tube formation in vitro and neovascularization in vivo, and secreted less of the proangiogenic ELR(+) CXC chemokines CXCL1, CXCL3, and CXCL5. Ex vivo SIRT1 activation restored EOC chemokine secretion and increased the in vitro and in vivo angiogenic activity of EOC conditioned medium derived from diabetic animals to levels similar to that derived from control animals. These findings suggest a pivotal role for SIRT1 in diabetes-induced EOC dysfunction and that its pharmacologic activation may provide a new strategy for the restoration of EOC-mediated repair mechanisms.

[Blood-brain barrier part III: therapeutic approaches to cross the blood-brain barrier and target the brain].

PubMed

Weiss, N; Miller, F; Cazaubon, S; Couraud, P-O

2010-03-01

Over the last few years, the blood-brain barrier has come to be considered as the main limitation for the treatment of neurological diseases caused by inflammatory, tumor or neurodegenerative disorders. In the blood-brain barrier, the close intercellular contact between cerebral endothelial cells due to tight junctions prevents the passive diffusion of hydrophilic components from the bloodstream into the brain. Several specific transport systems (via transporters expressed on cerebral endothelial cells) are implicated in the delivery of nutriments, ions and vitamins to the brain; other transporters expressed on cerebral endothelial cells extrude endogenous substances or xenobiotics, which have crossed the cerebral endothelium, out of the brain and into the bloodstream. Recently, several strategies have been proposed to target the brain, (i) by by-passing the blood-brain barrier by central drug administration, (ii) by increasing permeability of the blood-brain barrier, (iii) by modulating the expression and/or the activity of efflux transporters, (iv) by using the physiological receptor-dependent blood-brain barrier transport, and (v) by creating new viral or chemical vectors to cross the blood-brain barrier. This review focuses on the illustration of these different approaches. Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.

The Role of PGC-1α in Vascular Regulation: Implications for Atherosclerosis

PubMed Central

Kadlec, Andrew O.; Chabowski, Dawid S.; Ait-Aissa, Karima; Gutterman, David D.

2016-01-01

Mitochondrial dysfunction results in high levels of oxidative stress and mitochondrial damage, leading to disruption of endothelial homeostasis. Recent discoveries have clarified several pathways whereby mitochondrial dysregulation contributes to endothelial dysfunction and vascular disease burden. One such pathway centers around PGC-1α, a transcriptional coactivator linked to mitochondrial biogenesis and antioxidant defense, among other functions. Although primarily investigated for its therapeutic potential in obesity and skeletal muscle differentiation, the ability of PGC-1α to alter a multitude of cellular functions has sparked interest in its role in the vasculature. Within this context, recent studies demonstrate that PGC-1α plays a key role in endothelial cell and smooth muscle cell regulation through effects on oxidative stress, apoptosis, inflammation, and cell proliferation. The ability of PGC-1α to impact these parameters is relevant to vascular disease progression, particularly in relation to atherosclerosis. Upregulation of PGC-1α can prevent the development of, and even encourage regression of, atherosclerotic lesions. Therefore, PGC-1α is poised to serve as a promising target in vascular disease. This review details recent findings related to PGC-1α in vascular regulation, regulation of PGC-1α itself, the role of PGC-1α in atherosclerosis, and therapies that target this key protein. PMID:27312223

Endothelial chimerism and vascular sequestration protect pancreatic islet grafts from antibody-mediated rejection

PubMed Central

Chen, Chien-Chia; Pouliquen, Eric; Broisat, Alexis; Andreata, Francesco; Racapé, Maud; Bruneval, Patrick; Kessler, Laurence; Ahmadi, Mitra; Bacot, Sandrine; Saison-Delaplace, Carole; Marcaud, Marina; Van Huyen, Jean-Paul Duong; Loupy, Alexandre; Villard, Jean; Demuylder-Mischler, Sandrine; Morelon, Emmanuel; Tsai, Meng-Kun; Kolopp-Sarda, Marie-Nathalie; Koenig, Alice; Mathias, Virginie; Ghezzi, Catherine; Dubois, Valerie; Defrance, Thierry

2017-01-01

Humoral rejection is the most common cause of solid organ transplant failure. Here, we evaluated a cohort of 49 patients who were successfully grafted with allogenic islets and determined that the appearance of donor-specific anti-HLA antibodies (DSAs) did not accelerate the rate of islet graft attrition, suggesting resistance to humoral rejection. Murine DSAs bound to allogeneic targets expressed by islet cells and induced their destruction in vitro; however, passive transfer of the same DSAs did not affect islet graft survival in murine models. Live imaging revealed that DSAs were sequestrated in the circulation of the recipients and failed to reach the endocrine cells of grafted islets. We used murine heart transplantation models to confirm that endothelial cells were the only accessible targets for DSAs, which induced the development of typical microvascular lesions in allogeneic transplants. In contrast, the vasculature of DSA-exposed allogeneic islet grafts was devoid of lesions because sprouting of recipient capillaries reestablished blood flow in grafted islets. Thus, we conclude that endothelial chimerism combined with vascular sequestration of DSAs protects islet grafts from humoral rejection. The reduced immunoglobulin concentrations in the interstitial tissue, confirmed in patients, may have important implications for biotherapies such as vaccines and monoclonal antibodies. PMID:29202467

Responses of brain and non-brain endothelial cells to meningitis-causing Escherichia coli K1.

PubMed

Paul-Satyaseela, Maneesh; Xie, Yi; Di Cello, Francescopaolo; Kim, Kwang Sik

2006-03-31

Bacterial interaction with specific host tissue may contribute to its propensity to cause an infection in a particular site. In this study, we examined whether meningitis-causing Escherichia coli K1 interaction with human brain microvascular endothelial cells, which constitute the blood-brain barrier, differed from its interaction with non-brain endothelial cells derived from skin and umbilical cord. We showed that E. coli K1 association was significantly greater with human brain microvascular endothelial cells than with non-brain endothelial cells. In addition, human brain microvascular endothelial cells maintained their morphology and intercellular junctional resistance in response to E. coli K1. In contrast, non-brain endothelial cells exhibited decreased transendothelial electrical resistance and detachment from the matrix upon exposure to E. coli K1. These different responses of brain and non-brain endothelial cells to E. coli K1 may form the basis of E. coli K1's propensity to cause meningitis.

The Methods and Mechanisms to Differentiate Endothelial-Like Cells and Smooth Muscle Cells from Mesenchymal Stem Cells for Vascularization in Vaginal Reconstruction.

PubMed

Zhang, Hua; Zhang, Jingkun; Huang, Xianghua; Li, Yanan

2018-06-01

Endothelial cells and smooth muscle cells (SMCs) are important aspects of vascularization in vaginal reconstruction. Research has confirmed that mesenchymal stem cells could differentiate into endothelial-like cells and SMCs. But the methods were more complicated and the mechanism was unknown. In the current study, we induced the bone mesenchymal stem cells (BMSCs) to differentiate into endothelial-like cells and SMCs in vitro by differentiation medium and investigated the effect of Wnt/β-catenin signaling on the differentiation process of BMSCs. Results showed that the hypoxic environment combined with VEGF and bFGF could induce increased expression of endothelial-like cells markers VEGFR1, VEGFR2, and vWF. The SMCs derived from BMSCs induced by TGF-β1 and PDGF-AB significantly expressed SMC markers SMMHC11 and α-SMA. The data also showed that activation of Wnt/β-catenin signaling could promote the differentiation of BMSCs into endothelial-like cells and SMCs. Thus, we established endothelial-like cells and SMCs in vitro by more simple methods, presented the important role of hypoxic environment on the differentiation of BMSCs into endothelial-like cells, and confirmed that the Wnt/β-catenin signaling pathway has a positive impact on the differentiation of BMSCs into endothelial-like cells and SMCs. This is important for vascular reconstruction.

Endothelial nitric oxide synthase is dynamically expressed during bone marrow stem cell differentiation into endothelial cells.

PubMed

Liu, Zhenguo; Jiang, Yuehua; Hao, Hong; Gupta, Kalpna; Xu, Jian; Chu, Ling; McFalls, Edward; Zweier, Jay; Verfaillie, Catherine; Bache, Robert J

2007-09-01

This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells and to examine the functional status of the newly differentiated endothelial cells. Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem cells and were induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, nitric oxide synthase (NOS) activity, and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression reoccurred at day 7 during differentiation. Increasing eNOS mRNA, protein content, and activity were observed in the cells at days 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor-reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 occurred in these cells, which was inhibited by NOS inhibitor N(G)-nitro-L-arginine methyl ester. In conclusion, these data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro.

In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining.

PubMed

Zilla, P; Fasol, R; Dudeck, U; Siedler, S; Preiss, P; Fischlein, T; Müller-Glauser, W; Baitella, G; Sanan, D; Odell, J

1990-08-01

A rapid and reliable harvest and culture technique was developed to provide a sufficient number of autologous endothelial cells for the confluent in vitro lining of cardiovascular prostheses. Enzymatic endothelial cell detachment was achieved by the in situ application of collagenase to short vessel segments. This harvest technique resulted in a complete lack of contaminating smooth muscle cells in all of 124 cultures from nonhuman primates and 13 cultures from human adults. The use of a microgrid technique enabled the daily in situ quantification of available endothelial cells. To assess ideal plating densities after passage the population doubling time was continuously related to the cell density. Surprisingly, a low plating density of 1.5 X 10(3) endothelial cells/cm2 achieved 43% shorter cell cycles than the usual plating density of 1.0 X 10(4) endothelial cells/cm2. Moreover, low density plating enabled mass cultures after one single cell passage, thereby reducing the cell damaging effect of trypsin. When the growth characteristics of endothelial cells from five anatomically different vessel sites were compared, the external jugular vein--which would be easily accessible and dispensable in each patient--proved to be an excellent source for endothelial cell cultures. By applying in situ administration of collagenase, low density plating and microgrid follow-up to adult human saphenous vein endothelial cells, 14,000,000 first passage endothelial cells--sufficient for the in vitro lining of long vascular prostheses--were obtained 26.2 days after harvest. (95% confidence interval:22.3 to 32.2 days).

Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.

PubMed

Parker, William H; Qu, Zhi-chao; May, James M

2015-08-28

Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Endothelial Glycocalyx as a Shield Against Diabetic Vascular Complications: Involvement of Hyaluronan and Hyaluronidases.

PubMed

Dogné, Sophie; Flamion, Bruno; Caron, Nathalie

2018-07-01

The endothelial glycocalyx (EG), which covers the apical surface of the endothelial cells and floats into the lumen of the vessels, is a key player in vascular integrity and cardiovascular homeostasis. The EG is composed of PGs (proteoglycans), glycoproteins, glycolipids, and glycosaminoglycans, in particular hyaluronan (HA). HA seems to be implicated in most of the functions described for EG such as creating a space between blood and the endothelium, controlling vessel permeability, restricting leukocyte and platelet adhesion, and allowing an appropriate endothelial response to flow variation through mechanosensing. The amount of HA in the EG may be regulated by HYAL (hyaluronidase) 1, the most active somatic hyaluronidase. HYAL1 seems enriched in endothelial cells through endocytosis from the bloodstream. The role of the other main somatic hyaluronidase, HYAL2, in the EG is uncertain. Damage to the EG, accompanied by shedding of one or more of its components, is an early sign of various pathologies including diabetes mellitus. Shedding increases the blood or plasma concentration of several EG components, such as HA, heparan sulfate, and syndecan. The plasma levels of these molecules can then be used as sensitive markers of EG degradation. This has been shown in type 1 and type 2 diabetic patients. Recent experimental studies suggest that preserving the size and amount of EG HA in the face of diabetic insults could be a useful novel therapeutic strategy to slow diabetic complications. One way to achieve this goal, as suggested by a murine model of HYAL1 deficiency, may be to inhibit the function of HYAL1. The same approach may succeed in other pathological situations involving endothelial dysfunction and EG damage. © 2018 American Heart Association, Inc.

Reduced Ang2 expression in aging endothelial cells.

PubMed

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

2016-06-03

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. Copyright © 2016 Elsevier Inc. All rights reserved.

Neuropilins: expression and roles in the epithelium

PubMed Central

Wild, Jonathan R L; Staton, Carolyn A; Chapple, Keith; Corfe, Bernard M

2012-01-01

Summary Initially found expressed in neuronal and then later in endothelial cells, it is well established that the transmembrane glycoproteins neuropilin-1 (NRP1) and neuropilin-2 (NRP2) play essential roles in axonal growth and guidance and in physiological and pathological angiogenesis. Neuropilin expression and function in epithelial cells has received little attention when compared with neuronal and endothelial cells. Overexpression of NRPs is shown to enhance growth, correlate with invasion and is associated with poor prognosis in various tumour types, especially those of epithelial origin. The contribution of NRP and its ligands to tumour growth and metastasis has spurred a strong interest in NRPs as novel chemotherapy drug targets. Given NRP’s role as a multifunctional co-receptor with an ability to bind with disparate ligand families, this has sparked new areas of research implicating NRPs in diverse biological functions. Here, we review the growing body of research demonstrating NRP expression and role in the normal and neoplastic epithelium. PMID:22414290

Mitochondrial complex I deactivation is related to superoxide production in acute hypoxia.

PubMed

Hernansanz-Agustín, Pablo; Ramos, Elena; Navarro, Elisa; Parada, Esther; Sánchez-López, Nuria; Peláez-Aguado, Laura; Cabrera-García, J Daniel; Tello, Daniel; Buendia, Izaskun; Marina, Anabel; Egea, Javier; López, Manuela G; Bogdanova, Anna; Martínez-Ruiz, Antonio

2017-08-01

Mitochondria use oxygen as the final acceptor of the respiratory chain, but its incomplete reduction can also produce reactive oxygen species (ROS), especially superoxide. Acute hypoxia produces a superoxide burst in different cell types, but the triggering mechanism is still unknown. Herein, we show that complex I is involved in this superoxide burst under acute hypoxia in endothelial cells. We have also studied the possible mechanisms by which complex I could be involved in this burst, discarding reverse electron transport in complex I and the implication of PTEN-induced putative kinase 1 (PINK1). We show that complex I transition from the active to 'deactive' form is enhanced by acute hypoxia in endothelial cells and brain tissue, and we suggest that it can trigger ROS production through its Na + /H + antiporter activity. These results highlight the role of complex I as a key actor in redox signalling in acute hypoxia. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

cAMP signalling in the vasculature: the role of Epac (exchange protein directly activated by cAMP).

PubMed

Roberts, Owain Llŷr; Dart, Caroline

2014-02-01

The second messenger cAMP plays a central role in mediating vascular smooth muscle relaxation in response to vasoactive transmitters and in strengthening endothelial cell-cell junctions that regulate the movement of solutes, cells and macromolecules between the blood and the surrounding tissue. The vasculature expresses three cAMP effector proteins: PKA (protein kinase A), CNG (cyclic-nucleotide-gated) ion channels, and the most recently discovered Epacs (exchange proteins directly activated by cAMP). Epacs are a family of GEFs (guanine-nucleotide-exchange factors) for the small Ras-related GTPases Rap1 and Rap2, and are being increasingly implicated as important mediators of cAMP signalling, both in their own right and in parallel with the prototypical cAMP target PKA. In the present paper, we review what is currently known about the role of Epac within blood vessels, particularly with regard to the regulation of vascular tone, endothelial barrier function and inflammation.

Chlamydia pneumoniae and oxidative stress in cardiovascular disease: state of the art and prevention strategies.

PubMed

Di Pietro, Marisa; Filardo, Simone; De Santis, Fiorenzo; Mastromarino, Paola; Sessa, Rosa

2014-12-30

Chlamydia pneumoniae, a pathogenic bacteria responsible for respiratory tract infections, is known as the most implicated infectious agent in atherosclerotic cardiovascular diseases (CVDs). Accumulating evidence suggests that C. pneumoniae-induced oxidative stress may play a critical role in the pathogenesis of CVDs. Indeed, the overproduction of reactive oxygen species (ROS) within macrophages, endothelial cells, platelets and vascular smooth muscle cells (VSMCs) after C. pneumoniae exposure, has been shown to cause low density lipoprotein oxidation, foam cell formation, endothelial dysfunction, platelet adhesion and aggregation, and VSMC proliferation and migration, all responsible for the typical pathological changes of atherosclerotic plaque. The aim of this review is to improve our insight into C. pneumoniae-induced oxidative stress in order to suggest potential strategies for CVD prevention. Several antioxidants, acting on multi-enzymatic targets related to ROS production induced by C. pneumoniae, have been discussed. A future strategy for the prevention of C. pneumoniae-associated CVDs will be to target chlamydial HSP60, involved in oxidative stress.

Chlamydia pneumoniae and Oxidative Stress in Cardiovascular Disease: State of the Art and Prevention Strategies

PubMed Central

Di Pietro, Marisa; Filardo, Simone; De Santis, Fiorenzo; Mastromarino, Paola; Sessa, Rosa

2014-01-01

Chlamydia pneumoniae, a pathogenic bacteria responsible for respiratory tract infections, is known as the most implicated infectious agent in atherosclerotic cardiovascular diseases (CVDs). Accumulating evidence suggests that C. pneumoniae-induced oxidative stress may play a critical role in the pathogenesis of CVDs. Indeed, the overproduction of reactive oxygen species (ROS) within macrophages, endothelial cells, platelets and vascular smooth muscle cells (VSMCs) after C. pneumoniae exposure, has been shown to cause low density lipoprotein oxidation, foam cell formation, endothelial dysfunction, platelet adhesion and aggregation, and VSMC proliferation and migration, all responsible for the typical pathological changes of atherosclerotic plaque. The aim of this review is to improve our insight into C. pneumoniae-induced oxidative stress in order to suggest potential strategies for CVD prevention. Several antioxidants, acting on multi-enzymatic targets related to ROS production induced by C. pneumoniae, have been discussed. A future strategy for the prevention of C. pneumoniae-associated CVDs will be to target chlamydial HSP60, involved in oxidative stress. PMID:25561227

Antitumour and antiangiogenic activities of [Pt(O,O′‐acac)(γ‐acac)(DMS)] in a xenograft model of human renal cell carcinoma

PubMed Central

Vetrugno, C; Biagioni, F; Calabriso, N; Calierno, M T; Fornai, F; De Pascali, S A; Marsigliante, S; Fanizzi, F P

2016-01-01

Background and Purpose It is thought that the mechanism of action of anticancer chemotherapeutic agents is mainly due to a direct inhibition of tumour cell proliferation. In tumour specimens, the endothelial cell proliferation rate increases, suggesting that the therapeutic effects of anticancer agents could also be attributed to inhibition of tumour angiogenesis. Hence, we investigated the potential effects of [Pt(O,O′‐acac)(γ‐acac)(DMS)] ([Pt(DMS)]), a new platinum drug for non‐genomic targets, on human renal carcinoma and compared them with those of the well‐established anticancer drug, cisplatin. Experimental Approach Tumour growth, tumour cell proliferation and microvessel density were investigated in a xenograft model of renal cell carcinoma, developed by injecting Caki‐1 cells into BALB/c nude mice. The antiangiogenic potential of compounds was also investigated using HUVECs. Key Results Treatment of the Caki‐1 cells with cisplatin or [Pt(DMS)] resulted in a dose‐dependent inhibition of cell survival, but the cytotoxicity of [Pt(DMS)] was approximately fivefold greater than that of cisplatin. [Pt(DMS)] was much more effective than cisplatin at inhibiting tumour growth, proliferation and angiogenesis in vivo, as well as migration, tube formation and MMP1, MMP2 and MMP9 secretion of endothelial cells in vitro. Whereas, cisplatin exerted a greater cytotoxic effect on HUVECs, but did not affect tube formation or the migration of endothelial cells. In addition, treatment of the xenograft mice with [Pt(DMS)] decreased VEGF, MMP1 and MMP2 expressions in tumours. Conclusions and Implications The antiangiogenic and antitumour activities of [Pt(DMS)] provide a solid starting point for its validation as a suitable candidate for further pharmacological testing. PMID:27351124

PC12 Cells Differentiate into Chromaffin Cell-Like Phenotype in Coculture with Adrenal Medullary Endothelial Cells

NASA Astrophysics Data System (ADS)

Mizrachi, Yaffa; Naranjo, Jose R.; Levi, Ben-Zion; Pollard, Harvey B.; Lelkes, Peter I.

1990-08-01

Previously we described specific in vitro interactions between PC12 cells, a cloned, catecholamine-secreting pheochromocytoma cell line derived from the rat adrenal medulla, and bovine adrenal medullary endothelial cells. We now demonstrate that these interactions induce the PC12 cells to acquire physical and biochemical characteristics reminiscent of chromaffin cells. Under coculture conditions involving direct cell-cell contact, the endothelial cells and the PC12 cells reduced their rates of proliferation; upon prolonged coculture PC12 cells clustered into nests of cells similar to the organization of chromaffin cells seen in vivo. Within 3 days in coculture with endothelial cells, but not with unrelated control cells, PC12 cells synthesized increased levels of [Met]enkephalin. In addition, PC12 cells, growing on confluent endothelial monolayers, failed to extend neurites in response to nerve growth factor. Neither medium conditioned by endothelial cells nor fixed endothelial cells could by themselves induce all of these different phenomena in the PC12 cells. These results suggest that under coculture conditions PC12 cells change their state of differentiation toward a chromaffin cell-like phenotype. The rapid, transient increase in the expression of the protooncogene c-fos suggests that the mechanism(s) inducing the change in the state of differentiation in PC12 cells in coculture with the endothelial cells may be distinct from that described for the differentiation of PC12 cells--e.g., by glucocorticoids. We propose that similar interactions between endothelial cells and chromaffin cell precursors may occur during embryonic development and that these interactions might be instrumental for the organ-specific differentiation of the adrenal medulla in vivo.

Mesenchyme Homeobox 2 Enhances Migration of Endothelial Colony Forming Cells Exposed to Intrauterine Diabetes Mellitus.

PubMed

Gohn, Cassandra R; Blue, Emily K; Sheehan, BreAnn M; Varberg, Kaela M; Haneline, Laura S

2017-07-01

Diabetes mellitus (DM) during pregnancy has long-lasting implications for the fetus, including cardiovascular morbidity. Previously, we showed that endothelial colony forming cells (ECFCs) from DM human pregnancies have decreased vasculogenic potential. Here, we evaluate whether the molecular mechanism responsible for this phenotype involves the transcription factor, Mesenchyme Homeobox 2 (MEOX2). In human umbilical vein endothelial cells, MEOX2 upregulates cyclin-dependent kinase inhibitor expression, resulting in increased senescence and decreased proliferation. We hypothesized that dysregulated MEOX2 expression in neonatal ECFCs from DM pregnancies decreases network formation through increased senescence and altered cell cycle progression. Our studies show that nuclear MEOX2 is increased in ECFCs from DM pregnancies. To determine if MEOX2 is sufficient and/or required to induce impaired network formation, MEOX2 was overexpressed and depleted in ECFCs from control and DM pregnancies, respectively. Surprisingly, MEOX2 overexpression in control ECFCs resulted in increased network formation, altered cell cycle progression, and increased senescence. In contrast, MEOX2 knockdown in ECFCs from DM pregnancies led to decreased network formation, while cell cycle progression and senescence were unaffected. Importantly, migration studies demonstrated that MEOX2 overexpression increased migration, while MEOX2 knockdown decreased migration. Taken together, these data suggest that altered migration may be mediating the impaired vasculogenesis of ECFCs from DM pregnancies. While initially believed to be maladaptive, these data suggest that MEOX2 may serve a protective role, enabling increased vessel formation despite exposure to a DM intrauterine environment. J. Cell. Physiol. 232: 1885-1892, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A GCH1 haplotype confers sex-specific susceptibility to pain crises and altered endothelial function in adults with sickle cell anemia

PubMed Central

Belfer, Inna; Youngblood, Victoria; Darbari, Deepika S.; Wang, Zhengyuan; Diaw, Lena; Freeman, Lita; Desai, Krupa; Dizon, Michael; Allen, Darlene; Cunnington, Colin; Channon, Keith M.; Milton, Jacqueline; Hartley, Stephen W.; Nolan, Vikki; Kato, Gregory J.; Steinberg, Martin H.; Goldman, David; Taylor, James G.

2014-01-01

GTP cyclohydrolase (GCH1) is rate limiting for tetrahydrobiopterin (BH4) synthesis, where BH4 is a cofactor for nitric oxide (NO) synthases and aromatic hydroxylases. GCH1 polymorphisms are implicated in the pathophysiology of pain, but have not been investigated in African populations. We examined GCH1 and pain in sickle cell anemia where GCH1 rs8007267 was a risk factor for pain crises in discovery (n = 228; odds ratio [OR] 2.26; P = 0.009) and replication (n = 513; OR 2.23; P = 0.004) cohorts. In vitro, cells from sickle cell anemia subjects homozygous for the risk allele produced higher BH4. In vivo physiological studies of traits likely to be modulated by GCH1 showed rs8007267 is associated with altered endothelial dependent blood flow in females with SCA (8.42% of variation; P = 0.002). The GCH1 pain association is attributable to an African haplotype with where its sickle cell anemia pain association is limited to females (OR 2.69; 95% CI 1.21–5.94; P = 0.01) and has the opposite directional association described in Europeans independent of global admixture. The presence of a GCH1 haplotype with high BH4 in populations of African ancestry could explain the association of rs8007267 with sickle cell anemia pain crises. The vascular effects of GCH1 and BH4 may also have broader implications for cardiovascular disease in populations of African ancestry. PMID:24136375

Endotoxin, Toll-like Receptor-4, and Atherosclerotic Heart Disease

PubMed Central

Horseman, Michael A.; Surani, Salim; Bowman, John D.

2017-01-01

Background: Endotoxin is a lipopolysaccharide (LPS) constituent of the outer membrane of most gram negative bacteria. Ubiquitous in the environment, it has been implicated as a cause or con-tributing factor in several disparate disorders from sepsis to heatstroke and Type II diabetes mellitus. Starting at birth, the innate immune system develops cellular defense mechanisms against environmen-tal microbes that are in part modulated through a series of receptors known as toll-like receptors. Endo-toxin, often referred to as LPS, binds to toll-like receptor 4 (TLR4)/ myeloid differentiation protein 2 (MD2) complexes on various tissues including cells of the innate immune system, smooth muscle and endothelial cells of blood vessels including coronary arteries, and adipose tissue. Entry of LPS into the systemic circulation ultimately leads to intracellular transcription of several inflammatory mediators. The subsequent inflammation has been implicated in the development and progression atherosclerosis and subsequent coronary artery disease and heart failure. Objective: The potential roles of endotoxin and TLR4 are reviewed regarding their role in the pathogen-esis of atherosclerotic heart disease. Conclusion: Atherosclerosis is initiated by inflammation in arterial endothelial and subendothelial cells, and inflammatory processes are implicated in its progression to clinical heart disease. Endotoxin and TLR4 play a central role in the inflammatory process, and represent potential targets for therapeutic intervention. Therapy with HMG-CoA inhibitors may reduce the expression of TLR4 on monocytes. Other therapeutic interventions targeting TLR4 expression or function may prove beneficial in athero-sclerotic disease prevention and treatment.

Atrial Fibrillation Pacing Decreases Intravascular Shear Stress in a New Zealand White Rabbit Model: Implications in Endothelial Function

PubMed Central

Jen, Nelson; Yu, Fei; Lee, Juhyun; Wasmund, Steve; Dai, Xiaohu; Chen, Christina; Chawareeyawong, Pai; Yang, Yongmo; Li, Rongsong; Hamdan, Mohamed H.; Hsiai, Tzung

2012-01-01

Atrial fibrillation (AF) is characterized by multiple rapid and irregular atrial depolarization leading to rapid ventricular responses exceeding 100 beats per minute (bpm). We hypothesized that rapid and irregular pacing reduced intravascular shear stress (ISS) with implication to modulating endothelial responses. To simulate AF, we paced the left atrial appendage of New Zealand White (NZW) rabbits (n=4) at rapid and irregular intervals. Surface electrical cardiograms (ECG) were recorded for atrial and ventricular rhythm, and intravascular convective heat transfer was measured by micro thermal sensors, from which ISS was inferred. Rapid and irregular pacing decreased arterial systolic and diastolic pressures (baseline: 99/75 mmHg; rapid regular pacing: 92/73; rapid irregular pacing: 90/68; P < 0.001, n=4), temporal gradients (∂τ/∂t from 1275 ± 80 to 1056 ± 180 dyne/cm2·s), and reduced ISS (from baseline at 32.0 ± 2.4 to 22.7 ± 3.5 dyne/cm2). Computational fluid dynamics (CFD) code demonstrated that experimentally inferred ISS provided a close approximation to the computed wall shear stress (WSS) at a given catheter to vessel diameter ratio, shear stress range, and catheter position. In an in vitro flow system in which time-averaged shear stress was maintained at τavg=23 ±4 dyn·cm−2·s−1, we further demonstrated that rapid pulse rates at 150 bpm down-regulated endothelial nitric oxide (NO), promoted superoxide (O2·−) production, and increased monocyte binding to endothelial cells. These findings suggest that rapid pacing reduces ISS and ∂τ/∂t, and rapid pulse rates modulate endothelial responses. PMID:22983703

Angiopoietin-1 protects the endothelial cells against advanced glycation end product injury by strengthening cell junctions and inhibiting cell apoptosis.

PubMed

Zhao, Jingling; Chen, Lei; Shu, Bin; Tang, Jinming; Zhang, Lijun; Xie, Julin; Liu, Xusheng; Xu, Yingbin; Qi, Shaohai

2015-08-01

Endothelial dysfunction is a major characteristic of diabetic vasculopathy. Protection of the vascular endothelium is an essential aspect of preventing and treating diabetic vascular complications. Although Angiopoietin-1 (Ang-1) is an important endothelial-specific protective factor, whether Ang-1 protects vascular cells undergoing advanced glycation end product (AGE) injury has not been investigated. The aim of the present study was to determine the potential effects of Ang-1 on endothelial cells after exposure to AGE. We show here that Ang-1 prevented AGE-induced vascular leakage by enhancing the adherens junctions between endothelial cells, and this process was mediated by the phosphorylation and membrane localization of VE-cadherin. Furthermore, Ang-1 also protected endothelial cells from AGE-induced death by regulating phosphatidylinositol 3-kinase (PI3K)/Akt-dependent Bad phosphorylation. Our findings suggest that the novel protective mechanisms of Ang-1 on endothelium are achieved by strengthening endothelial cell junctions and reducing endothelial cell death after AGE injury. © 2014 Wiley Periodicals, Inc.

Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations.

PubMed

Zhao, Zhenjun; Johnson, Michael S; Chen, Biyi; Grace, Michael; Ukath, Jaysree; Lee, Vivienne S; McRobb, Lucinda S; Sedger, Lisa M; Stoodley, Marcus A

2016-06-01

OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation causes remarkable cellular changes in endothelial cells. Significant PS externalization is induced by radiation at doses of 15 Gy or higher, concomitant with a block in the cell cycle. Radiation-induced markers/targets may have high discriminating power to be harnessed in vascular targeting for AVM treatment.

The use of heparin in infertility and recurrent pregnancy loss: Are its antiviral properties at play?

PubMed

Eliassen, Eva; Marci, Roberto; Di Luca, Dario; Rizzo, Roberta

2017-05-01

Recently, human herpesvirus 6 (HHV-6) has been implicated in cases of poor pregnancy outcomes. The ability of HHV-6 to disrupt endothelial cell functioning may inhibit the creation of an appropriate uterine environment for implantation and fetal development, resulting in infertility and pregnancy loss, among other complications. Heparin has been used to treat pregnant women who are predisposed to thrombosis, and it has also been used in some women with infertility or recurrent pregnancy loss without associated thrombotic events. Positive pregnancy outcomes after heparin treatment in these groups indicate that heparin may alter the uterine environment to make it more suitable for implantation and fetal growth. In this paper, we propose that in some cases, heparin may accomplish this by inhibiting HHV-6 transfer to the endometrium and/or by offsetting the virally-mediated effects of endothelial cell injury. Copyright © 2017 Elsevier Ltd. All rights reserved.

Orphan nuclear receptor Nur77 is a novel negative regulator of endothelin-1 expression in vascular endothelial cells.

PubMed

Qin, Qing; Chen, Ming; Yi, Bing; You, Xiaohua; Yang, Ping; Sun, Jianxin

2014-12-01

Endothelin-1 (ET-1) produced by vascular endothelial cells plays essential roles in the regulation of vascular tone and development of cardiovascular diseases. The objective of this study is to identify novel regulators implicated in the regulation of ET-1 expression in vascular endothelial cells (ECs). By using quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), we show that either ectopic expression of orphan nuclear receptor Nur77 or pharmacological activation of Nur77 by 6-mercaptopurine (6-MP) substantially inhibits ET-1 expression in human umbilical vein endothelial cells (HUVECs), under both basal and thrombin-stimulated conditions. Furthermore, thrombin-stimulated ET expression is significantly augmented in both Nur77 knockdown ECs and aort from Nur77 knockout mice, suggesting that Nur77 is a negative regulator of ET-1 expression. Inhibition of ET-1 expression by Nur77 occurs at gene transcriptional levels, since Nur77 potently inhibits ET-1 promoter activity, without affecting ET-1 mRNA stability. As shown in electrophoretic mobility shift assay (EMSA), Nur77 overexpression markedly inhibits both basal and thrombin-stimulated transcriptional activity of AP-1. Mechanistically, we demonstrate that Nur77 specially interacts with c-Jun and inhibits AP-1 dependent c-Jun promoter activity, which leads to a decreased expression of c-Jun, a critical component involved in both AP-1 transcriptional activity and ET-1 expression in ECs. These findings demonstrate that Nur77 is a novel negative regulator of ET-1 expression in vascular ECs through an inhibitory interaction with the c-Jun/AP-1 pathway. Activation of Nur77 may represent a useful therapeutic strategy for preventing certain cardiovascular diseases, such as atherosclerosis and pulmonary artery hypertension. Copyright © 2014 Elsevier Ltd. All rights reserved.

Transcriptional profiling of CD31(+) cells isolated from murine embryonic stem cells.

PubMed

Mariappan, Devi; Winkler, Johannes; Chen, Shuhua; Schulz, Herbert; Hescheler, Jürgen; Sachinidis, Agapios

2009-02-01

Identification of genes involved in endothelial differentiation is of great interest for the understanding of the cellular and molecular mechanisms involved in the development of new blood vessels. Mouse embryonic stem (mES) cells serve as a potential source of endothelial cells for transcriptomic analysis. We isolated endothelial cells from 8-days old embryoid bodies by immuno-magnetic separation using platelet endothelial cell adhesion molecule-1 (also known as CD31) expressed on both early and mature endothelial cells. CD31(+) cells exhibit endothelial-like behavior by being able to incorporate DiI-labeled acetylated low-density lipoprotein as well as form tubular structures on matrigel. Quantitative and semi-quantitative PCR analysis further demonstrated the increased expression of endothelial transcripts. To ascertain the specific transcriptomic identity of the CD31(+) cells, large-scale microarray analysis was carried out. Comparative bioinformatic analysis reveals an enrichment of the gene ontology categories angiogenesis, blood vessel morphogenesis, vasculogenesis and blood coagulation in the CD31(+) cell population. Based on the transcriptomic signatures of the CD31(+) cells, we conclude that this ES cell-derived population contains endothelial-like cells expressing a mesodermal marker BMP2 and possess an angiogenic potential. The transcriptomic characterization of CD31(+) cells enables an in vitro functional genomic model to identify genes required for angiogenesis.

Culture and Characterization of Circulating Endothelial Progenitor Cells in Patients with Renal Cell Carcinoma.

PubMed

Gu, Wenyu; Sun, Wei; Guo, Changcheng; Yan, Yang; Liu, Min; Yao, Xudong; Yang, Bin; Zheng, Junhua

2015-07-01

Although emerging evidence demonstrates increased circulating endothelial progenitor cells in patients with solid tumors, to our knowledge it is still unknown whether such cells can be cultured from patients with highly angiogenic renal cell carcinoma. We cultured and characterized circulating endothelial progenitor cells from patients with renal cell carcinoma. The circulating endothelial progenitor cell level (percent of CD45(-)CD34(+) VEGF-R2(+) cells in total peripheral blood mononuclear cells) was quantified in 47 patients with renal cell carcinoma and 40 healthy controls. Peripheral blood mononuclear cells were then isolated from 33 patients with renal cell carcinoma and 30 healthy controls to culture and characterize circulating endothelial progenitor cells. The circulating endothelial progenitor cell level was significantly higher in patients with renal cell carcinoma than in healthy controls (0.276% vs 0.086%, p <0.001). A colony of circulating endothelial progenitor cells first emerged significantly earlier in patient than in control preparations (6.72 vs 14.67 days, p <0.001). The culture success rate (87.8% vs 40.0% of participants) and the number of colonies (10.06 vs 1.83) were significantly greater for patients than for controls (each p <0.001). The circulating endothelial progenitor cell level correlated positively with the number of patient colonies (r = 0.762, p <0.001). Cells cultured from patients and controls showed a similar growth pattern, immunophenotype, ability to uptake Ac-LDL and bind lectin, and form capillary tubes in vitro. However, significantly more VEGF-R2(+) circulating endothelial progenitor cells were found in preparations from patients with renal cell carcinoma than from healthy controls (21.1% vs 13.4%, p <0.001). Earlier emergence of circulating endothelial progenitor cell colonies, a higher cell culture success rate and more colonies were found for patients with renal cell carcinoma than for healthy controls. Results indicate the important significance of VEGF-R2(+) circulating endothelial progenitors in patients with renal cell carcinoma. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Dobesilate enhances endothelial nitric oxide synthase-activity in macro- and microvascular endothelial cells

PubMed Central

Suschek, Christoph; Kolb, Hubert; Kolb-Bachofen, Victoria

1997-01-01

Dobesilate is used for normalizing vascular dysfunction in a number of diseases. In search for an effect on endothelial NO production, macrovascular endothelial cells from rat aorta, microvascular endothelial cells from rat exocrine pancreatic tissue, and capillary endothelial cells from rat islets, were cultured in the presence or absence of Mg-Dobesilate. The activity of constitutive nitric oxide synthase (ecNOS) in resident cells as well as of inducible nitric oxide synthase (iNOS) in cytokine-activated cells was measured indirectly by recording the citrulline concentrations in culture supernatants.In each of the different endothelial cells Mg-Dobesilate incubation (0.25–1 mM) for 24 h led to a significant and concentration-dependent increase in ecNOS-activities. With cytokine-activated endothelial cell cultures only moderate effects were seen with little or no concentration-dependency. Addition of the NOS-inhibitor NG-monomethyl-L-arginine led to a significant suppression of citrulline formation in all cultures as an evidence for the enzyme specificity of these effects.iNOS- and ecNOS-specific reverse transcription and semi-quantitative polymerase chain reaction (RT–PCR) with RNA from resident or cytokine-activated endothelial cells gave no evidence for an increase in NOS-specific mRNA after Mg-Dobesilate-treatment. Furthermore, Dobesilate-mediated enhancement of NO synthesis in resting endothelial cells was not due to iNOS induction in these cells, as no iNOS-specific signal was found by RT–PCR. PMID:9421302

Approaches to improve angiogenesis in tissue-engineered skin.

PubMed

Sahota, Parbinder S; Burn, J Lance; Brown, Nicola J; MacNeil, Sheila

2004-01-01

A problem with tissue-engineered skin is clinical failure due to delays in vascularization. The aim of this study was to explore a number of simple strategies to improve angiogenesis/vascularization using a tissue-engineered model of skin to which small vessel human dermal microvascular endothelial cells were added. For the majority of these studies, a modified Guirguis chamber was used, which allowed the investigation of several variables within the same experiment using the same human dermis; cell type, angiogenic growth factors, the influence of keratinocytes and fibroblasts, mechanical penetration of the human dermis, the site of endothelial cell addition, and the influence of hypoxia were all examined. A qualitative scoring system was used to assess the impact of these factors on the penetration of endothelial cells throughout the dermis. Similar results were achieved using freshly isolated small vessel human dermal microvascular endothelial cells or an endothelial cell line and a minimum cell seeding density was identified. Cell penetration was not influenced by the addition of angiogenic growth factors (vascular endothelial growth factor and basic fibroblast growth factor); similarly, including epidermal keratinocytes or dermal fibroblasts did not encourage endothelial cell entry, and neither did mechanical introduction of holes throughout the dermis. Two factors were identified that significantly enhanced endothelial cell penetration into the dermis: hypoxia and the site of endothelial cell addition. Endothelial cells added from the papillary surface entered into the dermis much more effectively than when cells were added to the reticular surface of the dermis. We conclude that this model is valuable in improving our understanding of how to enhance vascularization of tissue-engineered grafts.

Hedgehog signaling in the murine melanoma microenvironment.

PubMed

Geng, Ling; Cuneo, Kyle C; Cooper, Michael K; Wang, Hong; Sekhar, Konjeti; Fu, Allie; Hallahan, Dennis E

2007-01-01

The Hedgehog intercellular signaling pathway regulates cell proliferation and differentiation. This pathway has been implicated to play a role in the pathogenesis of cancer and in embryonic blood vessel development. In the current study, Hedgehog signaling in tumor related vasculature and microenvironment was examined using human umbilical vein endothelial cells and B16F0 (murine melanoma) tumors models. Use of exogenous Sonic hedgehog (Shh) peptide significantly increased BrdU incorporation in endothelial cells in vitro by a factor of 2 (P < 0.001). The Hedgehog pathway antagonist cyclopamine effectively reduced Shh-induced proliferation to control levels. To study Hedgehog signaling in vivo a hind limb tumor model with the B16F0 cell line was used. Treatment with 25 mg/kg cyclopamine significantly attenuated BrdU incorporation in tumor cells threefold (P < 0.001), in tumor related endothelial cells threefold (P = 0.004), and delayed tumor growth by 4 days. Immunohistochemistry revealed that the Hedgehog receptor Patched was localized to the tumor stroma and that B16F0 cells expressed Shh peptide. Furthermore, mouse embryonic fibroblasts required the presence of B16F0 cells to express Patched in a co-culture assay system. These studies indicate that Shh peptide produced by melanoma cells induces Patched expression in fibroblasts. To study tumor related angiogenesis a vascular window model was used to monitor tumor vascularity. Treatment with cyclopamine significantly attenuated vascular formation by a factor of 2.5 (P < 0.001) and altered vascular morphology. Furthermore, cyclopamine reduced tumor blood vessel permeability to FITC labeled dextran while having no effect on normal blood vessels. These studies suggest that Hedgehog signaling regulates melanoma related vascular formation and function.

Laminar shear stress regulates endothelial kinin B1 receptor expression and function: potential implication in atherogenesis

PubMed Central

Duchene, Johan; Cayla, Cécile; Vessillier, Sandrine; Scotland, Ramona; Yamashiro, Kazuo; Lecomte, Florence; Syed, Irfan; Vo, Phuong; Marrelli, Alessandra; Pitzalis, Costantino; Cipollone, Francesco; Schanstra, Joost; Bascands, Jean-Loup; Hobbs, Adrian J; Perretti, Mauro; Ahluwalia, Amrita

2009-01-01

OBJECTIVE The pro-inflammatory phenotype induced by low laminar shear stress (LSS) is implicated in atherogenesis. The kinin B1 receptor (B1R), known to be induced by inflammatory stimuli, exerts many pro-inflammatory effects including vasodilatation and leukocyte recruitment. We investigated whether low LSS is a stimulus for endothelial B1R expression and function. METHODS AND RESULTS Human and mouse atherosclerotic plaques expressed high level of B1R mRNA and protein. In addition, B1R expression was upregulated in the aortic arch (low LSS region) of ApoE-/- mice fed a high fat diet compared to vascular regions of high LSS and animals fed normal chow. Of interest, a greater expression of B1R was noticed in endothelial cells from regions of low LSS in aortic arch of ApoE-/- mice. B1R was also upregulated in human umbilical vein endothelial cells (HUVEC) exposed to low LSS (0-2dyn/cm2) compared to physiological LSS (6-10dyn/cm2): an effect similarly evident in murine vascular tissue perfused ex vivo. Functionally, B1R activation increased prostaglandin and CXCL5 expression in cells exposed to low, but not physiological, LSS. IL-1β and ox-LDL induced B1R expression and function in HUVECs, a response substantially enhanced under low LSS conditions and inhibited by blockade of NFκB activation. CONCLUSION Herein, we show that LSS is a major determinant of functional B1R expression in endothelium. Furthermore, whilst physiological high LSS is a powerful repressor of this inflammatory receptor, low LSS at sites of atheroma are associated with substantial upregulation, identifying this receptor as a potential therapeutic target. CONDENSED ABSTRACT Low laminar shear stress (LSS) underlies the pro-inflammatory processes in atherogenesis. Herein, we demonstrate that whilst physiological LSS represses inflammatory kinin B1 receptor (B1R) expression/function, low atherogenic LSS is associated with profound upregulation of both in atherosclerosis in both humans and animal models, highlighting B1R as an exciting potential therapeutic target. PMID:19661485

Vascular Targeting of Nanocarriers: Perplexing Aspects of the Seemingly Straightforward Paradigm

PubMed Central

2015-01-01

Targeted nanomedicine holds promise to find clinical use in many medical areas. Endothelial cells that line the luminal surface of blood vessels represent a key target for treatment of inflammation, ischemia, thrombosis, stroke, and other neurological, cardiovascular, pulmonary, and oncological conditions. In other cases, the endothelium is a barrier for tissue penetration or a victim of adverse effects. Several endothelial surface markers including peptidases (e.g., ACE, APP, and APN) and adhesion molecules (e.g., ICAM-1 and PECAM) have been identified as key targets. Binding of nanocarriers to these molecules enables drug targeting and subsequent penetration into or across the endothelium, offering therapeutic effects that are unattainable by their nontargeted counterparts. We analyze diverse aspects of endothelial nanomedicine including (i) circulation and targeting of carriers with diverse geometries, (ii) multivalent interactions of carrier with endothelium, (iii) anchoring to multiple determinants, (iv) accessibility of binding sites and cellular response to their engagement, (v) role of cell phenotype and microenvironment in targeting, (vi) optimization of targeting by lowering carrier avidity, (vii) endocytosis of multivalent carriers via molecules not implicated in internalization of their ligands, and (viii) modulation of cellular uptake and trafficking by selection of specific epitopes on the target determinant, carrier geometry, and hydrodynamic factors. Refinement of these aspects and improving our understanding of vascular biology and pathology is likely to enable the clinical translation of vascular endothelial targeting of nanocarriers. PMID:24787360

Targeting superoxide dismutase to endothelial caveolae profoundly alleviates inflammation caused by endotoxin.

PubMed

Shuvaev, Vladimir V; Kiseleva, Raisa Yu; Arguiri, Evguenia; Villa, Carlos H; Muro, Silvia; Christofidou-Solomidou, Melpo; Stan, Radu V; Muzykantov, Vladimir R

2018-02-28

Inflammatory mediators binding to Toll-Like receptors (TLR) induce an influx of superoxide anion in the ensuing endosomes. In endothelial cells, endosomal surplus of superoxide causes pro-inflammatory activation and TLR4 agonists act preferentially via caveolae-derived endosomes. To test the hypothesis that SOD delivery to caveolae may specifically inhibit this pathological pathway, we conjugated SOD with antibodies (Ab/SOD, size ~10nm) to plasmalemmal vesicle-associated protein (Plvap) that is specifically localized to endothelial caveolae in vivo and compared its effects to non-caveolar target CD31/PECAM-1. Plvap Ab/SOD bound to endothelial cells in culture with much lower efficacy than CD31 Ab/SOD, yet blocked the effects of LPS signaling with higher efficiency than CD31 Ab/SOD. Disruption of cholesterol-rich membrane domains by filipin inhibits Plvap Ab/SOD endocytosis and LPS signaling, implicating the caveolae-dependent pathway(s) in both processes. Both Ab/SOD conjugates targeted to Plvap and CD31 accumulated in the lungs after IV injection in mice, but the former more profoundly inhibited LPS-induced pulmonary inflammation and elevation of plasma level of interferon-beta and -gamma and interleukin-27. Taken together, these results indicate that targeted delivery of SOD to specific cellular compartments may offer effective, mechanistically precise interception of pro-inflammatory signaling mediated by reactive oxygen species. Copyright © 2018 Elsevier B.V. All rights reserved.

Red Blood Cell Dysfunction Induced by High-Fat Diet

PubMed Central

Unruh, Dusten; Srinivasan, Ramprasad; Benson, Tyler; Haigh, Stephen; Coyle, Danielle; Batra, Neil; Keil, Ryan; Sturm, Robert; Blanco, Victor; Palascak, Mary; Franco, Robert S.; Tong, Wilson; Chatterjee, Tapan; Hui, David Y.; Davidson, W. Sean; Aronow, Bruce J.; Kalfa, Theodosia; Manka, David; Peairs, Abigail; Blomkalns, Andra; Fulton, David J.; Brittain, Julia E.; Weintraub, Neal L.; Bogdanov, Vladimir Y.

2015-01-01

Background High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC−/− mice. In RBCs from HFD-fed wild-type and DARC−/− mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic. PMID:26467254

Cigarette smoking impairs nitric oxide-mediated cerebral blood flow increase: Implications for Alzheimer's disease.

PubMed

Toda, Noboru; Okamura, Tomio

2016-08-01

Cerebral blood flow is mainly regulated by nitrergic (parasympathetic, postganglionic) nerves and nitric oxide (NO) liberated from endothelial cells in response to shear stress and stretch of vasculature, whereas sympathetic vasoconstrictor control is quite weak. On the other hand, peripheral vascular resistance and blood flow are mainly controlled by adrenergic vasoconstrictor nerves; endothelium-derived NO and nitrergic nerves play some roles as vasodilator factors. Cigarette smoking impairs NO synthesis in cerebral vascular endothelial cells and nitrergic nerves leading to interference with cerebral blood flow and glucose metabolism in the brain. Smoking-induced cerebral hypoperfusion is induced by impairment of synthesis and actions of NO via endothelial nitric oxide synthase (eNOS)/neuronal NOS (nNOS) inhibition and by increased production of oxygen radicals, resulting in decreased actions of NO on vascular smooth muscle. Nicotine acutely and chronically impairs the action of endothelial NO and also inhibits nitrergic nerve function in chronic use. Impaired cerebral blood supply promotes the synthesis of amyloid β that accelerates blood flow decrease. This vicious cycle is thought to be one of the important factors involving in Alzheimer's disease (AD). Quitting smoking is undoubtedly one of the important ways to prevent and delay the genesis or slow the progress of impaired cognitive function and AD. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

A Mutant Receptor Tyrosine Phosphatase, CD148, Causes Defects in Vascular Development

PubMed Central

Takahashi, Takamune; Takahashi, Keiko; St. John, Patricia L.; Fleming, Paul A.; Tomemori, Takuya; Watanabe, Toshio; Abrahamson, Dale R.; Drake, Christopher J.; Shirasawa, Takuji; Daniel, Thomas O.

2003-01-01

Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPη), participates in developmental vascularization. A mutant allele, CD148ΔCyGFP, was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions. PMID:12588999

Role of contact inhibition in the regulation of receptor-mediated uptake of low density lipoprotein in cultured vascular endothelial cells.

PubMed Central

Vlodavsky, I; Fielding, P E; Fielding, C J; Gospodarowicz, D

1978-01-01

Bovine vascular endothelial cells during logarithmic growth bind, internalize, and degrade low density lipoprotein (LDL) via a receptor-mediated pathway. However, contact-inhibited (confluent) monolayers bind but do not internalize LDL. This is in contrast to aortic smooth muscle cells or endothelial cells that have lost the property of contact inhibition. These cells internalize and degrade LDL at both high and low cell densities. The LDL receptors of smooth muscle and sparse endothelial cells down-regulate in response to LDL. In contrast, normal endothelial cells at confluency show little response. When contact inhibition in endothelial monolayers was locally released by wounding, and LDL was present, only cells released from contact inhibition accumulated LDL cholesterol. In smooth muscle cells under the same conditions, the entire culture interiorized lipid. It thus appears that in endothelial cells, unlike smooth muscle cells, contact inhibition is the major factor regulating cellular uptake of LDL cholesteryl ester. Reversal of contact inhibition by wounding provides a mechanism by which the endothelium could be the primary initiator of the atherosclerotic plaque. Images PMID:203937

Endothelial cell membrane vesicles in the study of organ preference of metastasis.

PubMed

Johnson, R C; Augustin-Voss, H G; Zhu, D Z; Pauli, B U

1991-01-01

Many malignancies exhibit distinct patterns of metastasis that appear to be mediated by receptor/ligand-like interactions between tumor cells and organ-specific vascular endothelium. In order to study endothelial cell surface molecules involved in the binding of metastatic cells, we developed a perfusion method to isolate outside-out membrane vesicles from the lumenal surface of rat lung microvascular endothelium. Lungs were perfused in situ for 4 h at 37 degrees C with a solution of 100 mM formaldehyde, 2 mM dithiothreitol in phosphate-buffered saline to induce endothelial cell vesiculation. Radioiodinated rat lung endothelial cell membrane vesicles bound lung-metastatic tumor cells (B16F10, R323OAC-MET) in significantly higher numbers than their low or nonmetastatic counterparts (B16F0, R323OAC-LR). In contrast, leg endothelial membrane vesicle showed no binding preference for either cell line. Neuraminidase treatment of vesicles abolished specificity of adhesion of lung-derived vesicles to lung metastatic tumor cells. These results demonstrate that in situ perfusion is an appropriate technique to obtain pure endothelial cell membrane vesicles containing functionally active adhesion molecules. The preferential binding of lung-derived endothelial cell membrane vesicles by lung metastatic tumor cells is evidence of the importance of endothelial cell adhesion molecules in the formation of metastases.

Cross talk between primary human renal tubular cells and endothelial cells in cocultures.

PubMed

Tasnim, Farah; Zink, Daniele

2012-04-15

Interactions between renal tubular epithelial cells and adjacent endothelial cells are essential for normal renal functions but also play important roles in renal disease and repair. Here, we investigated cocultures of human primary renal proximal tubular cells (HPTC) and human primary endothelial cells to address the cross talk between these cell types. HPTC showed improved proliferation, marker gene expression, and enzyme activity in cocultures. Also, the long-term maintenance of epithelia formed by HPTC was improved, which was due to the secretion of transforming growth factor-β1 and its antagonist α2-macroglobulin. HPTC induced endothelial cells to secrete increased amounts of these factors, which balanced each other functionally and only displayed in combination the observed positive effects. In addition, in the presence of HPTC endothelial cells expressed increased amounts of hepatocyte growth factor and vascular endothelial growth factor, which have well-characterized effects on renal tubular epithelial cells as well as on endothelial cells. Together, the results showed that HPTC stimulated endothelial cells to express a functionally balanced combination of various factors, which in turn improved the performance of HPTC. The results give new insights into the cross talk between renal epithelial and endothelial cells and suggest that cocultures could be also useful models for the analysis of cellular communication in renal disease and repair. Furthermore, the characterization of defined microenvironments, which positively affect HPTC, will be helpful for improving the performance of this cell type in in vitro applications including in vitro toxicology and kidney tissue engineering.

Nuclear Countermeasure Activity of TP508 Linked to Restoration of Endothelial Function and Acceleration of DNA Repair

PubMed Central

Olszewska-Pazdrak, Barbara; McVicar, Scott D.; Rayavara, Kempaiah; Moya, Stephanie M.; Kantara, Carla; Gammarano, Chris; Olszewska, Paulina; Fuller, Gerald M.; Sower, Laurie E.; Carney, Darrell H.

2016-01-01

There is increasing evidence that radiation-induced damage to endothelial cells and loss of endothelial function may contribute to both acute radiation syndromes and long-term effects of whole-body nuclear irradiation. Therefore, several drugs are being developed to mitigate the effects of nuclear radiation, most of these drugs will target and protect or regenerate leukocytes and platelets. Our laboratory has demonstrated that TP508, a 23-amino acid thrombin peptide, activates endothelial cells and stem cells to revascularize and regenerate tissues. We now show that TP508 can mitigate radiation-induced damage to endothelial cells in vitro and in vivo. Our in vitro results demonstrate that human endothelial cells irradiation attenuates nitric oxide (NO) signaling, disrupts tube formation and induces DNA double-strand breaks (DSB). TP508 treatment reverses radiation effects on NO signaling, restores tube formation and accelerates the repair of radiation-induced DSB. The radiation-mitigating effects of TP508 on endothelial cells were also seen in CD-1 mice where systemic injection of TP508 stimulated endothelial cell sprouting from aortic explants after 8 Gy irradiation. Systemic doses of TP508 that mitigated radiation-induced endothelial cell damage, also significantly increased survival of CD-1 mice when injected 24 h after 8.5 Gy exposure. These data suggest that increased survival observed with TP508 treatment may be due to its effects on vascular and microvascular endothelial cells. Our study supports the usage of a regenerative drug such as TP508 to activate endothelial cells as a countermeasure for mitigating the effects of nuclear radiation. PMID:27388041

Identification and functional analysis of endothelial tip cell-enriched genes.

PubMed

del Toro, Raquel; Prahst, Claudia; Mathivet, Thomas; Siegfried, Geraldine; Kaminker, Joshua S; Larrivee, Bruno; Breant, Christiane; Duarte, Antonio; Takakura, Nobuyuki; Fukamizu, Akiyoshi; Penninger, Josef; Eichmann, Anne

2010-11-11

Sprouting of developing blood vessels is mediated by specialized motile endothelial cells localized at the tips of growing capillaries. Following behind the tip cells, endothelial stalk cells form the capillary lumen and proliferate. Expression of the Notch ligand Delta-like-4 (Dll4) in tip cells suppresses tip cell fate in neighboring stalk cells via Notch signaling. In DLL4(+/-) mouse mutants, most retinal endothelial cells display morphologic features of tip cells. We hypothesized that these mouse mutants could be used to isolate tip cells and so to determine their genetic repertoire. Using transcriptome analysis of retinal endothelial cells isolated from DLL4(+/-) and wild-type mice, we identified 3 clusters of tip cell-enriched genes, encoding extracellular matrix degrading enzymes, basement membrane components, and secreted molecules. Secreted molecules endothelial-specific molecule 1, angiopoietin 2, and apelin bind to cognate receptors on endothelial stalk cells. Knockout mice and zebrafish morpholino knockdown of apelin showed delayed angiogenesis and reduced proliferation of stalk cells expressing the apelin receptor APJ. Thus, tip cells may regulate angiogenesis via matrix remodeling, production of basement membrane, and release of secreted molecules, some of which regulate stalk cell behavior.

Vascular endothelial growth factor-A enhances indoleamine 2,3-dioxygenase expression by dendritic cells and subsequently impacts lymphocyte proliferation

PubMed Central

Marti, Luciana Cavalheiro; Pavon, Lorena; Severino, Patricia; Sibov, Tatiana; Guilhen, Daiane; Moreira-Filho, Carlos Alberto

2013-01-01

Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity. PMID:24141959

Endothelial cells in the eyes of an immunologist.

PubMed

Young, M Rita

2012-10-01

Endothelial cell activation in the process of tumor angiogenesis and in various aspects of vascular biology has been extensively studied. However, endothelial cells also function in other capacities, including in immune regulation. Compared to the more traditional immune regulatory populations (Th1, Th2, Treg, etc.), endothelial cells have received far less credit as being immune regulators. Their regulatory capacity is multifaceted. They are critical in both limiting and facilitating the trafficking of various immune cell populations, including T cells and dendritic cells, out of the vasculature and into tissue. They also can be induced to stimulate immune reactivity or to be immune inhibitory. In each of these parameters (trafficking, immune stimulation and immune inhibition), their role can be physiological, whereby they have an active role in maintaining health. Alternatively, their role can be pathological, whereby they contribute to disease. In theory, endothelial cells are in an ideal location to recruit cells that can mediate immune reactivity to tumor tissue. Furthermore, they can activate the immune cells as they transmigrate across the endothelium into the tumor. However, what is seen is the absence of these protective effects of endothelial cells and, instead, the endothelial cells succumb to the defense mechanisms of the tumor, resulting in their acquisition of a tumor-protective role. To understand the immune regulatory potential of endothelial cells in protecting the host versus the tumor, it is useful to better understand the other circumstances in which endothelial cells modulate immune reactivities. Which of the multitude of immune regulatory roles that endothelial cells can take on seems to rely on the type of stimulus that they are encountering. It also depends on the extent to which they can be manipulated by potential dangers to succumb and contribute toward attack on the host. This review will explore the physiological and pathological roles of endothelial cells as they regulate immune trafficking, immune stimulation and immune inhibition in a variety of conditions and will then apply this information to their role in the tumor environment. Strategies to harness the immune regulatory potential of endothelial cells are starting to emerge in the non-tumor setting. Results from such efforts are expected to be applicable to being able to skew endothelial cells from having a tumor-protective role to a host-protective role.

Human trophoblast-derived hydrogen sulfide stimulates placental artery endothelial cell angiogenesis.

PubMed

Chen, Dong-Bao; Feng, Lin; Hodges, Jennifer K; Lechuga, Thomas J; Zhang, Honghai

2017-09-01

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (β-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The antiangiogenic activity of cleaved high molecular weight kininogen is mediated through binding to endothelial cell tropomyosin

PubMed Central

Zhang, Jing-Chuan; Doñate, Fernando; Qi, Xiaoping; Ziats, Nicholas P.; Juarez, Jose C.; Mazar, Andrew P.; Pang, Yuan-Ping; McCrae, Keith R.

2002-01-01

Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn2+-dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin. PMID:12196635

Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

PubMed

Herz, Katia; Becker, Alexandra; Shi, Chenyue; Ema, Masatsugo; Takahashi, Satoru; Potente, Michael; Hesse, Michael; Fleischmann, Bernd K; Wenzel, Daniela

2018-05-01

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP + signals specifically in Ki67 + /PECAM + endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent

PubMed Central

James, Daylon; Nam, Hyung-song; Seandel, Marco; Nolan, Daniel; Janovitz, Tyler; Tomishima, Mark; Studer, Lorenz; Lee, Gabsang; Lyden, David; Benezra, Robert; Zaninovic, Nikica; Rosenwaks, Zev; Rabbany, Sina Y; Rafii, Shahin

2010-01-01

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application. PMID:20081865

Intravenous Lipid Infusion Induces Endoplasmic Reticulum Stress in Endothelial Cells and Blood Mononuclear Cells of Healthy Adults.

PubMed

Tampakakis, Emmanouil; Tabit, Corey E; Holbrook, Monika; Linder, Erika A; Berk, Brittany D; Frame, Alissa A; Bretón-Romero, Rosa; Fetterman, Jessica L; Gokce, Noyan; Vita, Joseph A; Hamburg, Naomi M

2016-01-11

Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response may initially be protective, but when prolonged, have been implicated in atherogenesis in diabetic conditions. Triglycerides and free fatty acids (FFAs) are elevated in patients with diabetes and may contribute to ER stress. We sought to evaluate the effect of acute FFA elevation on ER stress in endothelial and circulating white cells. Twenty-one healthy subjects were treated with intralipid (20%; 45 mL/h) plus heparin (12 U/kg/h) infusion for 5 hours. Along with increased triglyceride and FFA levels, intralipid/heparin infusion reduced the calf reactive hyperemic response without a change in conduit artery flow-mediated dilation consistent with microvascular dysfunction. To investigate the short-term effects of elevated triglycerides and FFA, we measured markers of ER stress in peripheral blood mononuclear cells (PBMCs) and vascular endothelial cells (VECs). In VECs, activating transcription factor 6 (ATF6) and phospho-inositol requiring kinase 1 (pIRE1) proteins were elevated after infusion (both P<0.05). In PBMCs, ATF6 and spliced X-box-binding protein 1 (XBP-1) gene expression increased by 2.0- and 2.5-fold, respectively (both P<0.05), whereas CHOP and GADD34 decreased by ≈67% and 74%, respectively (both P<0.01). ATF6 and pIRE1 protein levels also increased (both P<0.05), and confocal microscopy revealed the nuclear localization of ATF6 after infusion, suggesting activation. Along with microvascular dysfunction, intralipid infusion induced an early protective ER stress response evidenced by activation of ATF6 and IRE1 in both leukocytes and endothelial cells. Our results suggest a potential link between metabolic disturbances and ER stress that may be relevant to vascular disease. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Directional cell migration in an extracellular pH gradient: a model study with an engineered cell line and primary microvascular endothelial cells.

PubMed

Paradise, Ranjani K; Whitfield, Matthew J; Lauffenburger, Douglas A; Van Vliet, Krystyn J

2013-02-15

Extracellular pH (pH(e)) gradients are characteristic of tumor and wound environments. Cell migration in these environments is critical to tumor progression and wound healing. While it has been shown previously that cell migration can be modulated in conditions of spatially invariant acidic pH(e) due to acid-induced activation of cell surface integrin receptors, the effects of pH(e) gradients on cell migration remain unknown. Here, we investigate cell migration in an extracellular pH(e) gradient, using both model α(v)β(3) CHO-B2 cells and primary microvascular endothelial cells. For both cell types, we find that the mean cell position shifts toward the acidic end of the gradient over time, and that cells preferentially polarize toward the acidic end of the gradient during migration. We further demonstrate that cell membrane protrusion stability and actin-integrin adhesion complex formation are increased in acidic pH(e), which could contribute to the preferential polarization toward acidic pH(e) that we observed for cells in pH(e) gradients. These results provide the first demonstration of preferential cell migration toward acid in a pH(e) gradient, with intriguing implications for directed cell migration in the tumor and wound healing environments. Copyright © 2012 Elsevier Inc. All rights reserved.

Antiapoptotic and antigenotoxic effects of N-acetylcysteine in human cells of endothelial origin.

PubMed

Aluigi, M G; De Flora, S; D'Agostini, F; Albini, A; Fassina, G

2000-01-01

N-Acetylcysteine (NAC) is a drug bearing multiple preventive properties that can inhibit genotoxicity and carcinogenicity. NAC also inhibits invasion and metastasis of malignant cells, as well as tumor take. We recently demonstrated the effects of NAC on Kaposi's sarcoma cells supernatant-induced invasion in vitro and angiogenesis in vivo. Many anticancer agents act through cytotoxicity of rapidly proliferating cells and several antineoplastic drugs induce apoptosis of cancer cells. Since endothelial cells are the target for the inhibition of angiogenesis, we wanted to verify that NAC, while inhibiting tumor vascularization and endothelial cell invasion would not induce endothelial cell apoptosis. We tested the ability of NAC to modulate apoptosis and cytogenetic damage in vitro and to promote differentiation on a reconstituted basement membrane (matrigel) in two endothelial cell lines (EAhy926 and HUVE). Treatment with NAC protected endothelial cells from TGF-beta-induced apoptosis and paraquat-induced cytogenetic damage. Therefore, NAC acts as an antiangiogenic agent and, at the same time, appears to prevent apoptosis and oxygen-related genotoxicity in endothelial cells.

Endothelial dysfunction in metabolic and vascular disorders.

PubMed

Polovina, Marija M; Potpara, Tatjana S

2014-03-01

Vascular endothelium has important regulatory functions in the cardiovascular system and a pivotal role in the maintenance of vascular health and metabolic homeostasis. It has long been recognized that endothelial dysfunction participates in the pathogenesis of atherosclerosis from early, preclinical lesions to advanced, thrombotic complications. In addition, endothelial dysfunction has been recently implicated in the development of insulin resistance and type 2 diabetes mellitus (T2DM). Considering that states of insulin resistance (eg, metabolic syndrome, impaired fasting glucose, impaired glucose tolerance, and T2DM) represent the most prevalent metabolic disorders and risk factors for atherosclerosis, it is of considerable scientific and clinical interest that both metabolic and vascular disorders have endothelial dysfunction as a common background. Importantly, endothelial dysfunction has been associated with adverse outcomes in patients with established cardiovascular disease, and a growing body of evidence indicates that endothelial dysfunction also imparts adverse prognosis in states of insulin resistance. In this review, we discuss the association of insulin resistance and T2DM with endothelial dysfunction and vascular disease, with a focus on the underlying mechanisms and prognostic implications of the endothelial dysfunction in metabolic and vascular disorders. We also address current therapeutic strategies for the improvement of endothelial dysfunction.

C/EBPβ and Nuclear Factor of Activated T Cells Differentially Regulate Adamts-1 Induction by Stimuli Associated with Vascular Remodeling

PubMed Central

Oller, Jorge; Alfranca, Arántzazu; Méndez-Barbero, Nerea; Villahoz, Silvia; Lozano-Vidal, Noelia; Martín-Alonso, Mara; Arroyo, Alicia G.; Escolano, Amelia; Armesilla, Angel Luis

2015-01-01

Emerging evidence indicates that the metalloproteinase Adamts-1 plays a significant role in the pathophysiology of vessel remodeling, but little is known about the signaling pathways that control Adamts-1 expression. We show that vascular endothelial growth factor (VEGF), angiotensin-II, interleukin-1β, and tumor necrosis factor α, stimuli implicated in pathological vascular remodeling, increase Adamts-1 expression in endothelial and vascular smooth muscle cells. Analysis of the intracellular signaling pathways implicated in this process revealed that VEGF and angiotensin-II upregulate Adamts-1 expression via activation of differential signaling pathways that ultimately promote functional binding of the NFAT or C/EBPβ transcription factors, respectively, to the Adamts-1 promoter. Infusion of mice with angiotensin-II triggered phosphorylation and nuclear translocation of C/EBPβ proteins in aortic cells concomitantly with an increase in the expression of Adamts-1, further underscoring the importance of C/EBPβ signaling in angiotensin-II-induced upregulation of Adamts-1. Similarly, VEGF promoted NFAT activation and subsequent Adamts-1 induction in aortic wall in a calcineurin-dependent manner. Our results demonstrate that Adamts-1 upregulation by inducers of pathological vascular remodeling is mediated by specific signal transduction pathways involving NFAT or C/EBPβ transcription factors. Targeting of these pathways may prove useful in the treatment of vascular disease. PMID:26217013

Nuclear Localization of Vascular Endothelial Growth Factor-D and Regulation of c-Myc–Dependent Transcripts in Human Lung Fibroblasts

PubMed Central

Pacheco-Rodriguez, Gustavo; Malide, Daniela; Meza-Carmen, Victor; Kato, Jiro; Cui, Ye; Padilla, Philip I.; Samidurai, Arun; Gochuico, Bernadette R.

2014-01-01

Lymphangiogenesis and angiogenesis are processes that are, in part, regulated by vascular endothelial growth factor (VEGF)-D. The formation of lymphatic structures has been implicated in multiple lung diseases, including pulmonary fibrosis. VEGF-D is a secreted protein produced by fibroblasts and macrophages, which induces lymphangiogenesis by signaling via VEGF receptor-3, and angiogenesis through VEGF receptor-2. VEGF-D contains a central VEGF homology domain, which is the biologically active domain, with flanking N- and C-terminal propeptides. Full-length VEGF-D (∼ 50 kD) is proteolytically processed in the extracellular space, to generate VEGF homology domain that contains the VEGF-D receptor–binding sites. Here, we report that, independent of its cell surface receptors, full-length VEGF-D accumulated in nuclei of fibroblasts, and that this process appears to increase with cell density. In nuclei, full-length VEGF-D associated with RNA polymerase II and c-Myc. In cells depleted of VEGF-D, the transcriptionally regulated genes appear to be modulated by c-Myc. These findings have potential clinical implications, as VEGF-D was found in fibroblast nuclei in idiopathic pulmonary fibrosis, a disease characterized by fibroblast proliferation. These findings are consistent with actions of full-length VEGF-D in cellular homeostasis in health and disease, independent of its receptors. PMID:24450584

Conventional and Electronic cigarettes dysregulate the expression of iron transporters and detoxifying enzymes at the brain vascular endothelium: In Vivo Evidence of a Gender-Specific Cellular Response to Chronic Cigarette Smoke Exposure.

PubMed

Kaisar, Mohammad A; Sivandzade, Farzane; Bhalerao, Aditya; Cucullo, Luca

2018-06-04

It is well established that tobacco smoking is associated with vascular endothelial dysfunction in a causative and dose dependent manner primarily related to the tobacco smoke (TS) content of reactive oxygen species (ROS), nicotine, and oxidative stress (OS) -driven inflammation. Preclinical studies have also shown that nicotine (the principal e-liquid's ingredient used in e-cigarettes (e-Cigs) can also cause OS, exacerbation of cerebral ischemia and secondary brain injury. Likewise, chronic e-Cig vaping could be prodromal to vascular endothelial dysfunctions. Herein, we provide direct evidence that similarly to TS, e-Cig promotes mitochondrial depolarization in primary brain vascular endothelial cells as well as the vascular endothelial cell line bEnd3. In addition, both TS and e-Cig exposure upregulated the transmembrane iron exporter Slc40a1 (crucial to maintain cellular iron and redox homeostasis) and that of porphyrin importer Abcb6 (linked to accelerated atherosclerosis). We then investigated in vivo whether gender plays a role in how chronic TS affect vascular endothelial functions. Our results clearly show chronic TS exposure differentially impacts the expression levels of Phase-II enzymes as well as the iron transporters previously investigated in vitro. Although the physiological implications of the gender-specific differential responses to TS are not fully clear, they do demonstrate that gender is a risk factor that needs to be investigated when assessing the potential impact of chronic smoking and perhaps e-Cig vaping. Copyright © 2018. Published by Elsevier B.V.

Dark Endothelial Spots After Descemet Membrane Endothelial Keratoplasty May Appear as Recurrent Fuchs Dystrophy or Herald Graft Failure or Rejection.

PubMed

Zygoura, Vasiliki; Baydoun, Lamis; Monnereau, Claire; Satué, Maria; Oellerich, Silke; Melles, Gerrit R J

2017-12-01

To evaluate the clinical significance of dark spots in the donor endothelial cell layer as observed with specular microscopy, in patients who underwent Descemet membrane endothelial keratoplasty (DMEK) for Fuchs endothelial dystrophy (FED). Specular microscopy images of 83 consecutive eyes up to 7 years after DMEK were retrospectively reviewed in a masked fashion for the presence of dark spots and morphologic changes in the endothelial cell layer and processed for endothelial cell density (ECD) measurements. A normal endothelial cell layer was found in 52/83 eyes (62.7%) (group 0). In the remaining 31/83 eyes, various dark discolorations with or without altered endothelial cell morphology were categorized into 4 groups. Dark spots were classified as artifacts in 10/83 (12.0%) eyes (group I) and as "superimposed" dots in 10/83 (12.0%) eyes (group II), that is, optical irregularities slightly anterior to a healthy endothelial cell layer. In 11/83 (13.3%) eyes, endothelial stress was characterized by dark grayish discolorations and/or nuclear activation (group III). Most of the latter eyes also had a significant ECD decrease; 3 of these eyes later developed secondary graft failure, of which one was preceded by allograft rejection. None of the eyes showed recurrent guttae typical for FED (group IV). Dark endothelial spots after DMEK for FED may not represent a recurrent disease, but tissue irregularities just anterior to the graft. However, if associated with changes in endothelial cell morphology, nuclear activation and/or ECD decrease, dark discolorations may reflect "cellular stress" heralding secondary graft failure or (subclinical) allograft rejection.

COPD as an endothelial disorder: endothelial injury linking lesions in the lungs and other organs? (2017 Grover Conference Series)

PubMed Central

Polverino, Francesca; Celli, Bartolome R.

2018-01-01

Chronic obstructive pulmonary disease (COPD) is characterized by chronic expiratory airflow obstruction that is not fully reversible. COPD patients develop varying degrees of emphysema, small and large airway disease, and various co-morbidities. It has not been clear whether these co-morbidities share common underlying pathogenic processes with the pulmonary lesions. Early research into the pathogenesis of COPD focused on the contributions of injury to the extracellular matrix and pulmonary epithelial cells. More recently, cigarette smoke-induced endothelial dysfunction/injury have been linked to the pulmonary lesions in COPD (especially emphysema) and systemic co-morbidities including atherosclerosis, pulmonary hypertension, and chronic renal injury. Herein, we review the evidence linking endothelial injury to COPD, and the pathways underlying endothelial injury and the “vascular COPD phenotype” including: (1) direct toxic effects of cigarette smoke on endothelial cells; (2) generation of auto-antibodies directed against endothelial cells; (3) vascular inflammation; (4) increased oxidative stress levels in vessels inducing increases in lipid peroxidation and increased activation of the receptor for advanced glycation end-products (RAGE); (5) reduced activation of the anti-oxidant pathways in endothelial cells; (6) increased endothelial cell release of mediators with vasoconstrictor, pro-inflammatory, and remodeling activities (endothelin-1) and reduced endothelial cell expression of mediators that promote vasodilation and homeostasis of endothelial cells (nitric oxide synthase and prostacyclin); and (7) increased endoplasmic reticular stress and the unfolded protein response in endothelial cells. We also review the literature on studies of drugs that inhibit RAGE signaling in other diseases (angiotensin-converting enzyme inhibitors and angiotensin receptor blockers), or vasodilators developed for idiopathic pulmonary arterial hypertension that have been tested on cell culture systems, animal models of COPD, and/or smokers and COPD patients. PMID:29468936

Cardiomyocyte exosomes regulate glycolytic flux in endothelium by direct transfer of GLUT transporters and glycolytic enzymes.

PubMed

Garcia, Nahuel A; Moncayo-Arlandi, Javier; Sepulveda, Pilar; Diez-Juan, Antonio

2016-03-01

Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship, which is essential for maintaining the metabolic requirements of the heart. Little is known about the mechanisms that regulate nutrient flow from ECs to associated CMs, especially in situations of acute stress when local active processes are required to regulate endothelial transport. We examined whether CM-derived exosomes can modulate glucose transport and metabolism in ECs. In conditions of glucose deprivation, CMs increase the synthesis and secretion of exosomes. These exosomes are loaded with functional glucose transporters and glycolytic enzymes, which are internalized by ECs, leading to increased glucose uptake, glycolytic activity, and pyruvate production in recipient cells. These findings establish CM-derived exosomes as key components of the cardio-endothelial communication system which, through intercellular protein complementation, would allow a rapid response from ECs to increase glucose transport and a putative uptake of metabolic fuels from blood to CMs. This CM-EC protein complementation process might have implications for metabolic regulation in health and disease. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

Endothelial-derived interleukin-6 induces cancer stem cell motility by generating a chemotactic gradient towards blood vessels.

PubMed

Kim, Hong Sun; Chen, Yu-Chih; Nör, Felipe; Warner, Kristy A; Andrews, April; Wagner, Vivian P; Zhang, Zhaocheng; Zhang, Zhixiong; Martins, Manoela D; Pearson, Alexander T; Yoon, Euisik; Nör, Jacques E

2017-11-21

Recent evidence suggests that the metastatic spread of head and neck squamous cell carcinomas (HNSCC) requires the function of cancer stem cells endowed with multipotency, self-renewal, and high tumorigenic potential. We demonstrated that cancer stem cells reside in perivascular niches and are characterized by high aldehyde dehydrogenase (ALDH) activity and high CD44 expression (ALDH high CD44 high ) in HNSCC. Here, we hypothesize that endothelial cell-secreted interleukin-6 (IL-6) contributes to tumor progression by enhancing the migratory phenotype and survival of cancer stem cells. Analysis of tissue microarrays generated from the invasive fronts of 77 HNSCC patients followed-up for up to 11 years revealed that high expression of IL-6 receptor (IL-6R) (p=0.0217) or co-receptor gp130 (p=0.0422) correlates with low HNSCC patient survival. We observed that endothelial cell-secreted factors induce epithelial to mesenchymal transition (EMT) and enhance invasive capacity of HNSCC cancer stem cells. Conditioned medium from CRISPR/Cas9-mediated IL-6 knockout primary human endothelial cells is less chemotactic for cancer stem cells in a microfluidics-based system than medium from control endothelial cells (p<0.05). Blockade of the IL-6 pathway with a humanized anti-IL-6R antibody (tocilizumab) inhibited endothelial cell-induced motility in vitro and decreased the fraction of cancer stem cells in vivo . Notably, xenograft HNSCC tumors vascularized with IL-6-knockout endothelial cells exhibited slower tumor growth and smaller cancer stem cell fraction. These findings demonstrate that endothelial cell-secreted IL-6 enhances the motility and survival of highly tumorigenic cancer stem cells, suggesting that endothelial cells can create a chemotactic gradient that enables the movement of carcinoma cells towards blood vessels.

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

PubMed Central

Xie, Liping; Liu, Zhen; Lu, Hui; Zhang, Wen; Mi, Qiongyu; Li, Xiaozhen; Tang, Yan; Chen, Qi; Ferro, Albert; Ji, Yong

2012-01-01

BACKGROUND AND PURPOSE One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O2•−) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism. EXPERIMENTAL APPROACH HUVECs were incubated with ox-LDL with/without pyridoxine, NG-nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O2•− was measured using lucigenin chemiluminescence, and O2•−-sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot. KEY RESULTS Ox-LDL significantly increased O2•− production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O2•− generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments. CONCLUSIONS AND IMPLICATIONS Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction. PMID:21797845

Endothelial cells genetically selected from differentiating mouse embryonic stem cells incorporate at sites of neovascularization in vivo.

PubMed

Marchetti, Sandrine; Gimond, Clotilde; Iljin, Kristiina; Bourcier, Christine; Alitalo, Kari; Pouysségur, Jacques; Pagès, Gilles

2002-05-15

Large scale purification of endothelial cells is of great interest as it could improve tissue transplantation, reperfusion of ischemic tissues and treatment of pathologies in which an endothelial cell dysfunction exists. In this study, we describe a novel genetic approach that selects for endothelial cells from differentiating embryonic stem (ES) cells. Our strategy is based on the establishment of ES-cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter, tie-1. Using EGFP as a reporter gene, we first confirmed the endothelial specificity of the tie-1 promoter in the embryoid body model and in cells differentiated in 2D cultures. Subsequently, tie-1-EGFP ES cells were used as recipients for the tie-1-driven puror transgene. The resulting stable clones were expanded and differentiated for seven days in the presence of VEGF before puromycin selection. As expected, puromycin-resistant cells were positive for EGFP and also expressed several endothelial markers, including CD31, CD34, VEGFR-1, VEGFR-2, Tie-1, VE-cadherin and ICAM-2. Release from the puromycin selection resulted in the appearance of alpha-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-beta1, two known inducers of smooth muscle cell differentiation. The hypothesis that endothelial cells or their progenitors may differentiate towards a smooth muscle cell phenotype was further supported by the presence of cells expressing both CD31 and alpha-smooth muscle actin markers. Finally, we show that purified endothelial cells can incorporate into the neovasculature of transplanted tumors in nude mice. Taken together, these results suggest that application of endothelial lineage selection to differentiating ES cells may become a useful approach for future pro-angiogenic and endothelial cell replacement therapies.

Angiogenesis mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1

NASA Astrophysics Data System (ADS)

Koch, Alisa E.; Halloran, Margaret M.; Haskell, Catherine J.; Shah, Manisha R.; Polverini, Peter J.

1995-08-01

ENDOTHELIAL adhesion molecules facilitate the entry of leukocytes into inflamed tissues. This in turn promotes neovascularization, a process central to the progression of rheumatoid arthritis, tumour growth and wound repair1. Here we test the hypothesis that soluble endothelial adhesion molecules promote angiogenesis2á¤-4. Human recombinant soluble E-selectin and soluble vascular cell adhesion molecule-1 induced chemotaxis of human endothelial cells in vitro and were angiogenic in rat cornea. Soluble E-selectin acted on endothelial cells in part through a sialyl Lewis-X-dependent mechanism, while soluble vascular cell adhesion molecule-1 acted on endothelial cells in part through a very late antigen (VLA)-4 dependent mechanism. The chemotactic activity of rheumatoid synovial fluid for endothelial cells, and also its angiogenic activity, were blocked by antibodies to either soluble E-selectin or soluble vascular cell adhesion molecule-1. These results suggest a novel function for soluble endothelial adhesion molecules as mediators of angiogenesis.

Gestational diabetes mellitus alters maternal and neonatal circulating endothelial progenitor cell subsets.

PubMed

Acosta, Juan C; Haas, David M; Saha, Chandan K; Dimeglio, Linda A; Ingram, David A; Haneline, Laura S

2011-03-01

The purpose of this study was to examine whether women with gestational diabetes mellitus (GDM) and their offspring have reduced endothelial progenitor cell subsets and vascular reactivity. Women with GDM, healthy control subjects, and their infants participated. Maternal blood and cord blood were assessed for colony-forming unit-endothelial cells and endothelial progenitor cell subsets with the use of polychromatic flow cytometry. Cord blood endothelial colony-forming cells were enumerated. Vascular reactivity was tested by laser Doppler imaging. Women with GDM had fewer CD34, CD133, CD45, and CD31 cells (circulating progenitor cells [CPCs]) at 24-32 weeks' gestation and 1-2 days after delivery, compared with control subjects. No differences were detected in colony-forming unit-endothelial cells or colony-forming unit-endothelial cells. In control subjects, CPCs were higher in the third trimester, compared with the postpartum period. Cord blood from GDM pregnancies had reduced CPCs. Vascular reactivity was not different between GDM and control subjects. The normal physiologic increase in CPCs during pregnancy is impaired in women with GDM, which may contribute to endothelial dysfunction and GDM-associated morbidities. Copyright © 2011 Mosby, Inc. All rights reserved.

Dermal Stem Cells Can Differentiate Down an Endothelial Lineage

PubMed Central

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

2012-01-01

In this study, we have demonstrated that cells of neural crest origin located in the dermal papilla (DP) exhibit endothelial marker expression and a functional activity. When grown in endothelial growth media, DP primary cultures upregulate expression of vascular endothelial growth factor receptor 1 (FLT1) mRNA and downregulate expression of the dermal stem cell marker α-smooth muscle actin. DP cells have demonstrated functional characteristics of endothelial cells, including the ability to form capillary-like structures on Matrigel, increase uptake of low-density lipoprotein and upregulate ICAM1 (CD54) in response to tumour necrosis factor alpha (TNF-α) stimulation. We confirmed that these observations were not due to contaminating endothelial cells, by using DP clones. We have also used the WNT1cre/ROSA26R and WNT1cre/YFP lineage-tracing mouse models to identify a population of neural crest-derived cells in DP cultures that express the endothelial marker PECAM (CD31); these cells also form capillary-like structures on Matrigel. Importantly, cells of neural crest origin that express markers of endothelial and mesenchymal lineages exist within the dermal sheath of the vibrissae follicle. PMID:22571645

Towards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes.

PubMed

Menzel, Sarah; Finocchiaro, Nicole; Donay, Christine; Thiebes, Anja Lena; Hesselmann, Felix; Arens, Jutta; Djeljadini, Suzana; Wessling, Matthias; Schmitz-Rode, Thomas; Jockenhoevel, Stefan; Cornelissen, Christian Gabriel

2017-01-01

In patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.

Expression of an insulin-regulatable glucose carrier in muscle and fat endothelial cells

NASA Astrophysics Data System (ADS)

Vilaró, Senen; Palacín, Manuel; Pilch, Paul F.; Testar, Xavier; Zorzano, Antonio

1989-12-01

INSULIN rapidly stimulates glucose use in the major target tissues, muscle and fat, by modulating a tissue-specific glucose transporter isoform1-6. Access of glucose to the target tissue is restricted by endothelial cells which line the walls of nonfenestrated capillaries of fat and muscle7. Thus, we examined whether the capillary endothelial cells are actively involved in the modulation of glucose availability by these tissues. We report here the abundant expression of the muscle/fat glucose transporter isoform in endothelial cells, using an immunocytochemical analysis with a monoclonal antibody specific for this isoform1. This expression is restricted to endothelial cells from the major insulin target tissues, and it is not detected in brain and liver where insulin does not activate glucose transport. The expression of the muscle/fat transporter isoform in endothelial cells is significantly greater than in the neighbouring muscle and fat cells. Following administration of insulin to animals in vivo, there occurs a rapid increase in the number of muscle/fat transporters present in the lumenal plasma membrane of the capillary endothelial cells. These results document that insulin promotes the translocation of the muscle/fat glucose transporter in endothelial cells. It is therefore likely that endothelial cells play an important role in the regulation of glucose use by the major insulin target tissues in normal and diseased states.

Calcium supplementation prevents endothelial cell activation: possible relevance to preeclampsia.

PubMed

Chen, Qi; Tong, Mancy; Wu, Man; Stone, Peter R; Snowise, Saul; Chamley, Lawrence W

2013-09-01

Preeclampsia is a leading cause of maternal and fetal mortality and morbidity. A hallmark of preeclampsia is endothelial cell dysfunction/activation in response to 'toxins' from the placenta. Necrotic trophoblastic debris (NTD) is one possible placental toxin and other activators of endothelial cells include inflammatory cytokines. Calcium supplementation appears to protect 'at-risk' women from developing preeclampsia but how is unclear. Placental explants were cultured with interleukin-6 (IL-6) in varied concentrations of calcium. The resultant trophoblastic debris was exposed to endothelial cells. Endothelial cells were exposed to activators including NTD, IL-6, and preeclamptic sera in the presence of varied concentrations of calcium and activation monitored by quantifying cell surface markers by ELISA. Raising the levels of calcium did not prevent the IL-6-induced shedding of NTD from placental explants but did prevent the activation of endothelial cells in response to IL-6, preeclamptic sera, or NTD. Reducing the level of calcium directly induced the activation of endothelial cells. Inhibiting nitric oxide synthetase ablated the ability of high calcium levels to protect endothelial cell activation. The activity of endothelial cell nitric oxide synthetase was blocked with L-N-nitroarginine methyl ester. Our results demonstrate calcium levels do not affect the shedding of trophoblastic debris but are important to endothelial cell activation and supplemental calcium may reverse the activation of the endothelium in preeclamptic women. These results may in part explain the benefits of calcium supplementation in the reduction of risk for developing preeclampsia and provide in-vitro mechanistic support for the use of calcium supplementation in at-risk women.

Flavorings in Tobacco Products Induce Endothelial Cell Dysfunction.

PubMed

Fetterman, Jessica L; Weisbrod, Robert M; Feng, Bihua; Bastin, Reena; Tuttle, Shawn T; Holbrook, Monica; Baker, Gregory; Robertson, Rose Marie; Conklin, Daniel J; Bhatnagar, Aruni; Hamburg, Naomi M

2018-06-14

Use of alternative tobacco products including electronic cigarettes is rapidly rising. The wide variety of flavored tobacco products available is of great appeal to smokers and youth. The flavorings added to tobacco products have been deemed safe for ingestion, but the cardiovascular health effects are unknown. The purpose of this study was to examine the effect of 9 flavors on vascular endothelial cell function. Freshly isolated endothelial cells from participants who use nonmenthol- or menthol-flavored tobacco cigarettes showed impaired A23187-stimulated nitric oxide production compared with endothelial cells from nonsmoking participants. Treatment of endothelial cells isolated from nonsmoking participants with either menthol (0.01 mmol/L) or eugenol (0.01 mmol/L) decreased A23187-stimulated nitric oxide production. To further evaluate the effects of flavoring compounds on endothelial cell phenotype, commercially available human aortic endothelial cells were incubated with vanillin, menthol, cinnamaldehyde, eugenol, dimethylpyrazine, diacetyl, isoamyl acetate, eucalyptol, and acetylpyrazine (0.1-100 mmol/L) for 90 minutes. Cell death, reactive oxygen species production, expression of the proinflammatory marker IL-6 (interleukin-6), and nitric oxide production were measured. Cell death and reactive oxygen species production were induced only at high concentrations unlikely to be achieved in vivo. Lower concentrations of selected flavors (vanillin, menthol, cinnamaldehyde, eugenol, and acetylpyridine) induced both inflammation and impaired A23187-stimulated nitric oxide production consistent with endothelial dysfunction. Our data suggest that short-term exposure of endothelial cells to flavoring compounds used in tobacco products have adverse effects on endothelial cell phenotype that may have relevance to cardiovascular toxicity. © 2018 American Heart Association, Inc.

Somatic GNAQ Mutation is Enriched in Brain Endothelial Cells in Sturge-Weber Syndrome.

PubMed

Huang, Lan; Couto, Javier A; Pinto, Anna; Alexandrescu, Sanda; Madsen, Joseph R; Greene, Arin K; Sahin, Mustafa; Bischoff, Joyce

2017-02-01

Sturge-Weber syndrome (SWS) is a rare congenital neurocutaneous disorder characterized by facial and extracraniofacial capillary malformations and capillary-venule malformations in the leptomeninges. A somatic mosaic mutation in GNAQ (c.548G>A; p.R183Q) was found in SWS brain and skin capillary malformations. Our laboratory showed endothelial cells in skin capillary malformations are enriched for the GNAQ mutation. The purpose of this study is to determine whether the GNAQ mutation is also enriched in endothelial cells in affected SWS brain. Two human SWS brain specimens were fractionated by fluorescence-activated cell sorting into hematopoietic (CD45), endothelial (CD31, VE-Cadherin, and vascular endothelial growth factor receptor 2), and perivascular (platelet-derived growth factor receptor beta) cells and cells negative for all markers. The sorted cell populations were analyzed for GNAQ p.R183Q mutation by droplet digital polymerase chain reaction. SWS patient-derived brain endothelial cells were selected by anti-CD31-coated magnetic beads and cultured in endothelial growth medium in vitro. The GNAQ p.R183Q mutation was present in brain endothelial cells in two SWS specimens, with mutant allelic frequencies of 34.7% and 24.0%. Cells negative for all markers also harbored the GNAQ mutation. The mutant allelic frequencies in these unidentified cells were 9.2% and 8.4%. SWS patient-derived brain endothelial cells with mutant allelic frequencies of 14.7% and 21% survived and proliferated in vitro. Our study provides evidence that GNAQ p.R183Q mutation is enriched in endothelial cells in SWS brain lesions and thereby reveals endothelial cells as a source of aberrant Gαq signaling. This will help to understand the pathophysiology of SWS, to discover biomarkers for predicting cerebral involvement, and to develop therapeutic targets to prevent neurological impairments in SWS. Copyright © 2016 Elsevier Inc. All rights reserved.

Loss of the endothelial glycocalyx is associated with increased E-selectin mediated adhesion of lung tumour cells to the brain microvascular endothelium.

PubMed

Rai, Srijana; Nejadhamzeeigilani, Zaynab; Gutowski, Nicholas J; Whatmore, Jacqueline L

2015-09-25

Arrest of metastasising lung cancer cells to the brain microvasculature maybe mediated by interactions between ligands on circulating tumour cells and endothelial E-selectin adhesion molecules; a process likely to be regulated by the endothelial glycocalyx. Using human cerebral microvascular endothelial cells and non-small cell lung cancer (NSCLC) cell lines, we describe how factors secreted by NSCLC cells i.e. cystatin C, cathepsin L, insulin-like growth factor-binding protein 7 (IGFBP7), vascular endothelial growth factor (VEGF) and tumour necrosis factor-alpha (TNF-α), damage the glycocalyx and enhance initial contacts between lung tumour and cerebral endothelial cells. Endothelial cells were treated with tumour secreted-proteins or lung tumour conditioned medium (CM). Surface levels of E-selectin were quantified by ELISA. Adhesion of A549 and SK-MES-1 cells was examined under flow conditions (1 dyne/cm(2)). Alterations in the endothelial glycocalyx were quantified by binding of fluorescein isothiocyanate-linked wheat germ agglutinin (WGA-FITC). A549 and SK-MES-1 CM and secreted-proteins significantly enhanced endothelial surface E-selectin levels after 30 min and 4 h and tumour cell adhesion after 30 min, 4 and 24 h. Both coincided with significant glycocalyx degradation; A549 and SK-MES-1 CM removing 55 ± 12 % and 58 ± 18.7 % of WGA-FITC binding, respectively. Inhibition of E-selectin binding by monoclonal anti-E-selectin antibody completely attenuated tumour cell adhesion. These data suggest that metastasising lung cancer cells facilitate their own adhesion to the brain endothelium by secreting factors that damage the endothelial glycocalyx, resulting in exposure of the previously shielded adhesion molecules and engagement of the E-selectin-mediated adhesion axis.

In Vitro Impact of Conditioned Medium From Demineralized Freeze-Dried Bone on Human Umbilical Endothelial Cells.

PubMed

Harnik, Branko; Miron, Richard J; Buser, Daniel; Gruber, Reinhard

2017-03-01

Angiogenesis is essential for the consolidation of bone allografts. The underlying molecular mechanism, however, remains unclear. Soluble factors released from demineralized freeze-dried bone target mesenchymal cells; however, their effect on endothelial cells has not been investigated so far. The aim of the present study was therefore to examine the effect of conditioned medium from demineralized freeze-dried bone on human umbilical endothelial cells in vitro. Conditioned medium was first prepared from demineralized freeze-dried bone following 24 hours incubation at room temperature to produce demineralized bone conditioned media. Thereafter, conditioned medium was used to stimulate human umbilical vein endothelial cells in vitro by determining the cell response based on viability, proliferation, expression of apoptotic genes, a Boyden chamber to determine cell migration, and the formation of branches. The authors report here that conditioned medium decreased viability and proliferation of endothelial cells. Neither of the apoptotic marker genes was significantly altered when endothelial cells were exposed to conditioned medium. The Boyden chamber revealed that endothelial cells migrate toward conditioned medium. Moreover, conditioned medium moderately stimulated the formation of branches. These findings support the concept that conditioned medium from demineralized freeze-dried bone targets endothelial cells by decreasing their proliferation and enhancing their motility under these in vitro conditions.

Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells

PubMed Central

Kobayashi, Hideki; Butler, Jason M.; O'Donnell, Rebekah; Kobayashi, Mariko; Ding, Bi-Sen; Bonner, Bryant; Chiu, Vi K.; Nolan, Daniel J.; Shido, Koji; Benjamin, Laura; Rafii, Shahin

2010-01-01

Endothelial cells establish an instructive vascular niche that reconstitutes haematopoietic stem and progenitor cells (HSPCs) through release of specific paracrine growth factors, known as angiocrine factors. However, the mechanism by which endothelial cells balance the rate of proliferation and lineage-specific differentiation of HSPCs is unknown. Here, we demonstrate that Akt activation in endothelial cells, through recruitment of mTOR, but not the FoxO pathway, upregulates specific angiocrine factors that support expansion of CD34−Flt3− KLS HSPCs with long-term haematopoietic stem cell (LT-HSC) repopulation capacity. Conversely, co-activation of Akt-stimulated endothelial cells with p42/44 MAPK shifts the balance towards maintenance and differentiation of the HSPCs. Selective activation of Akt1 in the endothelial cells of adult mice increased the number of colony forming units in the spleen and CD34−Flt3− KLS HSPCs with LT-HSC activity in the bone marrow, accelerating haematopoietic recovery. Therefore, the activation state of endothelial cells modulates reconstitution of HSPCs through the upregulation of angiocrine factors, with Akt–mTOR-activated endothelial cells supporting the self-renewal of LT-HSCs and expansion of HSPCs, whereas MAPK co-activation favours maintenance and lineage-specific differentiation of HSPCs. PMID:20972423

Hyperglycemia-induced PATZ1 negatively modulates endothelial vasculogenesis via repression of FABP4 signaling.

PubMed

Chen, Ren-An; Sun, Xiao-Mian; Yan, Chang-You; Liu, Li; Hao, Miao-Wang; Liu, Qiang; Jiao, Xi-Ying; Liang, Ying-Min

2016-09-02

Vascular endothelial dysfunction, a central hallmark of diabetes, predisposes diabetic patients to numerous cardiovascular complications. The POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1), is an important transcriptional regulatory factor and regulates divergent pathways depending on the cellular context, but its role in endothelial cells remains poorly understood. Herein, we report for the first time that endothelial PATZ1 expression was abnormally upregulated in diabetic endothelial cells (ECs) regardless of diabetes classification. This stimulatory effect was further confirmed in the high glucose-treated human umbilical vein endothelial cells (HUVECs). From a functional standpoint, transgenic overexpression of PATZ1 in endothelial colony forming cells (ECFCs) blunted angiogenesis in vivo and rendered endothelial cells unresponsive to established angiogenic factors. Mechanistically, PATZ1 acted as a potent transcriptional corepressor of fatty acid-binding protein 4 (FABP4), an essential convergence point for angiogenic and metabolic signaling pathways in ECs. Taken together, endothelial PATZ1 thus potently inhibits endothelial function and angiogenesis via inhibition of FABP4 expression, and abnormal induction of endothelial PATZ1 may contribute to multiple aspects of vascular dysfunction in diabetes. Copyright © 2016. Published by Elsevier Inc.

Toll-like receptor 4-induced endoplasmic reticulum stress contributes to endothelial dysfunction

USDA-ARS?s Scientific Manuscript database

Impairment of vasodilator action of insulin is associated with endothelial dysfunction and insulin resistance. Endoplasmic reticulum (ER) stress is implicated as one of the mechanisms for pathophysiology of various cardiometabolic syndromes, including insulin resistance and endothelial dysfunction. ...

Vascular Gene Expression in Nonneoplastic and Malignant Brain

PubMed Central

Madden, Stephen L.; Cook, Brian P.; Nacht, Mariana; Weber, William D.; Callahan, Michelle R.; Jiang, Yide; Dufault, Michael R.; Zhang, Xiaoming; Zhang, Wen; Walter-Yohrling, Jennifer; Rouleau, Cecile; Akmaev, Viatcheslav R.; Wang, Clarence J.; Cao, Xiaohong; St. Martin, Thia B.; Roberts, Bruce L.; Teicher, Beverly A.; Klinger, Katherine W.; Stan, Radu-Virgil; Lucey, Brenden; Carson-Walter, Eleanor B.; Laterra, John; Walter, Kevin A.

2004-01-01

Malignant gliomas are uniformly lethal tumors whose morbidity is mediated in large part by the angiogenic response of the brain to the invading tumor. This profound angiogenic response leads to aggressive tumor invasion and destruction of surrounding brain tissue as well as blood-brain barrier breakdown and life-threatening cerebral edema. To investigate the molecular mechanisms governing the proliferation of abnormal microvasculature in malignant brain tumor patients, we have undertaken a cell-specific transcriptome analysis from surgically harvested nonneoplastic and tumor-associated endothelial cells. SAGE-derived endothelial cell gene expression patterns from glioma and nonneoplastic brain tissue reveal distinct gene expression patterns and consistent up-regulation of certain glioma endothelial marker genes across patient samples. We define the G-protein-coupled receptor RDC1 as a tumor endothelial marker whose expression is distinctly induced in tumor endothelial cells of both brain and peripheral vasculature. Further, we demonstrate that the glioma-induced gene, PV1, shows expression both restricted to endothelial cells and coincident with endothelial cell tube formation. As PV1 provides a framework for endothelial cell caveolar diaphragms, this protein may serve to enhance glioma-induced disruption of the blood-brain barrier and transendothelial exchange. Additional characterization of this extensive brain endothelial cell gene expression database will provide unique molecular insights into vascular gene expression. PMID:15277233

Pericyte-derived sphingosine 1-phosphate induces the expression of adhesion proteins and modulates the retinal endothelial cell barrier.

PubMed

McGuire, Paul G; Rangasamy, Sampathkumar; Maestas, Joann; Das, Arup

2011-12-01

The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier. Human retinal microvascular endothelial cells were cocultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte-conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate. Sphingosine 1-phosphate aids in maintenance of microvascular stability by upregulating the expression of N-cadherin and VE-cadherin, and downregulating the expression of angiopoietin 2. Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of sphingosine 1-phosphate. Alteration of pericyte-derived sphingosine 1-phosphate production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability.

In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

PubMed

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

2016-01-01

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

Theobroma cacao increases cells viability and reduces IL-6 and sVCAM-1 level in endothelial cells induced by plasma from preeclamptic patients.

PubMed

Rahayu, Budi; Baktiyani, Siti Candra Windu; Nurdiana, Nurdiana

2016-01-01

This study aims to investigate whether an ethanolic extract of Theobroma cacao bean is able to increase cell viability and decrease IL-6 and sVCAM-1 in endothelial cells induced by plasma from preeclamptic patients. Endothelial cells were obtained from human umbilical vascular endothelial cells. At confluency, endothelial cells were divided into six groups, which included control (untreated), endothelial cells exposed to plasma from normal pregnancy, endothelial cells exposed to 2% plasma from preeclamptic patients (PP), endothelial cells exposed to PP in the presence of ethanolic extract of T. cacao (PP+TC) at the following three doses: 25, 50, and 100 ppm. The analysis was performed in silico using the Hex 8.0, LigPlus and LigandScout 3.1 software. Analysis on IL-6 and sVCAM-1 levels were done by enzyme linked immunosorbent assay (ELISA). We found that seven of them could bind to the protein NFκB (catechin, leucoanthocyanidin, niacin, phenylethylamine, theobromine, theophylline, and thiamin). This increase in IL-6 was significantly (P<0.05) attenuated by both the 50 and 100 ppm treatments of T. cacao extract. Plasma from PP significantly increased sVCAM-1 levels compared to untreated cells. This increase in sVCAM-1 was significantly attenuated by all doses of the extract. In conclusion, T. cacao extract prohibits the increase in IL-6 and sVCAM-1 in endothelial cells induced by plasma from preeclamptic patients. Therefore this may provide a herbal therapy for attenuating the endothelial dysfunction found in preeclampsia. Copyright © 2016 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.

New insights into antimetastatic and antiangiogenic effects of cannabinoids.

PubMed

Ramer, Robert; Hinz, Burkhard

2015-01-01

Cannabinoids exert antitumorigenic effects via multiple mechanisms. Of these, antimetastatic and antiangiogenic actions have attracted considerable interest in the past years. Regarding the underlying antimetastatic mechanism, several studies revealed cannabinoids to alter the gene expression of cancer cells toward a less-aggressive phenotype and to modulate their secretomic profile. Cannabinoids likewise modulate the release of factors from tumor cells that subsequently suppress the chemoattraction of vessel cells thereby conferring antiangiogenesis. Among the diverse mediators of cannabinoids' antitumorigenic action, the tissue inhibitor of matrix metalloproteinases-1, which is released from cancer cells upon cannabinoid treatment, has been implicated as a pivotal factor conferring both anti-invasive properties of cancer cells as well as antiangiogenic capacities of endothelial cells. In addition, cannabinoids have been shown to inhibit angiogenic capacities of endothelial cells directly via suppressing their proliferation, tube formation, and migration. This chapter reviews the cell- and substance-specific antitumorigenic mechanisms of cannabinoids with particular consideration of their antimetastatic/anti-invasive and antiangiogenic actions. In addition, beneficial interactions of cannabinoids with currently used chemotherapeutics as well as the influence of cannabinoids on tumor-immune surveillance are addressed. Collectively, the currently available data suggest cannabinoids as a potential tool in modern cancer pharmacotherapy. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantifying effects of cyclic stretch on cell-collagen substrate adhesiveness of vascular endothelial cells.

PubMed

Omidvar, Ramin; Tafazzoli-Shadpour, Mohammad; Mahmoodi-Nobar, Farbod; Azadi, Shohreh; Khani, Mohammad-Mehdi

2018-05-01

Vascular endothelium is continuously subjected to mechanical stimulation in the form of shear forces due to blood flow as well as tensile forces as a consequence of blood pressure. Such stimuli influence endothelial behavior and regulate cell-tissue interaction for an optimized functionality. This study aimed to quantify influence of cyclic stretch on the adhesive property and stiffness of endothelial cells. The 10% cyclic stretch with frequency of 1 Hz was applied to a layer of endothelial cells cultured on a polydimethylsiloxane substrate. Cell-substrate adhesion of endothelial cells was examined by the novel approach of atomic force microscope-based single-cell force spectroscopy and cell stiffness was measured by atomic force microscopy. Furthermore, the adhesive molecular bonds were evaluated using modified Hertz contact theory. Our results show that overall adhesion of endothelial cells with substrate decreased after cyclic stretch while they became stiffer. Based on the experimental results and theoretical modeling, the decrease in the number of molecular bonds after cyclic stretch was quantified. In conclusion, in vitro cyclic stretch caused alterations in both adhesive capacity and elastic modulus of endothelial cells through mechanotransductive pathways as two major determinants of the function of these cells within the cardiovascular system.

Dual inhibition of mTORC1 and mTORC2 perturbs cytoskeletal organization and impairs endothelial cell elongation.

PubMed

Tsuji-Tamura, Kiyomi; Ogawa, Minetaro

2018-02-26

Elongation of endothelial cells is an important process in vascular formation and is expected to be a therapeutic target for inhibiting tumor angiogenesis. We have previously demonstrated that inhibition of mTORC1 and mTORC2 impaired endothelial cell elongation, although the mechanism has not been well defined. In this study, we analyzed the effects of the mTORC1-specific inhibitor everolimus and the mTORC1/mTORC2 dual inhibitor KU0063794 on the cytoskeletal organization and morphology of endothelial cell lines. While both inhibitors equally inhibited cell proliferation, KU0063794 specifically caused abnormal accumulation of F-actin and disordered distribution of microtubules, thereby markedly impairing endothelial cell elongation and tube formation. The effects of KU0063794 were phenocopied by paclitaxel treatment, suggesting that KU0063794 might impair endothelial cell morphology through over-stabilization of microtubules. Although mTORC1 is a key signaling molecule in cell proliferation and has been considered a target for preventing angiogenesis, mTORC1 inhibitors have not been sufficient to suppress angiogenesis. Our results suggest that mTORC1/mTORC2 dual inhibition is more effective for anti-angiogenic therapy, as it impairs not only endothelial cell proliferation, but also endothelial cell elongation. Copyright © 2018 Elsevier Inc. All rights reserved.

Dengue Virus Infection of Mast Cells Triggers Endothelial Cell Activation ▿

PubMed Central

Brown, Michael G.; Hermann, Laura L.; Issekutz, Andrew C.; Marshall, Jean S.; Rowter, Derek; Al-Afif, Ayham; Anderson, Robert

2011-01-01

Vascular perturbation is a hallmark of severe forms of dengue disease. We show here that antibody-enhanced dengue virus infection of primary human cord blood-derived mast cells (CBMCs) and the human mast cell-like line HMC-1 results in the release of factor(s) which activate human endothelial cells, as evidenced by increased expression of the adhesion molecules ICAM-1 and VCAM-1. Endothelial cell activation was prevented by pretreatment of mast cell-derived supernatants with a tumor necrosis factor (TNF)-specific blocking antibody, thus identifying TNF as the endothelial cell-activating factor. Our findings suggest that mast cells may represent an important source of TNF, promoting vascular endothelial perturbation following antibody-enhanced dengue virus infection. PMID:21068256

Obesity-Induced Endoplasmic Reticulum Stress Causes Lung Endothelial Dysfunction and Promotes Acute Lung Injury.

PubMed

Shah, Dilip; Romero, Freddy; Guo, Zhi; Sun, Jianxin; Li, Jonathan; Kallen, Caleb B; Naik, Ulhas P; Summer, Ross

2017-08-01

Obesity is a significant risk factor for acute respiratory distress syndrome. The mechanisms underlying this association are unknown. We recently showed that diet-induced obese mice exhibit pulmonary vascular endothelial dysfunction, which is associated with enhanced susceptibility to LPS-induced acute lung injury. Here, we demonstrate that lung endothelial dysfunction in diet-induced obese mice coincides with increased endoplasmic reticulum (ER) stress. Specifically, we observed enhanced expression of the major sensors of misfolded proteins, including protein kinase R-like ER kinase, inositol-requiring enzyme α, and activating transcription factor 6, in whole lung and in primary lung endothelial cells isolated from diet-induced obese mice. Furthermore, we found that primary lung endothelial cells exposed to serum from obese mice, or to saturated fatty acids that mimic obese serum, resulted in enhanced expression of markers of ER stress and the induction of other biological responses that typify the lung endothelium of diet-induced obese mice, including an increase in expression of endothelial adhesion molecules and a decrease in expression of endothelial cell-cell junctional proteins. Similar changes were observed in lung endothelial cells and in whole-lung tissue after exposure to tunicamycin, a compound that causes ER stress by blocking N-linked glycosylation, indicating that ER stress causes endothelial dysfunction in the lung. Treatment with 4-phenylbutyric acid, a chemical protein chaperone that reduces ER stress, restored vascular endothelial cell expression of adhesion molecules and protected against LPS-induced acute lung injury in diet-induced obese mice. Our work indicates that fatty acids in obese serum induce ER stress in the pulmonary endothelium, leading to pulmonary endothelial cell dysfunction. Our work suggests that reducing protein load in the ER of pulmonary endothelial cells might protect against acute respiratory distress syndrome in obese individuals.

HDL-transferred microRNA-223 regulates ICAM-1 expression in endothelial cells

PubMed Central

Tabet, Fatiha; Vickers, Kasey C.; Cuesta Torres, Luisa F.; Wiese, Carrie B.; Shoucri, Bassem M.; Lambert, Gilles; Catherinet, Claire; Prado-Lourenco, Leonel; Levin, Michael G.; Thacker, Seth; Sethupathy, Praveen; Barter, Philip J.; Remaley, Alan T.; Rye, Kerry-Anne

2014-01-01

High-density lipoproteins (HDL) have many biological functions, including reducing endothelial activation and adhesion molecule expression. We recently reported that HDL transport and deliver functional microRNAs (miRNA). Here we show that HDL suppresses expression of intercellular adhesion molecule 1 (ICAM-1) through the transfer of miR-223 to endothelial cells. After incubation of endothelial cells with HDL, mature miR-223 levels are significantly increased in endothelial cells and decreased on HDL. However, miR-223 is not transcribed in endothelial cells and is not increased in cells treated with HDL from miR-223−/− mice. HDL inhibit ICAM-1 protein levels, but not in cells pretreated with miR-223 inhibitors. ICAM-1 is a direct target of HDL-transferred miR-223 and this is the first example of an extracellular miRNA regulating gene expression in cells where it is not transcribed. Collectively, we demonstrate that HDL’s anti-inflammatory properties are conferred, in part, through HDL-miR-223 delivery and translational repression of ICAM-1 in endothelial cells. PMID:24576947

Prostaglandin E2 induces expression of P-selectin (CD62P) on cultured human umbilical vein endothelial cells and enhances endothelial binding of CD4-T-cells.

PubMed

Hailer, N P; Oppermann, E; Leckel, K; Cinatl, J; Markus, B H; Blaheta, R A

2000-07-15

Interaction of endothelial P-selectin with sialyl Lewis(x)-glycoprotein or P-selectin glycoprotein ligand (PSGL)-1 on leukocytes represents an early step in leukocyte recruitment. Redistribution of P-selectin to the endothelial cell surface occurs rapidly after challenge with several proinflammatory agents, for example, histamine, leucopterins, or lipopolysaccharide. We present evidence that prostaglandin E2 (PGE2) is an efficient inductor of surface P-selectin on cultured human umbilical vein endothelial cells (HUVEC). The increase in P-selectin-immunoreactivity coincided with redistribution of cytoplasmic P-selectin-reactive granulae to the endothelial cell surface, as visualized by confocal laser microscopic examination. CD4-T-cell adhesion to PGE2-stimulated HUVEC was also enhanced by a factor of 4, and blocking mAb directed against the binding site of P-selectin almost completely abrogated this increase in CD4-T-cell adhesion. In summary, our findings show that liberation of PGE2 is an important inductor of P-selectin surface expression on endothelial cells, resulting in enhanced recruitment of inflammatory cells.

Protective effects of sulphonated formononetin in a rat model of cerebral ischemia and reperfusion injury.

PubMed

Zhu, Haibo; Zou, Libo; Tian, Jingwei; Lin, Fei; He, Jie; Hou, Jian

2014-03-01

Sodium formononetin-3'-sulphonate is a derivative of the plant isoflavone formononetin. The present study aimed to investigate the neuroprotective and angiogenesis effects of sodium formononetin-3'-sulphonate in vivo and in vitro. Treatment with sodium formononetin-3'-sulphonate (3, 7.5, 15, and 30 mg/kg, intravenous injection) could protect the brain from ischemia and reperfusion injury by improving neurological function, suppressing cell apoptosis, and increasing expression levels of vascular endothelial growth factor and platelet endothelial cell adhesion molecule 1 by middle cerebral artery occlusion. Treatment with sodium formononetin-3'-sulphonate (10 and 20 µg/mL) significantly increased cell migration, tube formation, and vascular endothelial growth factor and platelet endothelial cell adhesion molecule levels in human umbilical vein endothelial cells. Our results suggest that sodium formononetin-3'-sulphonate provides significant neuroprotective effects against cerebral ischemia and reperfusion injury in rats, and improves cerebrovascular angiogenesis in human umbilical vein endothelial cells. The protective mechanisms of sodium formononetin-3'-sulphonate may be attributed to the suppression of cell apoptosis and improved cerebrovascular angiogenesis by promoting vascular endothelial growth factor and platelet endothelial cell adhesion molecule expression. Georg Thieme Verlag KG Stuttgart · New York.

Involvement of PUMA in pericyte migration induced by methamphetamine.

PubMed

Zhang, Yanhong; Zhang, Yuan; Bai, Ying; Chao, Jie; Hu, Gang; Chen, Xufeng; Yao, Honghong

2017-07-01

Mounting evidence indicates that methamphetamine causes blood-brain barrier damage, with emphasis on endothelial cells. The role of pericytes in methamphetamine-induced BBB damage remains unknown. Our study demonstrated that methamphetamine increased the migration of pericytes from the endothelial basement membrane. However, the detailed mechanisms underlying this process remain poorly understood. Thus, we examined the molecular mechanisms involved in methamphetamine-induced pericyte migration. The results showed that exposure of C3H/10T1/2 cells and HBVPs to methamphetamine increased PUMA expression via activation of the sigma-1 receptor, MAPK and Akt/PI3K pathways. Moreover, methamphetamine treatment resulted in the increased migration of C3H/10T1/2 cells and HBVPs. Knockdown of PUMA in pericytes transduced with PUMA siRNA attenuated the methamphetamine-induced increase in cell migration through attenuation of integrin and tyrosine kinase mechanisms, implicating a role of PUMA in the migration of C3H/10T1/2 cells and HBVPs. This study has demonstrated that methamphetamine-mediated pericytes migration involves PUMA up-regulation. Thus, targeted studies of PUMA could provide insights to facilitate the development of a potential therapeutic approach for alleviation of methamphetamine-induced pericyte migration. Copyright © 2017. Published by Elsevier Inc.

Epigallocatechin 3-gallate inhibits 7-ketocholesterol-induced monocyte-endothelial cell adhesion.

PubMed

Yamagata, Kazuo; Tanaka, Noriko; Suzuki, Koichi

2013-07-01

7-Ketocholesterol (7KC) induces monocytic adhesion to endothelial cells, and induces arteriosclerosis while high-density lipoprotein (HDL) inhibits monocytic adhesion to the endothelium. Epigallocatechin 3-gallate (EGCG) was found to have a protective effect against arteriosclerosis. Therefore, the purpose of this study was to examine the possible HDL-like mechanisms of EGCG in endothelial cells by investigating whether EGCG inhibits 7KC-induced monocyte-endothelial cell adhesion by activating HDL-dependent signal transduction pathways. 7KC and/or EGCG were added to human endothelial cells (ISO-HAS), and the adhesion of pro-monocytic U937 cells was examined. The expression of genes associated with HDL effects such as Ca(2+)/calmodulin-dependent kinase II (CaMKKII), liver kinase B (LKD1), PSD-95/Dlg/ZO-1 kinase 1 (PDZK1), phosphatidylinositol 3-kinase (PI3K), intercellular adhesion molecule-1 (ICAM-1), monocyte chemotactic protein-1 (MCP-1), and endothelial nitric oxide synthase (eNOS) was examined by RT-PCR, and ICAM-1 protein expression was evaluated by western blot (WB). Production of reactive oxygen species (ROS) was examined with H2DCFDA. 7KC significantly induced adhesion of U937 cells to human endothelial cells while significantly increasing gene expressions of ICAM-1 and MCP-1 and decreasing eNOS and CaMKKII gene expressions. EGCG inhibited 7KC-induced monocytic adhesion to endothelial cells, and induced expression of eNOS and several genes involved in the CaMKKII pathway. Stimulation of endothelial cells with EGCG produced intracellular ROS, whereas treatment with N-acetylcysteine (NAC) blocked EGCG-induced expression of eNOS and CaMKKII. These results suggest that inhibition of monocyte-endothelial cell adhesion by EGCG is associated with CaMKKII pathway activation by ROS. Inhibition of 7KC-induced monocyte-endothelial cell adhesion induced by EGCG may function similarly to HDL. Copyright © 2013 Elsevier Inc. All rights reserved.

Apigenin and naringenin ameliorate PKCβII-associated endothelial dysfunction via regulating ROS/caspase-3 and NO pathway in endothelial cells exposed to high glucose.

PubMed

Qin, Weiwei; Ren, Bei; Wang, Shanshan; Liang, Shujun; He, Baiqiu; Shi, Xiaoji; Wang, Liying; Liang, Jingyu; Wu, Feihua

2016-10-01

Endothelial dysfunction is a key event in the progression of atherosclerosis with diabetes. Increasing cell apoptosis may lead to endothelial dysfunction. Apigenin and naringenin are two kinds of widely used flavones. In the present study, we investigated whether and how apigenin and naringenin reduced endothelial dysfunction induced by high glucose in endothelial cells. We showed that apigenin and naringenin protected against endothelial dysfunction via inhibiting phosphorylation of protein kinase C βII (PKCβII) expression and downstream reactive oxygen species (ROS) production in endothelial cells exposed to high glucose. Furthermore, we demonstrated that apigenin and naringenin reduced high glucose-increased apoptosis, Bax expression, caspase-3 activity and phosphorylation of NF-κB in endothelial cells. Moreover, apigenin and naringenin effectively restored high glucose-reduced Bcl-2 expression and Akt phosphorylation. Importantly, apigenin and naringenin significantly increased NO production in endothelial cells subjected to high glucose challenge. Consistently, high glucose stimulation impaired acetylcholine (ACh)-mediated vasodilation in the rat aorta, apigenin and naringenin treatment restored the impaired endothelium-dependent vasodilation via dramatically increasing eNOS activity and nitric oxide (NO) level. Taken together, our results manifest that apigenin and naringenin can ameliorate endothelial dysfunction via regulating ROS/caspase-3 and NO pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Ming-Chung; Department of Anesthesiology, Chi Mei Medical Center, Liouying, Tainan, Taiwan; Chen, Chia-Ling

An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-likemore » cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an increase in peritoneal vascular permeability.« less

Hypotonic shock stimulates ascorbate release from coronary artery endothelial cells by a Ca2+ -independent pathway.

PubMed

Davis, Kim A; Samson, Sue E; Wilson, John X; Grover, Ashok K

2006-10-24

In endothelial cells, anion channels open upon osmotic swelling during shear stress and hypotonic shock. Therefore, we examined the effects of hypotonic shock on release of the antioxidant anion ascorbate from pig coronary artery endothelial cells. Hypotonic shock potentiated ascorbate release from freshly isolated or cultured pig coronary artery endothelial cells; subsequently cultured endothelial cells were used. The hypotonic shock-induced increase in Asc release was rapid, depended on the degree of hypotonic shock, and not due to membrane leakiness. Stimulating P2Y2 like receptors in endothelial cells with ATP causes ascorbate release via a Ca2+ -mediated pathway. Hypotonic shock-induced release differed from the Ca2+-mediated Asc release because: (a) the increase in release with hypotonic shock was additive to that with ATP or A23187 (Ca2+ -ionophore), (b) apyrase, suramin or removing extracellular Ca2+ did not affect the hypotonic shock-stimulated release, (c) anion channel blockers inhibited the release by the two pathways differently, and (d) hypotonic shock increased the ascorbate release from endothelial cells and cultured smooth muscle cells whereas the Ca2+ -mediated ascorbate release occurred only in endothelial cells. Accumulation of ascorbate by endothelial cells was examined at extracellular ascorbate concentrations of 10 (Na+ -ascorbate symporter not saturated) and 5000 microM (Na+ -ascorbate symporter saturated). Hypotonic shock and A23187 decreased ascorbate accumulation at 10 microM ascorbate but increased it at 5000 microM. The effects of the two treatments were additive and also differed from each other with substitution of gluconate for extracellular chloride. Thus, ascorbate release from endothelial cells can be potentiated by two distinct pathways - hypotonic shock mediated and ATP/Ca2+ stimulated.

Role of mitochondrial electron transport complex I in coenzyme Q1 reduction by intact pulmonary arterial endothelial cells and the effect of hyperoxia.

PubMed

Merker, Marilyn P; Audi, Said H; Lindemer, Brian J; Krenz, Gary S; Bongard, Robert D

2007-09-01

The objective was to determine the impact of intact normoxic and hyperoxia-exposed (95% O(2) for 48 h) bovine pulmonary arterial endothelial cells in culture on the redox status of the coenzyme Q(10) homolog coenzyme Q(1) (CoQ(1)). When CoQ(1) (50 microM) was incubated with the cells for 30 min, its concentration in the medium decreased over time, reaching a lower level for normoxic than hyperoxia-exposed cells. The decreases in CoQ(1) concentration were associated with generation of CoQ(1) hydroquinone (CoQ(1)H(2)), wherein 3.4 times more CoQ(1)H(2) was produced in the normoxic than hyperoxia-exposed cell medium (8.2 +/- 0.3 and 2.4 +/- 0.4 microM, means +/- SE, respectively) after 30 min. The maximum CoQ(1) reduction rate for the hyperoxia-exposed cells, measured using the cell membrane-impermeant redox indicator potassium ferricyanide, was about one-half that of normoxic cells (11.4 and 24.1 nmol x min(-1) x mg(-1) cell protein, respectively). The mitochondrial electron transport complex I inhibitor rotenone decreased the CoQ(1) reduction rate by 85% in the normoxic cells and 44% in the hyperoxia-exposed cells. There was little or no inhibitory effect of NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors on CoQ(1) reduction. Intact cell oxygen consumption rates and complex I activities in mitochondria-enriched fractions were also lower for hyperoxia-exposed than normoxic cells. The implication is that intact pulmonary endothelial cells influence the redox status of CoQ(1) via complex I-mediated reduction to CoQ(1)H(2), which appears in the extracellular medium, and that the hyperoxic exposure decreases the overall CoQ(1) reduction capacity via a depression in complex I activity.

Vitamin D rescues dysfunction of fetal endothelial colony forming cells from individuals with gestational diabetes.

PubMed

Gui, J; Rohrbach, A; Borns, K; Hillemanns, P; Feng, L; Hubel, C A; von Versen-Höynck, F

2015-04-01

Gestational diabetes (GDM) is associated with long-term cardiovascular and metabolic diseases in offspring. However, the mechanisms are not well understood. We explored whether fetal exposure to a diabetic environment is associated with fetal endothelial progenitor cell dysfunction, and whether vitamin D can reverse the impairment. Nineteen women with uncomplicated pregnancies and 18 women with GDM were recruited before delivery. Time to first appearance of endothelial colony forming cell (ECFC) colonies and number of ECFC colonies formed from culture of cord peripheral blood mononuclear cells were determined. Angiogenesis-related functions of ECFCs in vitro were tested in the presence or absence of vitamin D. Fetal ECFCs from GDM pregnancies formed fewer colonies in culture (P = 0.04) and displayed reduced proliferation (P = 0.02), migration (P = 0.04) and tubule formation (P = 0.03) compared to uncomplicated pregnancies. Fetal ECFCs exposed to hyperglycemia in vitro exhibited less migration (P < 0.05) and less tubule formation (P < 0.05) than normoglycemic control. Vitamin D significantly improved the dysfunction of fetal ECFCs from pregnancies complicated by GDM or after exposure of healthy ECFCs to hyperglycemia. Fetal ECFCs from GDM pregnancies or ECFCs exposed to hyperglycemia in vitro exhibit reduced quantity and impaired angiogenesis-related functions. Vitamin D significantly rescues these functions. These findings may have implications for vascular function of infants exposed to a diabetic intrauterine environment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Distinct subcellular trafficking resulting from monomeric vs multimeric targeting to endothelial ICAM-1: implications for drug delivery.

PubMed

Ghaffarian, Rasa; Muro, Silvia

2014-12-01

Ligand-targeted, receptor-mediated endocytosis is commonly exploited for intracellular drug delivery. However, cells-surface receptors may follow distinct endocytic fates when bound by monomeric vs multimeric ligands. Our purpose was to study this paradigm using ICAM-1, an endothelial receptor involved in inflammation, to better understand its regulation and potential for drug delivery. Our procedure involved fluorescence microscopy of human endothelial cells to determine the endocytic behavior of unbound ICAM-1 vs ICAM-1 bound by model ligands: monomeric (anti-ICAM) vs multimeric (anti-ICAM biotin-streptavidin conjugates or anti-ICAM coated onto 100 nm nanocarriers). Our findings suggest that both monomeric and multimeric ligands undergo a similar endocytic pathway sensitive to amiloride (∼50% inhibition), but not inhibitors of clathrin-pits or caveoli. After 30 min, ∼60-70% of both ligands colocalized with Rab11a-compartments. By 3-5 h, ∼65-80% of multimeric anti-ICAM colocalized with perinuclear lysosomes with ∼60-80% degradation, while 70% of monomeric anti-ICAM remained associated with Rab11a at the cell periphery and recycled to and from the cell-surface with minimal (<10%) lysosomal colocalization and minimal (≤15%) degradation. In the absence of ligands, ICAM-1 also underwent amiloride-sensitive endocytosis with peripheral distribution, suggesting that monomeric (not multimeric) anti-ICAM follows the route of this receptor. In conclusion, ICAM-1 can mediate different intracellular itineraries, revealing new insight into this biological pathway and alternative avenues for drug delivery.

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.

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

Apicobasal polarity of brain endothelial cells

PubMed Central

Worzfeld, Thomas

2015-01-01

Normal brain homeostasis depends on the integrity of the blood–brain barrier that controls the access of nutrients, humoral factors, and immune cells to the CNS. The blood–brain barrier is composed mainly of brain endothelial cells. Forming the interface between two compartments, they are highly polarized. Apical/luminal and basolateral/abluminal membranes differ in their lipid and (glyco-)protein composition, allowing brain endothelial cells to secrete or transport soluble factors in a polarized manner and to maintain blood flow. Here, we summarize the basic concepts of apicobasal cell polarity in brain endothelial cells. To address potential molecular mechanisms underlying apicobasal polarity in brain endothelial cells, we draw on investigations in epithelial cells and discuss how polarity may go awry in neurological diseases. PMID:26661193

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

PubMed Central

Madathil, Bernadette K.; Anil Kumar, Pallickaveedu RajanAsari; Kumary, Thrikkovil Variyath

2014-01-01

Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate) polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty. PMID:25003113

Induction of endothelial cell proliferation by angiogenic factors released by activated monocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pakala, Rajbabu; Watanabe, Takuya; Benedict, Claude R

2002-06-01

Introduction: Cell-cell interaction is an essential component of atherosclerotic plaque development. Activated monocytes appear to play a central role in the development of atherosclerosis, not only through foam cell formation but also via the production of various growth factors that induce proliferation of different cell types that are involved in the plaque development. Using serum free co-culture method, we determined the effect of monocytes on endothelial cell proliferation. Methods: Endothelial cell proliferation is determined by the amount of [{sup 3}H]thymidine incorporated in to the DNA. Basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) levels inmore » the conditioned medium were determined by ELISA. Results: Conditioned medium from unactivated monocytes partially inhibited endothelial cell proliferation, whereas conditioned medium from activated monocytes promoted endothelial cell proliferation. The mitogenic effect of conditioned medium derived from activated monocytes is due to the presence of b-FGF, VEGF and IL-8. Neutralizing antibodies against b-FGF, VEGF and IL-8 partially reversed the mitogenic effect of conditioned medium derived from activated monocytes. When b-FGF, VEGF and IL-8 were immunoprecipitated from conditioned medium derived from activated monocytes, it is less mitogenic to endothelial cells. Conclusion: Activated monocytes may play an important role in the development of atherosclerotic plaque by producing endothelial cell growth factors.« less

The role of apoptosis in LDL transport through cultured endothelial cell monolayers

PubMed Central

Cancel, Limary M.; Tarbell, John M.

2009-01-01

We have previously shown that leaky junctions associated with dying or dividing cells are the dominant pathway for LDL transport under convective conditions, accounting for more than 90% of the transport [1]. To explore the role of apoptosis in the leaky junction pathway, TNFα and cycloheximide (TNFα/CHX) were used to induce an elevated rate of apoptosis in cultured bovine aortic endothelial cell (BAEC) monolayers and the convective fluxes of LDL and water were measured. Treatment with TNFα/CHX induced a 18.3-fold increase in apoptosis and a 4.4-fold increase in LDL permeability. Increases in apoptosis and permeability were attenuated by treatment with the caspase inhibitor Z-VAD-FMK. Water flux increased by 2.7-fold after treatment with TNFα/CHX, and this increase was not attenuated by treatment with Z-VAD-FMK. Immunostaining of the tight junction protein ZO-1 showed that TNFα/CHX treatment disrupts the tight junction in addition to inducing apoptosis. This disruption is present even when Z-VAD-FMK is used to inhibit apoptosis, and likely accounts for the increase in water flux. We found a strong correlation between the rate of apoptosis and the permeability of BAEC monolayers to LDL. These results demonstrate the potential of manipulating endothelial monolayer permeability by altering the rate of apoptosis pharmacollogicaly. This has implications for the treatment of atherosclerosis. PMID:19709659

Resolving the multifaceted mechanisms of the ferric chloride thrombosis model using an interdisciplinary microfluidic approach

PubMed Central

Ciciliano, Jordan C.; Sakurai, Yumiko; Myers, David R.; Fay, Meredith E.; Hechler, Beatrice; Meeks, Shannon; Li, Renhao; Dixon, J. Brandon; Lyon, L. Andrew; Gachet, Christian

2015-01-01

The mechanism of action of the widely used in vivo ferric chloride (FeCl3) thrombosis model remains poorly understood; although endothelial cell denudation is historically cited, a recent study refutes this and implicates a role for erythrocytes. Given the complexity of the in vivo environment, an in vitro reductionist approach is required to systematically isolate and analyze the biochemical, mass transfer, and biological phenomena that govern the system. To this end, we designed an “endothelial-ized” microfluidic device to introduce controlled FeCl3 concentrations to the molecular and cellular components of blood and vasculature. FeCl3 induces aggregation of all plasma proteins and blood cells, independent of endothelial cells, by colloidal chemistry principles: initial aggregation is due to binding of negatively charged blood components to positively charged iron, independent of biological receptor/ligand interactions. Full occlusion of the microchannel proceeds by conventional pathways, and can be attenuated by antithrombotic agents and loss-of-function proteins (as in IL4-R/Iba mice). As elevated FeCl3 concentrations overcome protective effects, the overlap between charge-based aggregation and clotting is a function of mass transfer. Our physiologically relevant in vitro system allows us to discern the multifaceted mechanism of FeCl3-induced thrombosis, thereby reconciling literature findings and cautioning researchers in using the FeCl3 model. PMID:25931587

Role of vascular endothelial cell growth factor in Ovarian Hyperstimulation Syndrome.

PubMed Central

Levin, E R; Rosen, G F; Cassidenti, D L; Yee, B; Meldrum, D; Wisot, A; Pedram, A

1998-01-01

Controlled ovarian hyperstimulation with gonadotropins is followed by Ovarian Hyperstimulation Syndrome (OHSS) in some women. An unidentified capillary permeability factor from the ovary has been implicated, and vascular endothelial cell growth/permeability factor (VEGF) is a candidate protein. Follicular fluids (FF) from 80 women who received hormonal induction for infertility were studied. FFs were grouped according to oocyte production, from group I (0-7 oocytes) through group IV (23-31 oocytes). Group IV was comprised of four women with the most severe symptoms of OHSS. Endothelial cell (EC) permeability induced by the individual FF was highly correlated to oocytes produced (r2 = 0.73, P < 0.001). Group IV FF stimulated a 63+/-4% greater permeability than FF from group I patients (P < 0. 01), reversed 98% by anti-VEGF antibody. Group IV fluids contained the VEGF165 isoform and significantly greater concentrations of VEGF as compared with group I (1,105+/-87 pg/ml vs. 353+/-28 pg/ml, P < 0. 05). Significant cytoskeletal rearrangement of F-actin into stress fibers and a destruction of ZO-1 tight junction protein alignment was caused by group IV FF, mediated in part by nitric oxide. These mechanisms, which lead to increased EC permeability, were reversed by the VEGF antibody. Our results indicate that VEGF is the FF factor responsible for increased vascular permeability, thereby contributing to the pathogenesis of OHSS. PMID:9835623

Aronia melanocarpa fruit extract exhibits anti-inflammatory activity in human aortic endothelial cells.

PubMed

Zapolska-Downar, D; Bryk, D; Małecki, M; Hajdukiewicz, K; Sitkiewicz, D

2012-08-01

Altered expression of cell adhesion molecules (CAMs) has been implicated in a variety of chronic inflammatory conditions, including atherosclerosis. Regulation of adhesion molecule expression by specific redox-sensitive mechanisms has been reported. Additionally, it has been observed that the extract of Aronia melanocarpa (A. Melanocarpa) fruits, rich in polyphenols, exhibits potent anti-oxidant properties and displays cardioprotective activity. Human aortic endothelial cells (HAECs) were pretreated with various concentrations (primarily 50 μg/mL) of Aronia Melanocarpa fruit extract prior to treatment with TNFα (10 ng/mL) for various periods of time. The surface protein and mRNA expression of ICAM-1 and VCAM-1 were determined using flow cytometry and real-time RT-PCR, respectively. Adhesion of peripheral blood mononuclear leucocytes (PBMLs) to TNFα-treated HAECs was evaluated by an adhesion assay. Activation of NF-κB was evaluated by measuring NF-κB p65 phosphorylation using flow cytometry. ROS production was determined by reduction in fluorescent 2',7'-dichlorofluorescein diacetate (DCFH-DA). Tested A. Melanocarpa extract significantly inhibited the expression of ICAM-1 and VCAM-1, attenuated the phosphorylation of NF-κB p65 and decreased intracellular ROS production in TNFα-treated HAECs. We conclude that A. Melanocarpa fruit extract exhibits anti-inflammatory effects in HAECs by inhibiting the expression of endothelial CAMs, activation of NF-κB and production of ROS.

[Endothelial keratoplasty: Descemet stripping (DSEK) using TAN EndoGlide™ device: case series].

PubMed

Pazos, Henrique Santiago Baltar; Pazos, Paula Fernanda Morais Ramalho Baltar; Nogueira Filho, Pedro Antônio; Grisolia, Ana Beatriz Diniz; Silva, André Berger Emiliano; Gomes, José Álvaro Pereira

2011-01-01

To report the results of Descemet stripping endothelial keratoplasty (DSEK) using the TAN EndoGlideTM device to facilitate the insertion of the endothelial membrane. Prospective clinical study that included nine patients presenting corneal edema secondary to endothelial dysfunction. Best corrected visual acuity, refraction, central corneal thickness, endothelial cell density and complications were analyzed after a six-month follow-up. There was a significant improvement in the corneal edema and visual acuity in 7 patients (77.78%). The best corrected visual acuity ranged between 20/40 and 20/200. The average density of endothelial cells in six months varied between 1,305 cells/mm² and 2,346 cells/mm² with an average loss of 33.14% cells. Detachment of part of the graft was observed in one eye (11.11%) and primary failure of the endothelial transplantation occurred in 2 eyes (22.22%). The device TAN EndoGlideTM facilitates the introduction of the graft in Descemet stripping endothelial keratoplasty.

Pericyte Derived Sphinogosine 1-Phosphate Induces the Expression of Adhesion Proteins and Modulates the Retinal Endothelial Cell Barrier

PubMed Central

McGuire, P.G.; Rangasamy, S.; Maestas, J.; Das, A.

2011-01-01

Objective The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier. Methods and Results Human retinal microvascular endothelial cells were co-cultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate (S1P). S1P aids in maintenance of microvascular stability by up-regulating the expression of N-cadherin and VE-cadherin, and down-regulating the expression of angiopoietin 2. Conclusion Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of S1P. Alteration of pericyte-derived S1P production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability. PMID:21940944

Endocytosis of Red Blood Cell Microparticles by Pulmonary Endothelial Cells is Mediated By Rab5.

PubMed

Kim, Young; Abplanalp, William A; Jung, Andrew D; Schuster, Rebecca M; Lentsch, Alex B; Gulbins, Erich; Caldwell, Charles C; Pritts, Timothy A

2018-03-01

Microparticles are submicron vesicles shed from aging erythrocytes as a characteristic feature of the red blood cell (RBC) storage lesion. Exposure of pulmonary endothelial cells to RBC-derived microparticles promotes an inflammatory response, but the mechanisms underlying microparticle-induced endothelial cell activation are poorly understood. In the present study, cultured murine lung endothelial cells (MLECs) were treated with microparticles isolated from aged murine packed RBCs or vehicle. Microparticle-treated cells demonstrated increased expression of the adhesion molecules ICAM and E-selectin, as well as the cytokine, IL-6. To identify mechanisms that mediate these effects of microparticles on MLECs, cells were treated with microparticles covalently bound to carboxyfluorescein succinimidyl ester (CFSE) and cellular uptake of microparticles was quantified via flow cytometry. Compared with controls, there was a greater proportion of CFSE-positive MLECs from 15 min up to 24 h, suggesting endocytosis of the microparticles by endothelial cells. Colocalization of microparticles with lysosomes was observed via immunofluorescence, indicating endocytosis and endolysosomal trafficking. This process was inhibited by endocytosis inhibitors. SiRNA knockdown of Rab5 signaling protein in endothelial cells resulted in impaired microparticle uptake as compared with nonsense siRNA-treated cells, as well as an attenuation of the inflammatory response to microparticle treatment. Taken together, these data suggest that endocytosis of RBC-derived microparticles by lung endothelial cells results in endothelial cell activation. This response seems to be mediated, in part, by the Rab5 signaling protein.

Corneal endothelial cell density after femtosecond thin-flap LASIK and PRK for myopia: a contralateral eye study.

PubMed

Smith, Ryan T; Waring, George O; Durrie, Daniel S; Stahl, Jason E; Thomas, Priscilla

2009-12-01

To compare the effect of femtosecond thinflap LASIK and photorefractive keratectomy (PRK) on postoperative endothelial cell density. In a prospective, randomized, contralateral, single-center clinical trial, 25 patients (mean age: 30+/-5 years [range: 21 to 38 years]) underwent PRK in one eye and thin-flap LASIK in the fellow eye for the correction of myopia using a wavefront-guided platform. The central corneal endothelial cell density was measured using the NIDEK Confoscan 4 preoperatively, and at 1 and 3 months postoperatively. Changes in endothelial cell density were analyzed over time between the two refractive techniques. In PRK, the average preoperative endothelial cell density was 3011+/-329 cells/mm(2), which decreased to 2951+/-327 cells/mm(2) at 1 month (P=.5736) and 2982+/-365 cells/mm(2) at 3 months (P=.6513). In thinflap LASIK, the average preoperative endothelial cell density was 2995+/-325 cells/mm(2), which decreased to 2977+/-358 cells/mm(2) at 1 month (P=.5756) and 2931+/-369 cells/mm(2) at 3 months (P=.4106). No statistically significant difference was found between the two groups at 1 (P=.7404) or 3 (P=.3208) months postoperatively. No statistically significant change was noted in endothelial cell density following either PRK or thin-flap LASIK for the treatment of myopia. Furthermore, no statistically significant difference was found between the two groups out to 3 months postoperatively, indicating that thin-flap LASIK is as safe as PRK with regards to endothelial health.

Molecular Disruption of Breast Tumor Angiogenesis

DTIC Science & Technology

2004-07-01

human breast cancer . During the period of this grant, we carried out studies to confirm that targeted ablation of PAI- 1 gene expression resulted in a...microvessel endothelial cells. Breast cancer -derived factors (TGF-P, EGF)were found to be important contributors of continued PAI-I expression and long...various culture model systems implicated both urokinase plasminogen activator (uPA) and its fast-acting type-i inhibitor (PAl-l) as necessary to achieve

Endothelium trans differentiated from Wharton's jelly mesenchymal cells promote tissue regeneration: potential role of soluble pro-angiogenic factors.

PubMed

Aguilera, Valeria; Briceño, Luis; Contreras, Hector; Lamperti, Liliana; Sepúlveda, Esperanza; Díaz-Perez, Francisca; León, Marcelo; Veas, Carlos; Maura, Rafael; Toledo, Jorge Roberto; Fernández, Paulina; Covarrubias, Ambart; Zuñiga, Felipe Andrés; Radojkovic, Claudia; Escudero, Carlos; Aguayo, Claudio

2014-01-01

Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton's jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton's jelly (hWMSCs) can accelerate tissue repair in vivo. Mesenchymal stem cells were isolated from human Wharton's jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO). hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures. These results demonstrate that mesenchymal stem cells isolated from Wharton's jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.

The endothelial sample size analysis in corneal specular microscopy clinical examinations.

PubMed

Abib, Fernando C; Holzchuh, Ricardo; Schaefer, Artur; Schaefer, Tania; Godois, Ronialci

2012-05-01

To evaluate endothelial cell sample size and statistical error in corneal specular microscopy (CSM) examinations. One hundred twenty examinations were conducted with 4 types of corneal specular microscopes: 30 with each BioOptics, CSO, Konan, and Topcon corneal specular microscopes. All endothelial image data were analyzed by respective instrument software and also by the Cells Analyzer software with a method developed in our lab. A reliability degree (RD) of 95% and a relative error (RE) of 0.05 were used as cut-off values to analyze images of the counted endothelial cells called samples. The sample size mean was the number of cells evaluated on the images obtained with each device. Only examinations with RE < 0.05 were considered statistically correct and suitable for comparisons with future examinations. The Cells Analyzer software was used to calculate the RE and customized sample size for all examinations. Bio-Optics: sample size, 97 ± 22 cells; RE, 6.52 ± 0.86; only 10% of the examinations had sufficient endothelial cell quantity (RE < 0.05); customized sample size, 162 ± 34 cells. CSO: sample size, 110 ± 20 cells; RE, 5.98 ± 0.98; only 16.6% of the examinations had sufficient endothelial cell quantity (RE < 0.05); customized sample size, 157 ± 45 cells. Konan: sample size, 80 ± 27 cells; RE, 10.6 ± 3.67; none of the examinations had sufficient endothelial cell quantity (RE > 0.05); customized sample size, 336 ± 131 cells. Topcon: sample size, 87 ± 17 cells; RE, 10.1 ± 2.52; none of the examinations had sufficient endothelial cell quantity (RE > 0.05); customized sample size, 382 ± 159 cells. A very high number of CSM examinations had sample errors based on Cells Analyzer software. The endothelial sample size (examinations) needs to include more cells to be reliable and reproducible. The Cells Analyzer tutorial routine will be useful for CSM examination reliability and reproducibility.

Circulating endothelial cells and their progenitors in acute myeloid leukemia

PubMed Central

Zahran, Asmaa Mohammed; Aly, Sanaa Shaker; Altayeb, Hanan Ahmed; Ali, Arwa Mohammed

2016-01-01

Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by the accumulation of immature myeloid progenitor cells in the bone marrow. Studies are required to investigate the prognostic and predictive value of surrogate biomarkers. Given the importance of angiogenesis in oncology in terms of pathogenesis as well as being a target for treatment, circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) are promising candidates to serve as such markers. The aim of the present study was to quantify CECs and EPCs in patients with AML at initial diagnosis and following induction chemotherapy, and to correlate these findings with the response to treatment in AML patients. The present study included 40 patients with de novo AML and 20 age- and gender-matched healthy controls. CECs and EPCs were evaluated by flow cytometry at initial diagnosis and after induction chemotherapy (3+7 protocol for AML other than M3 and all-trans-retinoic acid plus anthracycline for M3 disease). CECs and EPCs were significantly higher in AML patients at diagnosis and after induction chemotherapy than in controls. After induction chemotherapy, CECs and EPCs were significantly decreased compared with the levels at initial diagnosis. Patients who achieved complete response (n=28) had lower initial CEC and EPC levels compared with patients who did not respond to treatment. These results suggest that CEC levels are higher in AML patients and may correlate with disease status and treatment response. Further investigations are required to better determine the predictive value and implication of these cells in AML management. PMID:27602121

Gene expression profiling of rubella virus infected primary endothelial cells of fetal and adult origin.

PubMed

Geyer, Henriette; Bauer, Michael; Neumann, Jennifer; Lüdde, Amy; Rennert, Paul; Friedrich, Nicole; Claus, Claudia; Perelygina, Ludmilla; Mankertz, Annette

2016-02-02

Rubella virus (RV) infection is usually a mild illness in children and adults. However, maternal infection during the first trimester of pregnancy can lead to congenital rubella syndrome (CRS) in the infant. Fetuses with CRS show damage to the endothelium of the heart and blood vessels; thus, it has been speculated that the clinical manifestations associated with CRS may be a result of endothelial cells persistently infected with RV. Here, we compared the effects of RV infection on gene expression in primary endothelial cells of fetal (HUVEC) and of adult (HSaVEC) origin by transcriptional profiling. More than 75 % of the genes differentially regulated following RV infection were identical in both cell types. Gene Ontology (GO) analysis of these commonly regulated genes showed an enrichment of terms involved in cytokine production and cytokine regulation. Increased accumulation of inflammatory cytokines following RV infection was verified by protein microarray. Interestingly, the chemokine CCL14, which is implicated in supporting embryo implantation at the fetal-maternal interface, was down-regulated following RV infection only in HUVEC. Most noticeably, when analyzing the uniquely regulated transcripts for each cell type, GO term-based cluster analysis of the down-regulated genes of HUVEC revealed an enrichment of the GO terms "sensory organ development", "ear development" and "eye development". Since impairment in vision and hearing are the most prominent clinical manifestations observed in CRS patients, the here detected down-regulated genes involved in the development of sensory organs sheds light on the molecular mechanisms that may contribute to the teratogenic effect of RV.

Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca²⁺]i, nitric oxide, and gap formation in intact venules.

PubMed

Zhou, Xueping; He, Pingnian

2011-11-01

We have previously demonstrated that platelet-activating factor (PAF)-induced increases in microvessel permeability were associated with endothelial gap formation and that the magnitude of peak endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) and nitric oxide (NO) production at the single vessel level determines the degree of the permeability increase. This study aimed to examine whether the magnitudes of PAF-induced peak endothelial [Ca(2+)](i), NO production, and gap formation are correlated at the individual endothelial cell level in intact rat mesenteric venules. Endothelial gaps were quantified by the accumulation of fluorescent microspheres at endothelial clefts using confocal imaging. Endothelial [Ca(2+)](i) was measured on fura-2- or fluo-4-loaded vessels, and 4,5-diaminofluorescein (DAF-2) was used for NO measurements. The results showed that increases in endothelial [Ca(2+)](i), NO production, and gap formation occurred in all endothelial cells when vessels were exposed to PAF but manifested a spatial heterogeneity in magnitudes among cells in each vessel. PAF-induced peak endothelial [Ca(2+)](i) preceded the peak NO production by 0.6 min at the cellular level, and the magnitudes of NO production and gap formation linearly correlated with that of the peak endothelial [Ca(2+)](i) in each cell, suggesting that the initial levels of endothelial [Ca(2+)](i) determine downstream NO production and gap formation. These results provide direct evidence from intact venules that inflammatory mediator-induced increases in microvessel permeability are associated with the generalized formation of endothelial gaps around all endothelial cells. The spatial differences in the molecular signaling that were initiated by the heterogeneous endothelial Ca(2+) response contribute to the heterogeneity in permeability increases along the microvessel wall during inflammation.

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles.

PubMed

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel; Hilfiker, Andres

2016-01-01

Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle-cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN.

Contribution of endothelial cells to organogenesis: a modern reappraisal of an old Aristotelian concept

PubMed Central

Crivellato, E; Nico, B; Ribatti, D

2007-01-01

It is well established that many tissue-derived factors are involved in blood vessel formation, but evidence is now emerging that endothelial cells themselves represent a crucial source of instructive signals to non-vascular tissue cells during organ development. Thus, endothelial cell signalling is currently believed to promote fundamental cues for cell fate specification, embryo patterning, organ differentiation and postnatal tissue remodelling. This review article summarizes some of the recent advances in our understanding of the role of endothelial cells as effector cells in organ formation. PMID:17683480

Corneal endothelium: developmental strategies for regeneration

PubMed Central

Zavala, J; López Jaime, G R; Rodríguez Barrientos, C A; Valdez-Garcia, J

2013-01-01

The main treatment available for restoration of the corneal endothelium is keratoplasty. This procedure is faced with several difficulties, including the shortage of donor tissue, post-surgical complications associated with the use of drugs to prevent immune rejection, and a significant increase in the occurrence of glaucoma. Recently, surgical procedures such as Descemet's stripping endothelial keratoplasty have focused on the transplant of corneal endothelium, yielding better visual results but still facing the need for donor tissue. The emergent strategies in the field of cell biology and tissue cultivation of corneal endothelial cells aim at the production of transplantable endothelial cell sheets. Cell therapy focuses on the culture of corneal endothelial cells retrieved from the donor, in the donor's cornea, followed by transplantation into the recipient. Recently, research has focused on overcoming the challenge of harvesting human corneal endothelial cells and the generation of new biomembranes to be used as cell scaffolds in surgical procedures. The use of corneal endothelial precursors from the peripheral cornea has also demonstrated to be effective and represents a valuable tool for reducing the risk of rejection in allogeneic transplants. Several animal model reports also support the use of adult stem cells as therapy for corneal diseases. Current results represent important progresses in the development of new strategies based on alternative sources of tissue for the treatment of corneal endotheliopathies. Different databases were used to search literature: PubMed, Google Books, MD Consult, Google Scholar, Gene Cards, and NCBI Books. The main search terms used were: ‘cornea AND embryology AND transcription factors', ‘human endothelial keratoplasty AND risk factors', ‘(cornea OR corneal) AND (endothelium OR endothelial) AND cell culture', ‘mesenchymal stem cells AND cell therapy', ‘mesenchymal stem cells AND cornea', and ‘stem cells AND (cornea OR corneal) AND (endothelial OR endothelium)'. PMID:23470788

Nesting of colon and ovarian cancer cells in the endothelial niche is associated with alterations in glycan and lipid metabolism.

PubMed

Halama, Anna; Guerrouahen, Bella S; Pasquier, Jennifer; Satheesh, Noothan J; Suhre, Karsten; Rafii, Arash

2017-01-04

The metabolic phenotype of a cancer cell is determined by its genetic makeup and microenvironment, which dynamically modulates the tumor landscape. The endothelial cells provide both a promoting and protective microenvironment - a niche for cancer cells. Although metabolic alterations associated with cancer and its progression have been fairly defined, there is a significant gap in our understanding of cancer metabolism in context of its microenvironment. We deployed an in vitro co-culture system based on direct contact of cancer cells with endothelial cells (E4 + EC), mimicking the tumor microenvironment. Metabolism of colon (HTC15 and HTC116) and ovarian (OVCAR3 and SKOV3) cancer cell lines was profiled with non-targeted metabolic approaches at different time points in the first 48 hours after co-culture was established. We found significant, coherent and non-cell line specific changes in fatty acids, glycerophospholipids and carbohydrates over time, induced by endothelial cell contact. The metabolic patterns pinpoint alterations in hexosamine biosynthetic pathway, glycosylation and lipid metabolism as crucial for cancer - endothelial cells interaction. We demonstrated that "Warburg effect" is not modulated in the initial stage of nesting of cancer cell in the endothelial niche. Our study provides novel insight into cancer cell metabolism in the context of the endothelial microenvironment.

Platelet-independent adhesion of calcium-loaded erythrocytes to von Willebrand factor

PubMed Central

Bierings, Ruben; Meems, Henriet; Mul, Frederik P. J.; Geerts, Dirk; Vlaar, Alexander P. J.; Voorberg, Jan; Hordijk, Peter L.

2017-01-01

Adhesion of erythrocytes to endothelial cells lining the vascular wall can cause vaso-occlusive events that impair blood flow which in turn may result in ischemia and tissue damage. Adhesion of erythrocytes to vascular endothelial cells has been described in multiple hemolytic disorders, especially in sickle cell disease, but the adhesion of normal erythrocytes to endothelial cells has hardly been described. It was shown that calcium-loaded erythrocytes can adhere to endothelial cells. Because sickle erythrocyte adhesion to ECs can be enhanced by ultra-large von Willebrand factor multimers, we investigated whether calcium loading of erythrocytes could promote binding to endothelial cells via ultra-large von Willebrand factor multimers. We used (immunofluorescent) live-cell imaging of washed erythrocytes perfused over primary endothelial cells at venular flow rate. Using this approach, we show that calcium-loaded erythrocytes strongly adhere to histamine-stimulated primary human endothelial cells. This adhesion is mediated by ultra-large von Willebrand factor multimers. Von Willebrand factor knockdown or ADAMTS13 cleavage abolished the binding of erythrocytes to activated endothelial cells under flow. Platelet depletion did not interfere with erythrocyte binding to von Willebrand factor. Our results reveal platelet-independent adhesion of calcium-loaded erythrocytes to endothelium-derived von Willebrand factor. Erythrocyte adhesion to von Willebrand factor may be particularly relevant for venous thrombosis, which is characterized by the formation of erythrocyte-rich thrombi. PMID:28249049

Nerve Growth Factor-Induced Angiogenesis: 1. Endothelial Cell Tube Formation Assay.

PubMed

Lazarovici, Philip; Lahiani, Adi; Gincberg, Galit; Haham, Dikla; Fluksman, Arnon; Benny, Ofra; Marcinkiewicz, Cezary; Lelkes, Peter I

2018-01-01

Nerve growth factor (NGF) is a neurotrophin promoting survival, proliferation, differentiation, and neuroprotection in the embryonal and adult nervous system. NGF also induces angiogenic effects in the cardiovascular system, which may be beneficial in engineering new blood vessels and for developing novel anti-angiogenesis therapies for cancer. Angiogenesis is a cellular process characterized by a number of events, including endothelial cell migration, invasion, and assembly into capillaries. In vitro endothelial tube formation assays are performed using primary human umbilical vein endothelial cells, human aortic endothelial cells, and other human or rodent primary endothelial cells isolated from the vasculature of both tumors and normal tissues. Immortalized endothelial cell lines are also used for these assays. When seeded onto Matrigel, these cells reorganize to create tubelike structure, which may be used as models for studying some aspects of in vitro angiogenesis. Image acquisition by light and fluorescence microscopy and/or quantification of fluorescently labeled cells can be carried out manually or digitally, using commercial software and automated image processing. Here we detail materials, procedure, assay conditions, and cell labeling for quantification of endothelial cell tube formation. This model can be applied to study cellular and molecular mechanisms by which NGF or other neurotrophins promote angiogenesis. This model may also be useful for the development of potential angiogenic and/or anti-angiogenic drugs targeting NGF receptors.

Low oxygen tension enhances endothelial fate of human pluripotent stem cells.

PubMed

Kusuma, Sravanti; Peijnenburg, Elizabeth; Patel, Parth; Gerecht, Sharon

2014-04-01

A critical regulator of the developing or regenerating vasculature is low oxygen tension. Precise elucidation of the role of low oxygen environments on endothelial commitment from human pluripotent stem cells necessitates controlled in vitro differentiation environments. We used a feeder-free, 2-dimensional differentiation system in which we could monitor accurately dissolved oxygen levels during human pluripotent stem cell differentiation toward early vascular cells (EVCs). We found that oxygen uptake rate of differentiating human pluripotent stem cells is lower in 5% O2 compared with atmospheric conditions. EVCs differentiated in 5% O2 had an increased vascular endothelial cadherin expression with clusters of vascular endothelial cadherin+ cells surrounded by platelet-derived growth factor β+ cells. When we assessed the temporal effects of low oxygen differentiation environments, we determined that low oxygen environments during the early stages of EVC differentiation enhance endothelial lineage commitment. EVCs differentiated in 5% O2 exhibited an increased expression of vascular endothelial cadherin and CD31 along with their localization to the membrane, enhanced lectin binding and acetylated low-density lipoprotein uptake, rapid cord-like structure formation, and increased expression of arterial endothelial cell markers. Inhibition of reactive oxygen species generation during the early stages of differentiation abrogated the endothelial inductive effects of the low oxygen environments. Low oxygen tension during early stages of EVC derivation induces endothelial commitment and maturation through the accumulation of reactive oxygen species, highlighting the importance of regulating oxygen tensions during human pluripotent stem cell-vascular differentiation.

Differential procoagulant activity of microparticles derived from monocytes, granulocytes, platelets and endothelial cells: impact of active tissue factor.

PubMed

Shustova, Olga N; Antonova, Olga A; Golubeva, Nina V; Khaspekova, Svetlana G; Yakushkin, Vladimir V; Aksuk, Svetlana A; Alchinova, Irina B; Karganov, Mikhail Y; Mazurov, Alexey V

2017-07-01

: Microparticles released by activated/apoptotic cells exhibit coagulation activity as they express phosphatidylserine and some of them - tissue factor. We compared procoagulant properties of microparticles from monocytes, granulocytes, platelets and endothelial cells and assessed the impact of tissue factor in observed differences. Microparticles were sedimented (20 000g, 30 min) from the supernatants of activated monocytes, monocytic THP-1 cells, granulocytes, platelets and endothelial cells. Coagulation activity of microparticles was examined using plasma recalcification assay. The size of microparticles was evaluated by dynamic light scattering. Tissue factor activity was measured by its ability to activate factor X. All microparticles significantly accelerated plasma coagulation with the shortest lag times for microparticles derived from monocytes, intermediate - for microparticles from THP-1 cells and endothelial cells, and the longest - for microparticles from granulocytes and platelets. Average diameters of microparticles ranged within 400-600 nm. The largest microparticles were produced by endothelial cells and granulocytes, smaller - by monocytes, and the smallest - by THP-1 cells and platelets. The highest tissue factor activity was detected in microparticles from monocytes, lower activity - in microparticles from endothelial cells and THP-1 cells, and no activity - in microparticles from platelets and granulocytes. Anti-tissue factor antibodies extended coagulation lag times for microparticles from monocytes, endothelial cells and THP-1 cells and equalized them with those for microparticles from platelets and granulocytes. Higher coagulation activity of microparticles from monocytes, THP-1 cells and endothelial cells in comparison with microparticles from platelets and granulocytes is determined mainly by the presence of active tissue factor.

Proteasome inhibitors enhance endothelial thrombomodulin expression via induction of Krüppel-like transcription factors

PubMed Central

Hiroi, Toyoko; Deming, Clayton B.; Zhao, Haige; Hansen, Baranda S.; Arkenbout, Elisabeth K.; Myers, Thomas J.; McDevitt, Michael A.; Rade, Jeffrey J.

2009-01-01

Objective Impairment of the thrombomodulin-protein C anticoagulant pathway has been implicated in pathologic thrombosis associated with malignancy. Patients who receive proteasome inhibitors as part of their chemotherapeutic regimen appear to be at decreased risk for thromboembolic events. We investigated the effects of proteasome inhibitors on endothelial thrombomodulin expression and function. Methods and Results Proteasome inhibitors as a class markedly induced the expression thrombomodulin and enhanced the protein C activating capacity of endothelial cells. Thrombomodulin upregulation was independent of NF-κB signaling, a principal target of proteasome inhibitors, but was instead a direct consequence of increased expression of the Krüppel-like transcription factors, KLF2 and KLF4. These effects were confirmed in vivo, where systemic administration of a proteasome inhibitor enhanced thrombomodulin expression that was paralleled by changes in the expression of KLF2 and KLF4. Conclusions These findings identify a novel mechanism of action of proteasome inhibitors that may help to explain their clinically observed thromboprotective effects. PMID:19661484

Modified rice bran hemicellulose inhibits vascular endothelial growth factor-induced angiogenesis in vitro via VEGFR2 and its downstream signaling pathways

PubMed Central

ZHU, Xia; OKUBO, Aya; IGARI, Naoki; NINOMIYA, Kentaro; EGASHIRA, Yukari

2016-01-01

Angiogenesis is implicated in diverse pathological conditions such as cancer, rheumatoid arthritis, psoriasis, atherosclerosis, and retinal neovascularization. In the present study, we investigated the effects of modified rice bran hemicellulose (MRBH), a water-soluble hemicellulose preparation from rice bran treated with shiitake enzymes, on vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro and its mechanism. We found that MRBH significantly inhibited VEGF-induced tube formation in human umbilical vein endothelial cells (HUVECs) co-cultured with human dermal fibroblasts. We also observed that MRBH dose-dependently suppressed the VEGF-induced proliferation and migration of HUVECs. Furthermore, examination of the anti-angiogenic mechanism indicated that MRBH reduced not only VEGF-induced activation of VEGF receptor 2 but also of the downstream signaling proteins Akt, extracellular signal-regulated protein kinase 1/2, and p38 mitogen-activated protein kinase. These findings suggest that MRBH has in vitro anti-angiogenic effects that are partially mediated through the inhibition of VEGF signaling. PMID:28439487

Treating fat grafts with human endothelial progenitor cells promotes their vascularization and improves their survival in diabetes mellitus.

PubMed

Hamed, Saher; Ben-Nun, Ohad; Egozi, Dana; Keren, Aviad; Malyarova, Nastya; Kruchevsky, Danny; Gilhar, Amos; Ullmann, Yehuda

2012-10-01

Bone marrow-derived endothelial progenitor cells are required for vascularization of a fat graft to form a functional microvasculature within the graft and to facilitate its integration into the surrounding tissues. Organ transplantation carries a high risk of graft loss and rejection in patients with diabetes mellitus because endothelial progenitor cell function is impaired. The authors investigated the influence of endothelial progenitor cell treatment on the phenotype and survival of human fat grafts in immunocompromised mice with experimentally induced diabetes mellitus. The authors injected 1 ml of human fat tissue into the scalps of 14 nondiabetic and 28 diabetic immunocompromised mice, and then treated some of the grafts with endothelial progenitor cells that was isolated from the blood of a human donor. The phenotype of the endothelial progenitor cell-treated fat grafts from the 14 diabetic mice was compared with that of the untreated fat grafts from 14 nondiabetic and 14 diabetic mice, 18 days and 15 weeks after fat transplantation. Determination of graft phenotype included measurements of weight and volume, vascular endothelial growth factor levels, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase, and caspase 3 expression levels, and histologic analysis of the extent of vascularization. The untreated grafts from the diabetic mice were fully resorbed 15 weeks after fat transplantation. The phenotype of endothelial progenitor cell-treated fat grafts from the diabetic mice was similar to that of the untreated fat grafts from the nondiabetic mice. Endothelial progenitor cell treatment of transplanted fat can increase the survival of a fat graft by inducing its vascularization and decreasing the extent of apoptosis.

A fully automated cell segmentation and morphometric parameter system for quantifying corneal endothelial cell morphology.

PubMed

Al-Fahdawi, Shumoos; Qahwaji, Rami; Al-Waisy, Alaa S; Ipson, Stanley; Ferdousi, Maryam; Malik, Rayaz A; Brahma, Arun

2018-07-01

Corneal endothelial cell abnormalities may be associated with a number of corneal and systemic diseases. Damage to the endothelial cells can significantly affect corneal transparency by altering hydration of the corneal stroma, which can lead to irreversible endothelial cell pathology requiring corneal transplantation. To date, quantitative analysis of endothelial cell abnormalities has been manually performed by ophthalmologists using time consuming and highly subjective semi-automatic tools, which require an operator interaction. We developed and applied a fully-automated and real-time system, termed the Corneal Endothelium Analysis System (CEAS) for the segmentation and computation of endothelial cells in images of the human cornea obtained by in vivo corneal confocal microscopy. First, a Fast Fourier Transform (FFT) Band-pass filter is applied to reduce noise and enhance the image quality to make the cells more visible. Secondly, endothelial cell boundaries are detected using watershed transformations and Voronoi tessellations to accurately quantify the morphological parameters of the human corneal endothelial cells. The performance of the automated segmentation system was tested against manually traced ground-truth images based on a database consisting of 40 corneal confocal endothelial cell images in terms of segmentation accuracy and obtained clinical features. In addition, the robustness and efficiency of the proposed CEAS system were compared with manually obtained cell densities using a separate database of 40 images from controls (n = 11), obese subjects (n = 16) and patients with diabetes (n = 13). The Pearson correlation coefficient between automated and manual endothelial cell densities is 0.9 (p < 0.0001) and a Bland-Altman plot shows that 95% of the data are between the 2SD agreement lines. We demonstrate the effectiveness and robustness of the CEAS system, and the possibility of utilizing it in a real world clinical setting to enable rapid diagnosis and for patient follow-up, with an execution time of only 6 seconds per image. Copyright © 2018 Elsevier B.V. All rights reserved.

Could an endoneurial endothelial crosstalk between Wnt/β-catenin and Sonic Hedgehog pathways underlie the early disruption of the infra-orbital blood-nerve barrier following chronic constriction injury?

PubMed

Moreau, Nathan; Mauborgne, Annie; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette B; Villanueva, Luis; Pohl, Michel; Boucher, Yves

2017-01-01

Blood–nerve barrier disruption is pivotal in the development of neuroinflammation, peripheral sensitization, and neuropathic pain after peripheral nerve injury. Activation of toll-like receptor 4 and inactivation of Sonic Hedgehog signaling pathways within the endoneurial endothelial cells are key events, resulting in the infiltration of harmful molecules and immunocytes within the nerve parenchyma. However, we showed in a previous study that preemptive inactivation of toll-like receptor 4 signaling or sustained activation of Sonic Hedgehog signaling did not prevent the local alterations observed following peripheral nerve injury, suggesting the implication of another signaling pathway. Using a classical neuropathic pain model, the infraorbital nerve chronic constriction injury (IoN-CCI), we investigated the role of the Wnt/β-catenin pathway in chronic constriction injury-mediated blood–nerve barrier disruption and in its interactions with the toll-like receptor 4 and Sonic Hedgehog pathways. In the IoN-CCI model versus control, mRNA expression levels and/or immunochemical detection of major Wnt/Sonic Hedgehog pathway (Frizzled-7, vascular endothelial-cadherin, Patched-1 and Gli-1) and/or tight junction proteins (Claudin-1, Claudin-5, and Occludin) readouts were assessed. Vascular permeability was assessed by sodium fluorescein extravasation. IoN-CCI induced early alterations in the vascular endothelial-cadherin/β-catenin/Frizzled-7 complex, shown to participate in local blood–nerve barrier disruption via a β-catenin-dependent tight junction protein downregulation. Wnt pathway also mediated a crosstalk between toll-like receptor 4 and Sonic Hedgehog signaling within endoneurial endothelial cells. Nevertheless, preemptive inhibition of Wnt/β-catenin signaling before IoN-CCI could not prevent the downregulation of key Sonic Hedgehog pathway readouts or the disruption of the infraorbital blood–nerve barrier, suggesting that Sonic Hedgehog pathway inhibition observed following IoN-CCI is an independent event responsible for blood–nerve barrier disruption. A crosstalk between Wnt/β-catenin- and Sonic Hedgehog-mediated signaling pathways within endoneurial endothelial cells could mediate the chronic disruption of the blood–nerve barrier following IoN-CCI, resulting in increased irreversible endoneurial vascular permeability and neuropathic pain development.

Overexpression of stearoyl-CoA desaturase 1 in bone marrow mesenchymal stem cells enhance the expression of induced endothelial cells

PubMed Central

2014-01-01

Background Bone marrow mesenchymal stem cells (BM-MSCs) are capable of differentiating into endothelial cells in vitro and acquire major characteristics of mature endothelial-like expression of vWF and CD31. SFAs and lipid oxidation products have been linked with postprandial endothelial dysfunction. Consumption of SFAs impairs arterial endothelial function, while a Mediterranean-type MUFA-diet has a beneficial effect on endothelial function by producing a decrease in levels of vWF, TFPI and PAI-1. Stearoyl-CoA desaturase 1 (SCD1), which converts SFA to MUFA, is involved in the cellular biosynthesis of MUFAs from SFA substrates. High expression of SCD1 is corresponded with low rates of fatty acid oxidation, therefore it might reduce inflammatory responses and be beneficial for the growth of induced endothelial cells. Overexpression of SCD1 in BM-MSCs might increase the growth of induced endothelial cells. The goal of this research is to study the relationship between overexpression of SCD1 and the expression of induced endothelial cells in BM-MSCs in vitro. Methods The gene SCD1 was integrated into a lentiviral vector, and then 293 T cells were transfected by the connected product to produce a packaged virus. BM-MSCs were infected by the packaged virus. Cell culture and endothelial induction were performed. Fluorescent quantitative PCR of CD31, vWF and VE-cad was performed after 1 week and 2 weeks to test the growth of induced endothelial cells. Results The mRNA amount of CD31, vWF and VE-cad of the SCD1 overexpressed group was statistically higher than that of the empty vector (EV) group and that of the normal group after 1 week and 2 weeks, respectively (p < 0.05). Immunocytochemical staining of CD31 or vWF was detected by visualizing red color. Conclusions This study suggested that overexpression of SCD1 in BM-MSCs could increase the expression of induced endothelial cells in vitro. PMID:24650127

Human Herpesvirus-8-Transformed Endothelial Cells Have Functionally Activated Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor

PubMed Central

Masood, Rizwan; Cesarman, Ethel; Smith, D. Lynne; Gill, Parkash S.; Flore, Ornella

2002-01-01

Kaposi’s sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposi’s sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, -2, and -3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression. PMID:11786394

Effects of transplanted circulating endothelial progenitor cells and platelet microparticles in atherosclerosis development.

PubMed

Georgescu, Adriana; Alexandru, Nicoleta; Andrei, Eugen; Dragan, Emanuel; Cochior, Daniel; Dias, Sérgio

2016-08-01

Atherosclerosis is an inflammatory disease, in which risk factors such as hyperlipidemia and hypertension affect the arterial endothelium, resulting in dysfunction, cell damage or both. The number of circulating endothelial progenitor cells and microparticles provides invaluable outcome prediction for atherosclerosis disease. However, evidence for the therapeutic potential of endothelial progenitor cells and microparticles in atherosclerosis development is limited. Our study was designed to investigate the possible protective role of a cell therapy-based approach, using endothelial progenitor cells and the dual behaviour of circulating platelet microparticles, on atherosclerosis development in hypertensive-hypercholesterolemic hamster model. Consequently, control hamsters received four intravenous inoculations of: (1) 1×10(5) endothelial progenitor cells of healthy origins in one dose per month, during four months of diet-induced atherosclerosis, and after hypertensive-hypercholesterolemic diet for further four months; (2) in a second set of experiments, 1×10(5) endothelial progenitor cells of healthy origins or/and 1×10(5) platelet microparticles of atherosclerotic origins were inoculated every other month during hypertensive-hypercholesterolemic diet. Endothelial progenitor cell treatment had the following effects: (1) re-established plasmatic parameters: cholesterol and triglyceride concentrations, blood pressure, heart rate, cytokine and chemokine profiles, platelet microparticle pro-thrombotic activity and endothelial progenitor cell paracrine activity reflected by cytokine/chemokine detection; (2) reduced lipid, macrophage and microparticle accumulation in liver; (3) reduced atherosclerosis development, revealed by decreased lipid, macrophage and microparticle content of arterial wall; (4) induced the recruitment and incorporation of endothelial progenitor cells into liver and arterial wall; (5) improved arterial dysfunction by increasing contraction and relaxation; (6) reduced the protein expression of specific pro-inflammatory molecules in liver and arterial wall. Platelet microparticle transplantation aggravated the above-mentioned biomarkers and atherosclerosis process, which were partially reverted with co-inoculation of platelet microparticles and endothelial progenitor cells. With this study, we demonstrate in a hypertensive-hypercholesterolemic hamster model, that the endothelial progenitor cell-based therapy suppresses the development of atherosclerosis and reduces hepatic lipid and macrophage accumulation with the consequent alleviation of dyslipidaemia and hypertension. Our results support the notion that increasing the number of circulating endothelial progenitor cells by different ways could be a promising therapeutic tool for atherosclerosis. © 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

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

PubMed

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

PubMed Central

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J. R.; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A.; Petersen, Ole William; Magnusson, Magnus K.; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44high/CD24low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer. PMID:21915264

Monocyte trafficking to the brain with stress and inflammation: a novel axis of immune-to-brain communication that influences mood and behavior

PubMed Central

Wohleb, Eric S.; McKim, Daniel B.; Sheridan, John F.; Godbout, Jonathan P.

2015-01-01

HIGHLIGHTS Psychological stress activates neuroendocrine pathways that alter immune responses.Stress-induced alterations in microglia phenotype and monocyte priming leads to aberrant peripheral and central inflammation.Elevated pro-inflammatory cytokine levels caused by microglia activation and recruitment of monocytes to the brain contribute to development and persistent anxiety-like behavior.Mechanisms that mediate interactions between microglia, endothelial cells, and macrophages and how these contribute to changes in behavior are discussed.Sensitization of microglia and re-distribution of primed monocytes are implicated in re-establishment of anxiety-like behavior. Psychological stress causes physiological, immunological, and behavioral alterations in humans and rodents that can be maladaptive and negatively affect quality of life. Several lines of evidence indicate that psychological stress disrupts key functional interactions between the immune system and brain that ultimately affects mood and behavior. For example, activation of microglia, the resident innate immune cells of the brain, has been implicated as a key regulator of mood and behavior in the context of prolonged exposure to psychological stress. Emerging evidence implicates a novel neuroimmune circuit involving microglia activation and sympathetic outflow to the peripheral immune system that further reinforces stress-related behaviors by facilitating the recruitment of inflammatory monocytes to the brain. Evidence from various rodent models, including repeated social defeat (RSD), revealed that trafficking of monocytes to the brain promoted the establishment of anxiety-like behaviors following prolonged stress exposure. In addition, new evidence implicates monocyte trafficking from the spleen to the brain as key regulator of recurring anxiety following exposure to prolonged stress. The purpose of this review is to discuss mechanisms that cause stress-induced monocyte re-distribution in the brain and how dynamic interactions between microglia, endothelial cells, and brain macrophages lead to maladaptive behavioral responses. PMID:25653581

Timing of Galectin-1 Exposure Differentially Modulates Nipah Virus Entry and Syncytium Formation in Endothelial Cells

PubMed Central

Garner, Omai B.; Yun, Tatyana; Pernet, Olivier; Aguilar, Hector C.; Park, Arnold; Bowden, Thomas A.; Freiberg, Alexander N.

2014-01-01

ABSTRACT Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. IMPORTANCE Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by “bridging” the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell. PMID:25505064

Endothelial cell-initiated extravasation of cancer cells visualized in zebrafish

PubMed Central

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

2014-01-01

The extravasation of cancer cells, a key step for distant metastasis, is thought to be initiated by disruption of the endothelial barrier by malignant cancer cells. An endothelial covering-type extravasation of cancer cells in addition to conventional cancer cell invasion-type extravasation was dynamically visualized in a zebrafish hematogenous metastasis model. The inhibition of VEGF-signaling impaired the invasion-type extravasation via inhibition of cancer cell polarization and motility. Paradoxically, the anti-angiogenic treatment showed the promotion, rather than the inhibition, of the endothelial covering-type extravasation of cancer cells, with structural changes in the endothelial walls. These findings may be a set of clues to the full understanding of the metastatic process as well as the metastatic acceleration by anti-angiogenic reagents observed in preclinical studies. PMID:25551022

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

PubMed

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

2014-01-01

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

A versatile microfluidic platform for the study of cellular interactions between endothelial cells and neutrophils.

PubMed

Wu, Xiaojie; Newbold, Molly A; Gao, Zhe; Haynes, Christy L

2017-05-01

Endothelial migration is a critical physiological process during vascular angiogenesis, growth and development, as well as in various disease conditions, such as cancer and cardiovascular diseases. Neutrophil migration, known as the important characteristic of immune responses, is also recognized as a contributor to the diseases involving endothelial migration. Herein, the mutually dependent relationship between neutrophil recruitment and endothelial migration was studied on a microfluidic platform for the first time. An in vivo-like microenvironment is created inside microfluidic devices by embedding a gel scaffold into the micro-chambers. This approach, with controllable stable chemical gradients and the ability to quantitate interaction characteristics, overcomes the limitations of the current in vivo and in vitro assays for cell migration studies. The number of neutrophils migrating through the endothelial cell layer is heavily influenced by the concentration of vascular endothelial growth factor (VEGF) that induces endothelial cell migration in the gel scaffold, and is not as correlated to the concentration of chemokine solution used for initiating neutrophil migration. More importantly, neutrophil migration diminishes the effects of the drug that inhibits endothelial migration and this process is regulated by the concentration of chemokine molecules instead of VEGF concentration. The results presented herein demonstrate the complicated cellular interactions between endothelial cells and neutrophils: endothelial migration delicately regulates neutrophil migration while the presence of neutrophils stabilizes the structures of endothelial migration. This study provides deeper understanding of the dynamic cellular interactions between neutrophils and endothelial cells as well as the pathogenesis of relevant diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Pro-apoptotic BIM is an essential initiator of physiological endothelial cell death independent of regulation by FOXO3.

PubMed

Koenig, M N; Naik, E; Rohrbeck, L; Herold, M J; Trounson, E; Bouillet, P; Thomas, T; Voss, A K; Strasser, A; Coultas, L

2014-11-01

The growth of new blood vessels by angiogenesis is essential for normal development, but can also cause or contribute to the pathology of numerous diseases. Recent studies have shown that BIM, a pro-apoptotic BCL2-family protein, is required for endothelial cell apoptosis in vivo, and can contribute to the anti-angiogenic effect of VEGF-A inhibitors in certain tumor models. Despite its importance, the extent to which BIM is autonomously required for physiological endothelial apoptosis remains unknown and its regulation under such conditions is poorly defined. While the transcription factor FOXO3 has been proposed to induce Bim in response to growth factor withdrawal, evidence for this function is circumstantial. We report that apoptosis was reduced in Bim(-/-) primary endothelial cells, demonstrating a cell-autonomous role for BIM in endothelial death following serum and growth factor withdrawal. In conflict with in vitro studies, BIM-dependent endothelial death in vivo did not require FOXO3. Moreover, endothelial apoptosis proceeded normally in mice lacking FOXO-binding sites in the Bim promoter. Bim mRNA was upregulated in endothelial cells starved of serum and growth factors and this was accompanied by the downregulation of miRNAs of the miR-17∼92 cluster. Bim mRNA levels were also elevated in miR-17∼92(+/-) endothelial cells cultured under steady-state conditions, suggesting that miR-17∼92 cluster miRNAs may contribute to regulating overall Bim mRNA levels in endothelial cells.

Intestinal and peri-tumoral lymphatic endothelial cells are resistant to radiation-induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sung, Hoon Ki; Department of Anatomy, Yeung Nam University Medical School, Daegu 705-717; Morisada, Tohru

2006-06-30

Radiation therapy is a widely used cancer treatment, but it is unable to completely block cancer metastasis. The lymphatic vasculature serves as the primary route for metastatic spread, but little is known about how lymphatic endothelial cells respond to radiation. Here, we show that lymphatic endothelial cells in the small intestine and peri-tumor areas are highly resistant to radiation injury, while blood vessel endothelial cells in the small intestine are relatively sensitive. Our results suggest the need for alternative therapeutic modalities that can block lymphatic endothelial cell survival, and thus disrupt the integrity of lymphatic vessels in peri-tumor areas.

Patients with Dry Eye Disease and Low Subbasal Nerve Density are at High Risk for an Accelerated Corneal Endothelial Cell Loss

PubMed Central

Kheirkhah, Ahmad; Satitpitakul, Vannarut; Hamrah, Pedram; Dana, Reza

2016-01-01

Purpose To evaluate the changes in corneal endothelial cell density (CECD) over time in patients with dry eye disease (DED) and to correlate the endothelial cell loss with corneal subbasal nerve density. Methods This retrospective study included 40 eyes of 20 patients with DED. Laser in vivo confocal microscopy had been performed in the central cornea of both eyes at an initial visit and repeated after a mean follow-up of 33.2 ± 10.2 months. The densities of corneal endothelial cells and subbasal nerves were measured in both visits and compared with 13 eyes of 13 normal age-matched controls. Results At the initial visit, the DED group had lower densities of corneal endothelial cells (2620 ± 386 cells/mm2) and subbasal nerves (17.8 ± 7.5 mm/mm2) compared with the control group (2861 ± 292 cells/mm2 and 22.8 ± 3.0 mm/mm2, with P=0.08 and P=0.01, respectively). At the end of follow-up, although there was no significant change in subbasal nerve density (16.7 ± 7.2 mm/mm2, P=0.43), the mean CECD significantly decreased to 2465 ± 391 cells/mm2 (P=0.01), with a mean corneal endothelial cell loss of 2.1 ± 3.6% per year. The endothelial cell loss showed a statistically significant negative correlation with the initial subbasal nerve density (Rs= −0.55, P=0.02). Conclusion Patients with DED have an accelerated corneal endothelial cell loss which is more than what has been reported in the literature for normal aging. Those with lower subbasal nerve density, in particular, are at a higher risk for endothelial cell loss over time. PMID:28060067

N-acetylcysteine attenuates TNF-alpha-induced human vascular endothelial cell apoptosis and restores eNOS expression.

PubMed

Xia, Zhengyuan; Liu, Min; Wu, Yong; Sharma, Vijay; Luo, Tao; Ouyang, Jingping; McNeill, John H

2006-11-21

The circulatory inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in pathological conditions, such as diabetes, which initiate or exacerbate vascular endothelial injury. Both nitric oxide (NO) and reactive oxygen species may play a dual role (i.e., inhibiting or promoting) in TNF-alpha-induced endothelial cell apoptosis. We investigated the effects of the antioxidant N-acetylcysteine on TNF-alpha-induced apoptosis in human vascular endothelial cell (cell line ECV304) apoptosis, NO production and lipid peroxidation. Cultured vascular endothelial cell (ECV304) were either not treated (control), or treated with TNF-alpha (40 ng/ml) alone or TNF-alpha in the presence of N-acetylcysteine at 30 mmol/l or 1 mmol/l, respectively, for 24 h. Cell viability was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was assessed by flow cytometry. TNF-alpha-induced endothelial cell apoptosis was associated with increased inducible NO synthase but reduced endothelial NO synthase (eNOS) protein expression. NO production and the levels of the lipid peroxidation product malondialdehyde were concomitantly increased. Treatment with NAC at 30 mmol/l restored eNOS expression and further increased NO production as compared to TNF-alpha alone, resulting in improved cell viability and reduced apoptosis. This was accompanied by increased superoxide dismutase activity, increased glutathione peroxidase production and reduced malondialdehyde levels. N-acetylcysteine at 1 mmol/l, however, did not have significant effects on TNF-alpha-induced endothelial cell apoptosis and cell viability despite it slightly enhanced glutathione peroxidase production. N-acetylcysteine attenuation of TNF-alpha-induced human vascular endothelial cell apoptosis is associated with the restoration of eNOS expression.

[Isolation and culture of bovine choriocapillary endothelial cells using paramagnetic beads coated with Lycopersicon esculentum].

PubMed

Swiech-Zubilewicz, A; Soubrane, G; Mascarelli, F

2000-01-01

To establish a pure culture of choriocapillary endothelial cells as a model of angiogenesis in vitro. Bovine choriocapillary endothelial cells (BCEC) were obtained by the method described by Hoffmann et al. (6) using the polystyrene paramagnetic beads coated with Lycopersicon esculentum, which attach specifically to the rest of fucose on the surface of microvascular endothelial cells. The endothelial characteristic of the cultured cells was evaluated by immunocytochemistry using anti von Willebrand factor and anti-CD 31 antibodies. Proliferation and survival of BCEC were tested using haemacytometer of Mallasez. The purity of obtained BCEC culture was confirmed by positive immunocytochemical staining with anti von Willebrand and anti factor CD 31 antibodies in more than 95% of cells. The proliferation of cells in Endothelial Cell Medium resulted in twofold increase of number of cells during 4-day observation period. After reaching the confluence, the cells continued to proliferate with increase of the cell number by 60% during 4-day observation. The use of paramagnetic beads coated with specific lectine provide a pure isolation of BCEC, which can be maintained in culture with preservation of their characteristic.

Endothelial microparticle-mediated transfer of MicroRNA-126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles.

PubMed

Jansen, Felix; Yang, Xiaoyan; Hoelscher, Marion; Cattelan, Arianna; Schmitz, Theresa; Proebsting, Sebastian; Wenzel, Daniela; Vosen, Sarah; Franklin, Bernardo S; Fleischmann, Bernd K; Nickenig, Georg; Werner, Nikos

2013-10-29

Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Endothelial microparticles promote vascular endothelial repair by delivering functional miR-126 into recipient cells. In pathological hyperglycemic conditions, EMP-mediated miR-126-induced EC repair is altered.

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

PubMed

Brumm, Andrew J; Nunez, Stefanie; Doroudchi, Mehdi M; Kawaguchi, Riki; Duan, Jinhzu; Pellegrini, Matteo; Lam, Larry; Carmichael, S Thomas; Deb, Arjun; Hinman, Jason D

2017-08-01

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

Ligand-dependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2

PubMed Central

Eichmann, Anne; Corbel, Catherine; Nataf, Valérie; Vaigot, Pierre; Bréant, Christiane; Le Douarin, Nicole M.

1997-01-01

The existence of a common precursor for endothelial and hemopoietic cells, termed the hemangioblast, has been postulated since the beginning of the century. Recently, deletion of the endothelial-specific vascular endothelial growth factor receptor 2 (VEGFR2) by gene targeting has shown that both endothelial and hemopoietic cells are absent in homozygous null mice. This observation suggested that VEGFR2 could be expressed by the hemangioblast and essential for its further differentiation along both lineages. However, it was not possible to exclude the hypothesis that hemopoietic failure was a secondary effect resulting from the absence of an endothelial cell microenvironment. To distinguish between these two hypotheses, we have produced a mAb directed against the extracellular domain of avian VEGFR2 and isolated VEGFR2+ cells from the mesoderm of chicken embryos at the gastrulation stage. We have found that in clonal cultures, a VEGFR2+ cell gives rise to either a hemopoietic or an endothelial cell colony. The developmental decision appears to be regulated by the binding of two different VEGFR2 ligands. Thus, endothelial differentiation requires VEGF, whereas hemopoietic differentiation occurs in the absence of VEGF and is significantly reduced by soluble VEGFR2, showing that this process could be mediated by a second, yet unidentified, VEGFR2 ligand. These observations thus suggest strongly that in the absence of the VEGFR2 gene product, the precursors of both hemopoietic and vascular endothelial lineages cannot survive. These cells therefore might be the initial targets of the VEGFR2 null mutation. PMID:9144204

Establishment and characterization of an angiosarcoma-derived cell line, AS-M.

PubMed

Krump-Konvalinkova, Vera; Bittinger, Fernando; Olert, Jürgen; Bräuninger, Wolfgang; Brunner, Joachim; Kirkpatrick, C James

2003-01-01

A novel human endothelial cell line, AS-M, has been established from a cutaneous angiosarcoma on the scalp. The cells expressing platelet endothelial cell adhesion molecule-1 (CD31) were isolated using magnetic beads and subsequently cultured for a year. To date, the cells have undergone more than 100 population doublings (PDs). The AS-M cells manifested endothelial characteristics, such as active uptake of acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil-Ac-LDL), capacity to bind the Ulex europeaus agglutin-I (UEA-I), and expression of von Willebrand factor (vWF) and CD31. The single cell-derived clone, AS-M.5, showed a constitutive expression of CD31, vWF, angiotensin-converting enzyme (ACE), endoglin (CD105), and the endothelial cell receptor tyrosine kinases KDR and Tie-1. Similarly to freshly isolated endothelial cells, the AS-M.5 responded to induction by bacterial lipopolysaccharide (LPS) by increased transcription of cell adhesion molecules and cytokines. The AS-M.5 cultures required endothelial growth supplements for optimal growth and long-term propagation in vitro. However, in contrast to normal endothelial cells, p53 gene products were detected in nuclei of AS-M.5 cells. Cytogenetic analyses consistently revealed a hypodiploid karyotype with complete loss of one homologue of several chromosomes and a homogeneous pattern of distinct karyotypic changes. Although the AS-M.5 presented characteristics suggestive of tumor cells, they did not develop into tumors when inoculated subcutaneously into nude mice. The cell line AS-M.5 could be a useful model system to study endothelial pathobiology in vitro.

Establishment of a translational endothelial cell model using directed differentiation of induced pluripotent stem cells from Cynomolgus monkey.

PubMed

Thoma, Eva C; Heckel, Tobias; Keller, David; Giroud, Nicolas; Leonard, Brian; Christensen, Klaus; Roth, Adrian; Bertinetti-Lapatki, Cristina; Graf, Martin; Patsch, Christoph

2016-10-25

Due to their broad differentiation potential, pluripotent stem cells (PSCs) offer a promising approach for generating relevant cellular models for various applications. While human PSC-based cellular models are already advanced, similar systems for non-human primates (NHPs) are still lacking. However, as NHPs are the most appropriate animals for evaluating the safety of many novel pharmaceuticals, the availability of in vitro systems would be extremely useful to bridge the gap between cellular and animal models. Here, we present a NHP in vitro endothelial cell system using induced pluripotent stem cells (IPSCs) from Cynomolgus monkey (Macaca fascicularis). Based on an adapted protocol for human IPSCs, we directly differentiated macaque IPSCs into endothelial cells under chemically defined conditions. The resulting endothelial cells can be enriched using immuno-magnetic cell sorting and display endothelial marker expression and function. RNA sequencing revealed that the differentiation process closely resembled vasculogenesis. Moreover, we showed that endothelial cells derived from macaque and human IPSCs are highly similar with respect to gene expression patterns and key endothelial functions, such as inflammatory responses. These data demonstrate the power of IPSC differentiation technology to generate defined cell types for use as translational in vitro models to compare cell type-specific responses across species.

Gata4-Dependent Differentiation of c-Kit+ Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the Heart.

PubMed

Maliken, Bryan D; Kanisicak, Onur; Karch, Jason; Khalil, Hadi; Fu, Xing; Boyer, Justin G; Prasad, Vikram; Zheng, Yi; Molkentin, Jeffery D

2018-04-17

Background -While c-Kit + adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells (CPCs). Methods - Kit allele-dependent lineage tracing and fusion analysis was performed in mice following simultaneous Gata4 and Gata6 cell-type specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit + cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2 CreERT2 transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. Results -Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2 CreERT2 transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artefact of greater leukocyte-cardiomyocyte cellular fusion due to defective endothelial cell differentiation in the absence of Gata4 Conclusions -Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit + cells using Kit allele lineage tracing appears to be an artefact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit + endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and capillary network integrity.

Infection of endothelial cells by common human viruses.

PubMed

Friedman, H M

1989-01-01

Common human viruses were evaluated for their ability to replicate in the endothelial cells of human umbilical vein and bovine thoracic aorta in vitro. Infection occurred with most viruses. The susceptibilities of endothelial cells derived from bovine aorta, pulmonary artery, and vena cava were compared. Among the viruses studied, no differences were noted in the ability to grow in endothelial cells from these three large vessels. One virus, herpes simplex virus type 1, was evaluated for its ability to produce persistent infection of endothelial cells. Infection developed and persisted for up to 3 months. After the first week, productive infection was found in less than 1% of cells. Nevertheless, the infection markedly affected the growth and morphology of the endothelial monolayer. Infection with any of several different viruses was noted to alter endothelial cell functions, including adherence of granulocytes, production of colony-stimulating factor, and synthesis of matrix protein. In addition, herpes simplex virus type 1 induced receptors for the Fc portion of IgG and for complement component C3b. These findings indicate that common human viruses can profoundly affect the biology of the endothelium.

Pulmonary endothelial pavement patterns.

PubMed Central

Kibria, G; Heath, D; Smith, P; Biggar, R

1980-01-01

The appearance of the endothelial pavement pattern was studied in the pulmonary trunk, pulmonary veins, aorta, and inferior vena cava of the rat by means of silver staining of the cell borders. The endothelial cell in each of the four blood vessels was found to have its own distinctive shape, fusiform and pointed in the direction of blood flow in the case of the aorta and larger and more rectangular in the pulmonary trunk and pulmonary veins. Detailed quantitation of the dimensions and surface area of the endothelial cells in each blood vessel was carried out by a photographic technique. Pulmonary hypertension was induced in one group of rats by feeding them on Crotalaria spectabilis seeds. The endothelial pavement pattern in their pulmonary trunks became disrupted with many of the cells assuming a fusiform shape reminiscent of aortic endothelium. Many small, new endothelial cells formed in the pulmonary trunk suggesting division of cells to line the enlarging blood vessels. In contrast the endothelial cells of the inferior vena cava merely increased in size to cope with the dilatation of this vein. Images PMID:7385090

Femtosecond laser cutting of endothelial grafts: comparison of endothelial and epithelial applanation.

PubMed

Bernard, Aurélien; He, Zhiguo; Gauthier, Anne Sophie; Trone, Marie Caroline; Baubeau, Emmanuel; Forest, Fabien; Dumollard, Jean Marc; Peocʼh, Michel; Thuret, Gilles; Gain, Philippe

2015-02-01

Stromal surface quality of endothelial lamellae cut for endothelial keratoplasty with a femtosecond laser (FSL) with epithelial applanation remains disappointing. Applanation of the endothelial side of the cornea, mounted inverted on an artificial chamber, has therefore been proposed to improve cut quality. We compared lamellar quality after FSL cutting using epithelial versus endothelial applanation. Lamellae were cut with an FSL from organ-cultured corneas. After randomization, 7 were cut with epithelial applanation and 7 with endothelial applanation. Lamellae of 50-, 75-, and 100-μm thickness were targeted. Thickness was measured by optical coherence tomography before and immediately after cutting. Viable endothelial cell density was quantified immediately after cutting using triple labeling with Hoechst/ethidium/calcein-AM coupled with image analysis with ImageJ. The stromal surface was evaluated by 9 masked observers using semiquantitative scoring of scanning electronic microscopy images. Histology of 2 samples was also analyzed before lamellar detachment. Precision (difference in target/actual thickness) and thickness regularity [coefficient of variation (CV) of 10 measurements] were significantly better with endothelial applanation (precision: 18 μm; range, 10-30; CV: 11%; range, 8-12) than with epithelial applanation (precision: 84 μm; range, 54-107; P = 0.002; CV: 24%; range, 13-47; P = 0.001). Endothelial applanation provided thinner lamellae. However, viable endothelial cell density was significantly lower after endothelial applanation (1183 cells/mm2; range, 787-1725 versus 1688 cells/mm2; range, 1288-2025; P = 0.018). FSL cutting of endothelial lamellae using endothelial applanation provides thinner more regular grafts with more predictable thickness than with conventional epithelial applanation but strongly reduces the pool of viable endothelial cells.

Mechanism of action of rapalogues: the antiangiogenic hypothesis.

PubMed

Faivre, Sandrine; Raymond, Eric

2008-11-01

mTOR interacts with multiple proteins involved in major signal transduction pathways controlling cell growth, proliferation, and apoptosis. mTOR is acknowledged to play major roles in cellular interplays between cancer and stroma cells, including endothelial cells. Rapalogues demonstrated antitumour activity in several hypervascularized tumours in clinical trials. Whether rapalogues directly affect cancer cells or other stroma cells in tumours remains poorly understood. Knowing whether rapalogues act directly against cancer cells and/or could be considered as antiangiogenic agents has major implications in terms of medical indications and may help to further improve their drug development. Herein, we hypothesize that current rapalogues demonstrating activity in hypervascularized tumours may primarily act through antiangiogenic effects in patients, a hypothesis that certainly requires further translational investigations.

Hydraulic Conductivity of Smooth Muscle Cell-Initiated Arterial Cocultures

PubMed Central

Mathura, Rishi A.; Russell-Puleri, Sparkle; Cancel, Limary M.; Tarbell, John M.

2015-01-01

The purpose of the study was to examine the effects of arterial coculture conditions on the transport properties of several in vitro endothelial cell (EC) – smooth muscle cell (SMC) – porous filter constructs in which SMC were grown to confluence first and then EC were inoculated. This order of culturing simulates the environment of a blood vessel wall after endothelial layer damage due to stenting, vascular grafting or other vascular wall insult. For all coculture configurations examined, we observed that hydraulic conductivity (Lp) values were significantly higher than predicted by a resistances-in-series (RIS) model accounting for the Lp of EC and SMC measured separately. The greatest increases were observed when EC were plated directly on top of a confluent SMC layer without an intervening filter, presumably mediated by direct EC – SMC contacts that were observed under confocal microscopy. The results are the opposite of a previous study that showed Lp was significantly reduced compared to an RIS model when EC were grown to confluency first. The physiological, pathophysiological and tissue engineering implications of these results are discussed. PMID:26265460

Hydraulic Conductivity of Smooth Muscle Cell-Initiated Arterial Cocultures.

PubMed

Mathura, Rishi A; Russell-Puleri, Sparkle; Cancel, Limary M; Tarbell, John M

2016-05-01

The purpose of the study was to examine the effects of arterial coculture conditions on the transport properties of several in vitro endothelial cell (EC)-smooth muscle cell (SMC)-porous filter constructs in which SMC were grown to confluence first and then EC were inoculated. This order of culturing simulates the environment of a blood vessel wall after endothelial layer damage due to stenting, vascular grafting or other vascular wall insult. For all coculture configurations examined, we observed that hydraulic conductivity (L(p)) values were significantly higher than predicted by a resistances-in-series (RIS) model accounting for the L(p) of EC and SMC measured separately. The greatest increases were observed when EC were plated directly on top of a confluent SMC layer without an intervening filter, presumably mediated by direct EC-SMC contacts that were observed under confocal microscopy. The results are the opposite of a previous study that showed L(p) was significantly reduced compared to an RIS model when EC were grown to confluency first. The physiological, pathophysiological and tissue engineering implications of these results are discussed.

Omega-3 Fatty Acids Modulate TRPV4 Function through Plasma Membrane Remodeling.

PubMed

Caires, Rebeca; Sierra-Valdez, Francisco J; Millet, Jonathan R M; Herwig, Joshua D; Roan, Esra; Vásquez, Valeria; Cordero-Morales, Julio F

2017-10-03

Dietary consumption of ω-3 polyunsaturated fatty acids (PUFAs), present in fish oils, is known to improve the vascular response, but their molecular targets remain largely unknown. Activation of the TRPV4 channel has been implicated in endothelium-dependent vasorelaxation. Here, we studied the contribution of ω-3 PUFAs to TRPV4 function by precisely manipulating the fatty acid content in Caenorhabditis elegans. By genetically depriving the worms of PUFAs, we determined that the metabolism of ω-3 fatty acids is required for TRPV4 activity. Functional, lipid metabolome, and biophysical analyses demonstrated that ω-3 PUFAs enhance TRPV4 function in human endothelial cells and support the hypothesis that lipid metabolism and membrane remodeling regulate cell reactivity. We propose a model whereby the eicosanoid's epoxide group location increases membrane fluidity and influences the endothelial cell response by increasing TRPV4 channel activity. ω-3 PUFA-like molecules might be viable antihypertensive agents for targeting TRPV4 to reduce systemic blood pressure. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

Endothelial cell density after photorefractive keratectomy for moderate myopia using a 213 nm solid-state laser system.

PubMed

Tsiklis, Nikolaos S; Kymionis, George D; Pallikaris, Aristofanis I; Diakonis, Vasilios F; Ginis, Harilaos S; Kounis, George A; Panagopoulou, Sophia I; Pallikaris, Ioannis G

2007-11-01

To evaluate whether photorefractive keratectomy (PRK) for moderate myopia using a solid-state laser with a wavelength of 213 nm alters the corneal endothelial cell density. University refractive surgery center. The corneal endothelium was analyzed preoperatively and 1, 6, and 12 months postoperatively using corneal confocal microscopy (modified HRT II with a Rostock Cornea Module, Heidelberg Engineering) in 60 eyes (30 patients). Patients were randomized to have myopic PRK using a 213 nm wavelength solid-state laser (study group) or a conventional 193 nm wavelength excimer laser (control group). Three endothelial images were acquired in each of 30 preoperative normal eyes to evaluate the repeatability of endothelial cell density measurements. Repeated-measures analysis of variance was used to compare the variations in endothelial cell density between the 2 lasers and the changes in endothelial cell density over time. There were no statistically significant differences in sex, age, corneal pachymetry, attempted correction, preoperative endothelial cell density, or postoperative refractive outcomes (uncorrected visual acuity, best spectacle-corrected visual acuity, and spherical equivalent refraction) between the 2 groups (P>.05). The coefficient of repeatability of endothelial cell density was 131 cells/mm(2). The measured endothelial cell count per 1.0 mm(2) did not significantly change up to 1 year postoperatively in either group (both P>.05). No statistically significant difference was found between the 2 groups in any postoperative interval (P>.05). Photorefractive keratectomy for moderate myopia using a 213 nm wavelength solid-state laser or a conventional 193 nm wavelength excimer laser did not significantly affect corneal endothelial density during the 1-year postoperative period.

A Pilot Study Linking Endothelial Injury in Lungs and Kidneys in Chronic Obstructive Pulmonary Disease

PubMed Central

Laucho-Contreras, Maria E.; Petersen, Hans; Bijol, Vanesa; Sholl, Lynette M.; Choi, Mary E.; Divo, Miguel; Pinto-Plata, Victor; Chetta, Alfredo; Tesfaigzi, Yohannes; Celli, Bartolomé R.

2017-01-01

Rationale: Patients with chronic obstructive pulmonary disease (COPD) frequently have albuminuria (indicative of renal endothelial cell injury) associated with hypoxemia. Objectives: To determine whether (1) cigarette smoke (CS)-induced pulmonary and renal endothelial cell injury explains the association between albuminuria and COPD, (2) CS-induced albuminuria is linked to increases in the oxidative stress–advanced glycation end products (AGEs) receptor for AGEs (RAGE) pathway, and (3) enalapril (which has antioxidant properties) limits the progression of pulmonary and renal injury by reducing activation of the AGEs–RAGE pathway in endothelial cells in both organs. Methods: In 26 patients with COPD, 24 ever-smokers without COPD, 32 nonsmokers who underwent a renal biopsy or nephrectomy, and in CS-exposed mice, we assessed pathologic and ultrastructural renal lesions, and measured urinary albumin/creatinine ratios, tissue oxidative stress levels, and AGEs and RAGE levels in pulmonary and renal endothelial cells. The efficacy of enalapril on pulmonary and renal lesions was assessed in CS-exposed mice. Measurements and Main Results: Patients with COPD and/or CS-exposed mice had chronic renal injury, increased urinary albumin/creatinine ratios, and increased tissue oxidative stress and AGEs-RAGE levels in pulmonary and renal endothelial cells. Treating mice with enalapril attenuated CS-induced increases in urinary albumin/creatinine ratios, tissue oxidative stress levels, endothelial cell AGEs and RAGE levels, pulmonary and renal cell apoptosis, and the progression of chronic renal and pulmonary lesions. Conclusions: Patients with COPD and/or CS-exposed mice have pulmonary and renal endothelial cell injury linked to increased endothelial cell AGEs and RAGE levels. Albuminuria could identify patients with COPD in whom angiotensin-converting enzyme inhibitor therapy improves renal and lung function by reducing endothelial injury. PMID:28085500

Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells.

PubMed

Becher, Tobias; Schulze, Torsten J; Schmitt, Melanie; Trinkmann, Frederik; El-Battrawy, Ibrahim; Akin, Ibrahim; Kälsch, Thorsten; Borggrefe, Martin; Stach, Ksenija

2017-01-15

Lipid lowering therapy constitutes the basis of cardiovascular disease therapy. The purpose of this study was to investigate effects of ezetimibe, a selective inhibitor of intestinal cholesterol absorption, on platelets and endothelial cells in an in vitro endothelial cell model. After a 24h incubation period with ezetimibe (concentrations 1, 50, 100 and 1000ng/ml), human umbilical vein endothelial cells (HUVEC) were stimulated for 1h with lipopolysaccharide (LPS) and were then incubated in direct contact with activated platelets. Following this, the expression of CD40L and CD62P on platelets, and the expression of ICAM-1, VCAM-1, uPAR, and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analysed by enzyme linked immunosorbent assay for soluble MCP-1, IL-6 and MMP-1. The increased expression of uPAR on endothelial cells by proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets was significantly reduced through pre-incubation with 100ng/ml and 1000ng/ml ezetimibe (p<0.05). Platelets directly incubated with ezetimibe but without endothelial cell contact showed significantly reduced CD62P and CD40L surface expression (p<0.05). Ezetimibe had no significant effects on HUVEC expression of MT1-MMP, ICAM-1 and VCAM-1 and on CD40L expression on platelets in direct contact with endothelial cells. Levels of soluble IL-6 in HUVEC supernatants were significantly lower after pre-incubation with ezetimibe. In this in vitro analysis, ezetimibe directly attenuates platelet activation and has significant endothelial cell mediated effects on selected markers of atherosclerosis. Copyright © 2016. Published by Elsevier Ireland Ltd.

Apoptosis regulator through modulating IAP expression (ARIA) controls the PI3K/Akt pathway in endothelial and endothelial progenitor cells.

PubMed

Koide, Masahiro; Ikeda, Koji; Akakabe, Yoshiki; Kitamura, Youhei; Ueyama, Tomomi; Matoba, Satoaki; Yamada, Hiroyuki; Okigaki, Mitsuhiko; Matsubara, Hiroaki

2011-06-07

Endothelial and endothelial progenitor cells (ECs and EPCs) play a fundamental role in angiogenesis that is essential for numerous physiological and pathological processes. The phosphatase and tensin homolog (PTEN)/ phosphoinositide 3-kinase (PI3K) pathway has been implicated in angiogenesis, but the mechanism in the regulation of this pathway in ECs and EPCs is poorly understood. Here we show that ARIA (apoptosis regulator through modulating IAP expression), a transmembrane protein that we recently identified, regulates the PTEN/PI3K pathway in ECs and EPCs and controls developmental and postnatal angiogenesis in vivo. We found that ARIA is abundantly expressed in EPCs and regulates their angiogenic functions by modulating PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling. Genetic deletion of ARIA caused nonfatal bleeding during embryogenesis, in association with increased small vessel density and altered expression of various vascular growth factors including angiopoietins and VEGF receptors. Postnatal neovascularization induced by critical limb ischemia was substantially enhanced in ARIA-null mice, in conjunction with more bone marrow (BM)-derived ECs detected in ischemic muscles. Administration of PI3K or NO synthase inhibitor completely abolished the enhanced neovascularization in ARIA(-/-) mice. Mechanistically, we identified that ARIA interacts with PTEN at the intracellular domain independently of the PTEN phosphorylation in its C-terminal tail. Overexpressed ARIA increased PTEN in the membrane fraction, whereas ARIA-silencing reduced the membrane-associated PTEN, resulting in modified PI3K/Akt signaling. Taken together, our findings establish a previously undescribed mode of regulation of the PTEN/PI3K/Akt pathway by ARIA, and reveal a unique mechanism in the control of angiogenesis. These functions of ARIA might offer a unique therapeutic potential.

Angiogenesis in the reparatory mucosa of the mandibular edentulous ridge is driven by endothelial tip cells.

PubMed

Stănescu, Ruxandra; Didilescu, Andreea Cristiana; Jianu, Adelina Maria; Rusu, M C

2012-01-01

Sprouting angiogenesis is led by specialized cell--the endothelial tip cells (ETCs) which can be targeted by pro- or anti-angiogenic therapies. We aimed to perform a qualitative study in order to assess the guidance by tip cells of the endothelial sprouts in the repairing mucosa of the edentulous mandibular crest. Mucosa of the mandibular edentulous ridge was collected from six adult patients, prior to healing abutment placement (second surgery). Slides were prepared and immunostained with antibodies for CD34 and Ki67. The abundant vasculature of the lamina propria was observed on slides and the CD34 antibodies labeled endothelial tip cells in various stages of the endothelial sprouts. Ki67 identified positive endothelial cells, confirming the proliferative status of the microvascular bed. According to the results, the in situ sprouting angiogenesis is driven by tip cells in the oral mucosa of the edentulous ridge and these cells can be targeted by various therapies, as required by the local pathologic or therapeutic conditions.

Effects of ADMA upon Gene Expression: An Insight into the Pathophysiological Significance of Raised Plasma ADMA

PubMed Central

Smith, Caroline L; Anthony, Shelagh; Hubank, Mike; Leiper, James M; Vallance, Patrick

2005-01-01

Background Asymmetric dimethylarginine (ADMA) is a naturally occurring inhibitor of nitric oxide synthesis that accumulates in a wide range of diseases associated with endothelial dysfunction and enhanced atherosclerosis. Clinical studies implicate plasma ADMA as a major novel cardiovascular risk factor, but the mechanisms by which low concentrations of ADMA produce adverse effects on the cardiovascular system are unclear. Methods and Findings We treated human coronary artery endothelial cells with pathophysiological concentrations of ADMA and assessed the effects on gene expression using U133A GeneChips (Affymetrix). Changes in several genes, including bone morphogenetic protein 2 inducible kinase (BMP2K), SMA-related protein 5 (Smad5), bone morphogenetic protein receptor 1A, and protein arginine methyltransferase 3 (PRMT3; also known as HRMT1L3), were confirmed by Northern blotting, quantitative PCR, and in some instances Western blotting analysis to detect changes in protein expression. To determine whether these changes also occurred in vivo, tissue from gene deletion mice with raised ADMA levels was examined. More than 50 genes were significantly altered in endothelial cells after treatment with pathophysiological concentrations of ADMA (2 μM). We detected specific patterns of changes that identify pathways involved in processes relevant to cardiovascular risk and pulmonary hypertension. Changes in BMP2K and PRMT3 were confirmed at mRNA and protein levels, in vitro and in vivo. Conclusion Pathophysiological concentrations of ADMA are sufficient to elicit significant changes in coronary artery endothelial cell gene expression. Changes in bone morphogenetic protein signalling, and in enzymes involved in arginine methylation, may be particularly relevant to understanding the pathophysiological significance of raised ADMA levels. This study identifies the mechanisms by which increased ADMA may contribute to common cardiovascular diseases and thereby indicates possible targets for therapies. PMID:16190779

Establishment of pancreatic microenvironment model of ER stress: Quercetin attenuates β-cell apoptosis by invoking nitric oxide-cGMP signaling in endothelial cells.

PubMed

Suganya, Natarajan; Mani, Krishna Priya; Sireesh, Dornadula; Rajaguru, Palanisamy; Vairamani, Mariappanadar; Suresh, Thiruppathi; Suzuki, Takayoshi; Chatterjee, Suvro; Ramkumar, Kunka Mohanram

2018-05-01

The involvement of endoplasmic reticulum (ER) stress in endothelial dysfunction and diabetes-associated complications has been well documented. Inhibition of ER stress represents a promising therapeutic strategy to attenuate endothelial dysfunction in diabetes. Recent attention has focused on the development of small molecule inhibitors of ER stress to maintain endothelial homeostasis in diabetes. Here we have developed a reliable, robust co-culture system that allows a study on the endothelial cells and pancreatic β-cells crosstalk under ER stress and validated using a known ER stress modulator, quercetin. Furthermore, sensitizing of endothelial cells by quercetin (25 μM) confers protection of pancreatic β-cells against ER stress through nitric oxide (NO ∙ ) signaling. In addition, increased intracellular insulin and NO ∙ -mediated cyclic 3',5'-guanosine monophosphate (cGMP) levels in pancreatic β-cells further confirmed the mechanism of protection under co-culture system. In addition, the potential protein targets of quercetin against ER stress in the endothelial cells were investigated through proteomic profiling and its phosphoprotein targets through Bioplex analysis. On the whole, the developed in vitro co-culture set up can serve as a platform to study the signaling network between the endothelial and pancreatic β-cells as well as provides a mechanistic insight for the validation of novel ER stress modulators. Copyright © 2018 Elsevier Inc. All rights reserved.

Kisspeptin-10 induces endothelial cellular senescence and impaired endothelial cell growth.

PubMed

Usui, Sayaka; Iso, Yoshitaka; Sasai, Masahiro; Mizukami, Takuya; Mori, Hiroyoshi; Watanabe, Takuya; Shioda, Seiji; Suzuki, Hiroshi

2014-07-01

The KPs (kisspeptins) are a family of multifunctional peptides with established roles in cancer metastasis, puberty and vasoconstriction. The effects of KPs on endothelial cells have yet to be determined. The aim of the present study was to investigate the effects of KP-10 on endothelial cell growth and the mechanisms underlying those effects. The administration of recombinant KP-10 into the hindlimbs of rats with ischaemia significantly impaired blood flow recovery, as shown by laser Doppler, and capillary growth, as shown using histology, compared with the controls. HUVECs (human umbilical vein endothelial cells) express the KP receptor and were treated with KP-10 in culture studies. KP-10 inhibited endothelial cell tube formation and proliferation in a significant and dose-dependent manner. The HUVECs treated with KP exhibited the senescent phenotype, as determined using a senescence-associated β-galactosidase assay, cell morphology analysis, and decreased Sirt1 (sirtuin 1) expression and increased p53 expression shown by Western blot analysis. Intriguingly, a pharmacological Rho kinase inhibitor, Y-27632, was found to increase the proliferation of HUVECs and to reduce the number of senescent phenotype cells affected by KP-10. In conclusion, KP-10 suppressed endothelial cells growth both in vivo and in vitro in the present study. The adverse effect of KP on endothelial cells was attributable, at least in part, to the induction of cellular senescence.

Systemic administration of thrombin peptide TP508 enhances VEGF-stimulated angiogenesis and attenuates effects of chronic hypoxia

PubMed Central

Olszewska-Pazdrak, Barbara; Carney, Darrell H.

2015-01-01

Revascularization of chronic wounds and ischemic tissue is attenuated by endothelial dysfunction and the inability of angiogenic factors to stimulate angiogenesis. We recently showed that TP508, a nonproteolytic thrombin peptide, increases perfusion and NO-dependent vasodilation in hearts with chronic ischemia and stimulates NO production by endothelial cells. In this study, we investigated systemic in vivo effects of TP508 on VEGF-stimulated angiogenesis in vitro using aortic explants in normoxic and hypoxic conditions. Mice were injected with saline or TP508 and 24h later aortas were removed and cultured to quantify endothelial sprouting. TP508 injection increased endothelial sprouting and potentiated the in vitro response to VEGF. Exposure of control explants to hypoxia inhibited basal and VEGF-stimulated endothelial cell sprouting. This effect of hypoxia was significantly prevented by TP508 injection. Thus, TP508 systemic administration increases responsiveness of aortic endothelial cells to VEGF and diminishes the effect of chronic hypoxia on endothelial cell sprouting. Studies using human endothelial cells in culture suggest that protective effects of TP508 during hypoxia may involve stimulation of endothelial cell NO production. These data suggest potential clinical benefit of using a combination of systemic TP508 and local VEGF as a therapy for revascularization of ischemic tissue. PMID:23594718

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vallon, Mario, E-mail: m.vallon@arcor.de; Rohde, Franziska; Janssen, Klaus-Peter

2010-02-01

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

GPER is involved in the stimulatory effects of aldosterone in breast cancer cells and breast tumor-derived endothelial cells

PubMed Central

Rigiracciolo, Damiano Cosimo; Scarpelli, Andrea; Lappano, Rosamaria; Pisano, Assunta; Santolla, Maria Francesca; Avino, Silvia; De Marco, Paola; Bussolati, Benedetta; Maggiolini, Marcello; De Francesco, Ernestina Marianna

2016-01-01

Aldosterone induces relevant effects binding to the mineralcorticoid receptor (MR), which acts as a ligand-gated transcription factor. Alternate mechanisms can mediate the action of aldosterone such as the activation of epidermal growth factor receptor (EGFR), MAPK/ERK, transcription factors and ion channels. The G-protein estrogen receptor (GPER) has been involved in the stimulatory effects of estrogenic signalling in breast cancer. GPER has been also shown to contribute to certain responses to aldosterone, however the role played by GPER and the molecular mechanisms implicated remain to be fully understood. Here, we evaluated the involvement of GPER in the stimulatory action exerted by aldosterone in breast cancer cells and breast tumor derived endothelial cells (B-TEC). Competition assays, gene expression and silencing studies, immunoblotting and immunofluorescence experiments, cell proliferation and migration were performed in order to provide novel insights into the role of GPER in the aldosterone-activated signalling. Our results demonstrate that aldosterone triggers the EGFR/ERK transduction pathway in a MR- and GPER-dependent manner. Aldosterone does not bind to GPER, it however induces the direct interaction between MR and GPER as well as between GPER and EGFR. Next, we ascertain that the up-regulation of the Na+/H+ exchanger-1 (NHE-1) induced by aldosterone involves MR and GPER. Biologically, both MR and GPER contribute to the proliferation and migration of breast and endothelial cancer cells mediated by NHE-1 upon aldosterone exposure. Our data further extend the current knowledge on the molecular mechanisms through which GPER may contribute to the stimulatory action elicited by aldosterone in breast cancer. PMID:26646587

GPER is involved in the stimulatory effects of aldosterone in breast cancer cells and breast tumor-derived endothelial cells.

PubMed

Rigiracciolo, Damiano Cosimo; Scarpelli, Andrea; Lappano, Rosamaria; Pisano, Assunta; Santolla, Maria Francesca; Avino, Silvia; De Marco, Paola; Bussolati, Benedetta; Maggiolini, Marcello; De Francesco, Ernestina Marianna

2016-01-05

Aldosterone induces relevant effects binding to the mineralcorticoid receptor (MR), which acts as a ligand-gated transcription factor. Alternate mechanisms can mediate the action of aldosterone such as the activation of epidermal growth factor receptor (EGFR), MAPK/ERK, transcription factors and ion channels. The G-protein estrogen receptor (GPER) has been involved in the stimulatory effects of estrogenic signalling in breast cancer. GPER has been also shown to contribute to certain responses to aldosterone, however the role played by GPER and the molecular mechanisms implicated remain to be fully understood. Here, we evaluated the involvement of GPER in the stimulatory action exerted by aldosterone in breast cancer cells and breast tumor derived endothelial cells (B-TEC). Competition assays, gene expression and silencing studies, immunoblotting and immunofluorescence experiments, cell proliferation and migration were performed in order to provide novel insights into the role of GPER in the aldosterone-activated signalling. Our results demonstrate that aldosterone triggers the EGFR/ERK transduction pathway in a MR- and GPER-dependent manner. Aldosterone does not bind to GPER, it however induces the direct interaction between MR and GPER as well as between GPER and EGFR. Next, we ascertain that the up-regulation of the Na+/H+ exchanger-1 (NHE-1) induced by aldosterone involves MR and GPER. Biologically, both MR and GPER contribute to the proliferation and migration of breast and endothelial cancer cells mediated by NHE-1 upon aldosterone exposure. Our data further extend the current knowledge on the molecular mechanisms through which GPER may contribute to the stimulatory action elicited by aldosterone in breast cancer.

Downregulation of endothelial adhesion molecules by dimethylfumarate, but not monomethylfumarate, and impairment of dynamic lymphocyte-endothelial cell interactions.

PubMed

Wallbrecht, Katrin; Drick, Nora; Hund, Anna-Carina; Schön, Michael P

2011-12-01

Although fumaric acid esters (FAE) have a decade-long firm place in the therapeutic armamentarium for psoriasis, their pleiotropic mode of action is not yet fully understood. While most previous studies have focused on the effects of FAE on leucocytes, we have addressed their activity on macro- and microvascular endothelial cells. As detected both on mRNA and protein levels, dimethylfumarate effected a profound reduction of TNFα-induced expression of E-selectin (CD62E), ICAM-1 (CD54) and VCAM-1 (CD106) on two different endothelial cell populations in a concentration-dependent manner. This reduction of several endothelial adhesion molecules was accompanied by a dramatic diminution of both rolling and firm adhesive interactions between endothelial cells and lymphocytes in a dynamic flow chamber system. Dimethylfumarate, at a concentration of 50 μm, reduced lymphocyte rolling on endothelial cells by 85.9% (P<0.001 compared to untreated controls), and it diminished the number of adherent cells by 88% (P<0.001). In contrast, monomethylfumarate (MMF) influenced neither surface expression of adhesion molecules nor interactions between endothelial cells and lymphocytes. These observations demonstrate that endothelial cells, in addition to the known effects on leucocytes, undergo profound functional changes in response to dimethylfumarate. These changes are accompanied by severely impaired dynamic interactions with lymphocytes, which constitute the critical initial step of leucocyte recruitment to inflamed tissues in psoriasis and other TNF-related inflammatory disorders. © 2011 John Wiley & Sons A/S.

G-protein-coupled receptor 30 mediates the effects of estrogen on endothelial cell tube formation in vitro.

PubMed

Zhou, Liyuan; Chen, Hong; Mao, Xun; Qi, Hongbo; Baker, Philip N; Zhang, Hua

2017-06-01

The placenta is the exchange organ between the mother and the fetus. The inadequate function of this organ is associated with a number of pregnancy disorders. Hypoxia and oxidative stress during placental development may induce endothelial dysfunction, resulting in the reduction in the perfusion of the placenta. During pregnancy, the levels of estrogen are increased. Decreased estrogen levels have been reported in women with preeclampsia. However, whether estrogen is involved in placental angiogenesis remains unclear. In this study, we aimed to investigate the effects of estrogen on endothelial cell tube formation and to elucidate the underlying mechanisms. For this purpose, human umbilical vein endothelial cells (HUVECs) were cultured with 17‑β‑estradiol under conditions of hypoxia/reoxygenation (H/R). The total pipe length of the tube‑like structure on endothelial cells was measured. The expression levels of G‑protein‑coupled receptor 30 (GPR30) and endothelial nitric oxide synthase (eNOS) and Akt were also measured in the endothelial cells following treatment with 17‑β‑estradiol under H/R conditions by western blot analysis and immunostaining. We found that the total pipe length of the tube‑like structure on endothelial cells was significantly reduced. This reduction was reversed by treatment with 17‑β‑estradiol. The expression of GPR30 in endothelial cells was significantly increased following treatment with 17‑β‑estradiol under H/R conditions. Furthermore, the levels of eNOS and Akt in endothelial cells were also significantly increased following treatment with 17-β-estradiol under H/R conditions. The activation of eNOS was inhibited by wortmannin, an inhibitor of PI3K/Akt. Our data thus demonstrate that estrogen prevents the failure of endothelial cell tube formation induced by H/R. GPR30 plays an important role in these protective effects through the activation of eNOS and Akt in endothelial cells. Our data suggest that increased levels of estrogen are important for placental angiogenesis.

Endothelial glycocalyx: permeability barrier and mechanosensor.

PubMed

Curry, F E; Adamson, R H

2012-04-01

Endothelial cells are covered with a polysaccharide rich layer more than 400 nm thick, mechanical properties of which limit access of circulating plasma components to endothelial cell membranes. The barrier properties of this endothelial surface layer are deduced from the rate of tracer penetration into the layer and the mechanics of red and white cell movement through capillary microvessels. This review compares the mechanosensor and permeability properties of an inner layer (100-150 nm, close to the endothelial membrane) characterized as a quasi-periodic structure which accounts for key aspects of transvascular exchange and vascular permeability with those of the whole endothelial surface layers. We conclude that many of the barrier properties of the whole surface layer are not representative of the primary fiber matrix forming the molecular filter determining transvascular exchange. The differences between the properties of the whole layer and the inner glycocalyx structures likely reflect dynamic aspects of the endothelial surface layer including tracer binding to specific components, synthesis and degradation of key components, activation of signaling pathways in the endothelial cells when components of the surface layer are lost or degraded, and the spatial distribution of adhesion proteins in microdomains of the endothelial cell membrane.

Inhibition of tumor necrosis factor-{alpha}-induced expression of adhesion molecules in human endothelial cells by the saponins derived from roots of Platycodon grandiflorum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Ji Young; Kim, Dong Hee; Kim, Hyung Gyun

2006-01-15

Adhesion molecules play an important role in the development of atherogenesis and are produced by endothelial cells after being stimulated with various inflammatory cytokines. This study examined the effect of saponins that were isolated from the roots of Platycodon grandiflorum A. DC (Campanulaceae), Changkil saponins (CKS), on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. CKS significantly inhibited the TNF{alpha}-induced increase in monocyte adhesion to endothelial cells as well as decreased the protein and mRNA expression levels of vascular adhesion molecule-1 and intercellular cell adhesion molecule-1 on endothelial cells. Furthermore, CKS significantly inhibited themore » TNF{alpha}-induced production of intracellular reactive oxygen species (ROS) and activation of NF-{kappa}B by preventing I{kappa}B degradation and inhibiting I{kappa}B kinase activity. Overall, CKS has anti-atherosclerotic and anti-inflammatory activity, which is least in part the result of it reducing the cytokine-induced endothelial adhesion to monocytes by inhibiting intracellular ROS production, NF-{kappa}B activation, and cell adhesion molecule expression in endothelial cells.« less

Cerebral vasculopathy in children with sickle cell anemia.

PubMed

Fasano, Ross M; Meier, Emily R; Hulbert, Monica L

2015-01-01

Sickle cell anemia (SCA)-associated cerebral vasculopathy and moyamoya is a unique entity reflecting the abnormal interactions between sickled red blood cells (RBCs) and the cerebral arterial endothelium. Endothelial injury, coagulation activation, and the inflammatory response generated by sickled RBCs are implicated in the development of cerebral vasculopathy, but the pathophysiology remains incompletely understood. SCA-specific screening and treatment guidelines have successfully reduced the incidence of overt strokes in this high-risk population. However, despite aggressive hematological management, many children with cerebral vasculopathy due to SCA have progressive vasculopathy and recurrent strokes; therefore, more effective therapies, such as revascularization surgery and curative hematopoietic stem cell transplant, are urgently needed. Copyright © 2014 Elsevier Inc. All rights reserved.

Anti-inflammatory effect of intravenous immunoglobulin in comparison with dexamethasone in vitro: implication for treatment of Kawasaki disease.

PubMed

Makata, Haruyuki; Ichiyama, Takashi; Uchi, Ryutaro; Takekawa, Tsuyoshi; Matsubara, Tomoyo; Furukawa, Susumu

2006-08-01

High-dose intravenous immunoglobulin (IVIG) is a well-established standard therapy for Kawasaki disease (KD) that reduces the risk of developing coronary artery aneurysms. On the other hand, some reports have recommended an alternative therapy with steroids for KD patients. In this study we investigated the anti-inflammatory effect of IVIG in comparison with dexamethasone at clinical doses in vitro. High-dose IVIG inhibited tumor necrosis factor-alpha (TNF-alpha)-induced activation of nuclear factor-kappaB (NF-kappaB) to a greater degree than dexamethasone in human monocytic U937 cells and human coronary arterial endothelial cells (HCAEC), but not in human T lymphocytic Jurkat cells. IVIG was more potent than dexamethasone in reducing the expression of CD16 (FcgammaRIII) in human monocytic THP-1 cells stimulated with lipopolysaccharide and in Jurkat cells stimulated with dimethyl sulfoxide. In HCAEC exposed to TNF-alpha, IVIG and dexamethasone inhibited interleukin-6 production to a similar degree, whereas the expression of E-selectin was inhibited more strongly by IVIG. Our results show that high-dose IVIG inhibits the activation of monocytes/macrophages and coronary arterial endothelial cells more strongly than that of T cells, whereas dexamethasone inhibits the activation of all three cell types. These findings suggest that IVIG or dexamethasone therapy should be chosen to match the types of cells that are activated during acute KD.

Administration of tauroursodeoxycholic acid prevents endothelial dysfunction caused by an oral glucose load

PubMed Central

Walsh, Lauren K.; Restaino, Robert M.; Neuringer, Martha; Manrique, Camila; Padilla, Jaume

2017-01-01

Postprandial hyperglycemia leads to a transient impairment in endothelial function; however, the mechanisms remain largely unknown. Previous work in cell culture models demonstrate that high glucose results in endoplasmic reticulum (ER) stress and, in animal studies, ER stress has been implicated as a cause of endothelial dysfunction. Herein we tested the hypothesis that acute oral administration of tauroursodeoxycholic acid (TUDCA, 1500mg), a chemical chaperone known to alleviate ER stress, would prevent hyperglycemia-induced endothelial dysfunction. In 12 young healthy subjects (seven men, five women), brachial artery flow-mediated dilation (FMD) was assessed at baseline, 1 hour, and 2 hours post an oral glucose challenge. Subjects were tested on two separate visits in a single-blind randomized crossover design: after oral ingestion of TUDCA or placebo capsules. FMD was reduced from baseline during hyperglycemia under the placebo condition (−32% at 1 hr and −28% at 2 hr post oral glucose load; p<0.05 from baseline) but not under the TUDCA condition (−4% at 1 hr and +0.3% at 2 hr post oral glucose load; p>0.05 from baseline). Postprandial plasma glucose and insulin were not altered by TUDCA ingestion. Plasma oxidative stress markers 3-nitrotyrosine and TBARs remained unaltered throughout the oral glucose challenge in both conditions. These results suggest that hyperglycemia-induced endothelial dysfunction can be mitigated by oral administration of TUDCA, thus supporting the hypothesis that ER stress may contribute to endothelial dysfunction during postprandial hyperglycemia. PMID:27503949

Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro.

PubMed

Peh, Gary S L; Toh, Kah-Peng; Ang, Heng-Pei; Seah, Xin-Yi; George, Benjamin L; Mehta, Jodhbir S

2013-05-03

Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Our results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.

Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice1

PubMed Central

Sasaki, Takamitsu; Kitadai, Yasuhiko; Nakamura, Toru; Kim, Jang-Seong; Tsan, Rachel Z; Kuwai, Toshio; Langley, Robert R; Fan, Dominic; Kim, Sun-Jin; Fidler, Isaiah J

2007-01-01

The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α) and vascular endothelial growth factor (VEGF) but were negative for EGFR, human epidermal growth factor receptor 2 (HER2), and VEGFR. Double immunofluorescence staining revealed that tumor-associated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR), and phosphorylated VEGFR (pVEGFR). Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase) or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01); this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001). AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, and increased the level of apoptosis in both tumor-associated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer. PMID:18084614

Endothelial keratoplasty with infant donor tissue

PubMed Central

Kobayashi, Akira; Yokogawa, Hideaki; Yamazaki, Natsuko; Masaki, Toshinori; Sugiyama, Kazuhisa

2014-01-01

Here we report a case of endothelial keratoplasty with infant donor tissue obtained after brain death. A 52-year-old man with endothelial dysfunction of unknown cause in the right eye underwent non-Descemet stripping automated endothelial keratoplasty (nDSAEK) with tissue from an infant donor (2 years). Intraoperative and postoperative complications were recorded. Best corrected visual acuity and donor central endothelial cell density were recorded preoperatively and postoperatively. Infant donor tissue preparation with a microkeratome set at 300 μm was successful; the donor tissue was extremely elastic and soft compared with adult tissue. The central endothelial cell density of the infant donor tissue was as high as 4,291 cells/mm2. No complications were observed during donor tissue (8.0 mm in diameter) insertion with the double-glide technique (Busin glide with intraocular lens sheet glide) or any of the other procedures. Best corrected visual acuity improved from 1.7 logMAR (logarithm of the minimum angle of resolution; 0.02 decimal visual acuity) preoperatively to 0.2 logMAR (0.6) after 6 months and 0.1 logMAR (0.8) after 1 year. The central endothelial cell density after 6 months was 4,098 cells/mm2 (representing a 4.5% cell loss from preoperative donor cell measurements), and the central endothelial cell density after 1 year was 4,032 cells/mm2 (6.0% decrease). Infant donor tissue may be preferably used for DSAEK/nDASEK, since it may not be suitable for penetrating keratoplasty or Descemet membrane endothelial keratoplasty. PMID:25246761

Stable knock-down of the sphingosine 1-phosphate receptor S1P1 influences multiple functions of human endothelial cells.

PubMed

Krump-Konvalinkova, Vera; Yasuda, Satoshi; Rubic, Tina; Makarova, Natalia; Mages, Jörg; Erl, Wolfgang; Vosseler, Claudia; Kirkpatrick, C James; Tigyi, Gabor; Siess, Wolfgang

2005-03-01

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein-coupled receptors S1P1-5 and as a second messenger. Because S1P1 knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P1 in endothelial cells is needed. All human endothelial cells analyzed expressed abundant S1P1 transcripts. We permanently silenced (by RNA interference) the expression of S1P1 in the human endothelial cell lines AS-M.5 and ISO-HAS.1. The S1P1 knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress-mediated injury. New S1P1-dependent gene targets were identified in human endothelial cells. S1P1 silencing decreased the expression of platelet-endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-alpha. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P1 silencing. Long-term silencing of S1P1 enabled us for the first time to demonstrate the involvement of S1P1 in key functions of endothelial cells and to identify new S1P1-dependent gene targets.

Nesting of colon and ovarian cancer cells in the endothelial niche is associated with alterations in glycan and lipid metabolism

PubMed Central

Halama, Anna; Guerrouahen, Bella S.; Pasquier, Jennifer; Satheesh, Noothan J.; Suhre, Karsten; Rafii, Arash

2017-01-01

The metabolic phenotype of a cancer cell is determined by its genetic makeup and microenvironment, which dynamically modulates the tumor landscape. The endothelial cells provide both a promoting and protective microenvironment – a niche for cancer cells. Although metabolic alterations associated with cancer and its progression have been fairly defined, there is a significant gap in our understanding of cancer metabolism in context of its microenvironment. We deployed an in vitro co-culture system based on direct contact of cancer cells with endothelial cells (E4+EC), mimicking the tumor microenvironment. Metabolism of colon (HTC15 and HTC116) and ovarian (OVCAR3 and SKOV3) cancer cell lines was profiled with non-targeted metabolic approaches at different time points in the first 48 hours after co-culture was established. We found significant, coherent and non-cell line specific changes in fatty acids, glycerophospholipids and carbohydrates over time, induced by endothelial cell contact. The metabolic patterns pinpoint alterations in hexosamine biosynthetic pathway, glycosylation and lipid metabolism as crucial for cancer – endothelial cells interaction. We demonstrated that “Warburg effect” is not modulated in the initial stage of nesting of cancer cell in the endothelial niche. Our study provides novel insight into cancer cell metabolism in the context of the endothelial microenvironment. PMID:28051182

Endothelial cell responses in terms of adhesion, proliferation, and morphology to stiffness of polydimethylsiloxane elastomer substrates.

PubMed

Ataollahi, Forough; Pramanik, Sumit; Moradi, Ali; Dalilottojari, Adel; Pingguan-Murphy, Belinda; Wan Abas, Wan Abu Bakar; Abu Osman, Noor Azuan

2015-07-01

Extracellular environments can regulate cell behavior because cells can actively sense their mechanical environments. This study evaluated the adhesion, proliferation and morphology of endothelial cells on polydimethylsiloxane (PDMS)/alumina (Al2 O3 ) composites and pure PDMS. The substrates were prepared from pure PDMS and its composites with 2.5, 5, 7.5, and 10 wt % Al2 O3 at a curing temperature of 50°C for 4 h. The substrates were then characterized by mechanical, structural, and morphological analyses. The cell adhesion, proliferation, and morphology of cultured bovine aortic endothelial (BAEC) cells on substrate materials were evaluated by using resazurin assay and 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine perchlorate-acetylated LDL (Dil-Ac-LDL) cell staining, respectively. The composites (PDMS/2.5, 5, 7.5, and 10 wt % Al2 O3 ) exhibited higher stiffness than the pure PDMS substrate. The results also revealed that stiffer substrates promoted endothelial cell adhesion and proliferation and also induced spread morphology in the endothelial cells compared with lesser stiff substrates. Statistical analysis showed that the effect of time on cell proliferation depended on stiffness. Therefore, this study concludes that the addition of different Al2 O3 percentages to PDMS elevated substrate stiffness which in turn increased endothelial cell adhesion and proliferation significantly and induced spindle shape morphology in endothelial cells. © 2014 Wiley Periodicals, Inc.

Inducible Knockdown of Endothelial Protein Tyrosine Phosphatase-1B Promotes Neointima Formation in Obese Mice by Enhancing Endothelial Senescence.

PubMed

Jäger, Marianne; Hubert, Astrid; Gogiraju, Rajinikanth; Bochenek, Magdalena L; Münzel, Thomas; Schäfer, Katrin

2018-02-01

Protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of receptor tyrosine kinase signaling. In this study, we determined the importance of PTP1B expressed in endothelial cells for the vascular response to arterial injury in obesity. Morphometric analysis of vascular lesions generated by 10% ferric chloride (FeCl 3 ) revealed that tamoxifen-inducible endothelial PTP1B deletion (Tie2.ER T2 -Cre × PTP1B fl/fl ; End.PTP1B knockout, KO) significantly increased neointima formation, and reduced numbers of (endothelial lectin-positive) luminal cells in End.PTP1B-KO mice suggested impaired lesion re-endothelialization. Significantly higher numbers of proliferating cell nuclear antigen (PCNA)-positive proliferating cells as well as smooth muscle actin (SMA)-positive or vascular cell adhesion molecule-1 (VCAM1)-positive activated smooth muscle cells or vimentin-positive myofibroblasts were detected in neointimal lesions of End.PTP1B-KO mice, whereas F4/80-positive macrophage numbers did not differ. Activated receptor tyrosine kinase and transforming growth factor-beta (TGFβ) signaling and oxidative stress markers were also significantly more abundant in End.PTP1B-KO mouse lesions. Genetic knockdown or pharmacological inhibition of PTP1B in endothelial cells resulted in increased expression of caveolin-1 and oxidative stress, and distinct morphological changes, elevated numbers of senescence-associated β-galactosidase-positive cells, and increased expression of tumor suppressor protein 53 (p53) or the cell cycle inhibitor cyclin-dependent kinase inhibitor-2A (p16INK4A) suggested senescence, all of which could be attenuated by small interfering RNA (siRNA)-mediated downregulation of caveolin-1. In vitro, senescence could be prevented and impaired re-endothelialization restored by preincubation with the antioxidant Trolox. Our results reveal a previously unknown role of PTP1B in endothelial cells and provide mechanistic insights how PTP1B deletion or inhibition may promote endothelial senescence. Absence of PTP1B in endothelial cells impairs re-endothelialization, and the failure to induce smooth muscle cell quiescence or to protect from circulating growth factors may result in neointimal hyperplasia. Antioxid. Redox Signal. 00, 000-000.

A functional requirement for astroglia in promoting blood vessel development in the early postnatal brain.

PubMed

Ma, Shang; Kwon, Hyo Jun; Huang, Zhen

2012-01-01

Astroglia are a major cell type in the brain and play a key role in many aspects of brain development and function. In the adult brain, astrocytes are known to intimately ensheath blood vessels and actively coordinate local neural activity and blood flow. During development of the neural retina, blood vessel growth follows a meshwork of astrocytic processes. Several genes have also been implicated in retinal astrocytes for regulating vessel development. This suggests a role of astrocytes in promoting angiogenesis throughout the central nervous system. To determine the roles that astrocytes may play during brain angiogenesis, we employ genetic approaches to inhibit astrogliogenesis during perinatal corticogenesis and examine its effects on brain vessel development. We find that conditional deletion from glial progenitors of orc3, a gene required for DNA replication, dramatically reduces glial progenitor cell number in the subventricular zone and astrocytes in the early postnatal cerebral cortex. This, in turn, results in severe reductions in both the density and branching frequency of cortical blood vessels. Consistent with a delayed growth but not regression of vessels, we find neither significant net decreases in vessel density between different stages after normalizing for cortical expansion nor obvious apoptosis of endothelial cells in these mutants. Furthermore, concomitant with loss of astroglial interactions, we find increased endothelial cell proliferation, enlarged vessel luminal size as well as enhanced cytoskeletal gene expression in pericytes, which suggests compensatory changes in vascular cells. Lastly, we find that blood vessel morphology in mutant cortices recovers substantially at later stages, following astrogliosis. These results thus implicate a functional requirement for astroglia in promoting blood vessel growth during brain development.

Role of smooth muscle cells on endothelial cell cytosolic free calcium in porcine coronary arteries.

PubMed

Budel, S; Schuster, A; Stergiopoulos, N; Meister, J J; Bény, J L

2001-09-01

We tested the hypothesis that the cytosolic free calcium concentration in endothelial cells is under the influence of the smooth muscle cells in the coronary circulation. In the left descending branch of porcine coronary arteries, cytosolic free calcium concentration ([Ca(2+)](i)) was estimated by determining the fluorescence ratio of two calcium probes, fluo 4 and fura red, in smooth muscle and endothelial cells using confocal microscopy. Acetylcholine and potassium, which act directly on smooth muscle cells to increase [Ca(2+)](i), were found to indirectly elevate [Ca(2+)](i) in endothelial cells; in primary cultures of endothelial cells, neither stimulus affected [Ca(2+)](i), yet substance P increased the fluorescence ratio twofold. In response to acetylcholine and potassium, isometric tension developed by arterial strips with intact endothelium was attenuated by up to 22% (P < 0.05) compared with strips without endothelium. These findings suggest that stimuli that increase smooth muscle [Ca(2+)](i) can indirectly influence endothelial cell function in porcine coronary arteries. Such a pathway for negative feedback can moderate vasoconstriction and diminish the potential for vasospasm in the coronary circulation.

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles

PubMed Central

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel

2016-01-01

Summary Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle–cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN. PMID:27826507

Sildenafil Inhibits the Proliferation of Cultured Human Endothelial Cells

PubMed Central

Erdogan, Ali; Luedders, Doerte Wiebke; Muenz, Benedikt Manuel; Schaefer, Christian Alexander; Tillmanns, Harald; Wiecha, Johannes; Kuhlmann, Christoph Ruediger Wolfram

2007-01-01

The proliferation of endothelial cells plays a crucial role in the development of intraplaque angiogenesis (IPA). IPA is a major source of intraplaque hemorrhage and therefore contributes to the destabilization of atherosclerotic plaques. Therefore, the aim of the present study was to examine, whether sildenafil inhibits endothelial cell growth. The proliferation of human endothelial cells derived from umbilical cord veins (HUVEC) was examined on DNA level by measurements of (3H)-thymidine incorporation. Cell viability was analyzed using trypan blue staining. The proliferation of cultured human endothelial cells was significantly decreased by 1 μmol/l (-48.4%) and 10 μmol/l (-89.6%) sildenafil (n=10, p<0.05). This was not a cytotoxic effect, because cell viability was only reduced at sildenafil concentrations of 50 μmol/l or greater. In addition sildenafil significantly reduced endothelial proliferation induced by bFGF (n=10, p<0.05). The presented results demonstrate an antiangiogenic effect of sildenafil that might be useful in the prevention of atherosclerotic plaque vascularization. PMID:23675029

Association of Plasmodium falciparum with Human Endothelial Cells in vitro

PubMed Central

Utter, Christopher; Serrano, Adelfa E.; Glod, John W.; Leibowitz, Michael J.

2017-01-01

Endothelial abnormalities play a critical role in the pathogenesis of malaria caused by the human pathogen, Plasmodium falciparum. In serious infections and especially in cerebral malaria, red blood cells infected with the parasite are sequestered in small venules in various organs, resulting in endothelial activation and vascular occlusion, which are believed to be largely responsible for the morbidity and mortality caused by this infection, especially in children. We demonstrate that after incubation with infected red blood cells (iRBCs), cultured human umbilical vein endothelial cells (HUVECs) contain parasite protein, genomic DNA, and RNA, as well as intracellular vacuoles with apparent parasite-derived material, but not engulfed or adherent iRBCs. The association of this material with the HUVECs is observed over 96 hours after removal of iRBCs. This phenomenon may occur in endothelial cells in vivo by the process of trogocytosis, in which transfer of material between cells depends on direct cell contact. This process may contribute to the endothelial activation and disruption involved in the pathogenesis of cerebral malaria. PMID:28656007

Acidosis Activation of the Proton-Sensing GPR4 Receptor Stimulates Vascular Endothelial Cell Inflammatory Responses Revealed by Transcriptome Analysis

PubMed Central

Dong, Lixue; Li, Zhigang; Leffler, Nancy R.; Asch, Adam S.; Chi, Jen-Tsan; Yang, Li V.

2013-01-01

Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions due to hypoxia, glycolytic cell metabolism and deficient blood perfusion. However, the molecular mechanisms by which cells sense and respond to the acidic microenvironment are not well understood. GPR4 is a proton-sensing receptor expressed in endothelial cells and other cell types. The receptor is fully activated by acidic extracellular pH but exhibits lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To delineate the function and signaling pathways of GPR4 activation by acidosis in endothelial cells, we compared the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4. The results demonstrated that acidosis activation of GPR4 in HUVEC substantially increased the expression of a number of inflammatory genes such as chemokines, cytokines, adhesion molecules, NF-κB pathway genes, and prostaglandin-endoperoxidase synthase 2 (PTGS2 or COX-2) and stress response genes such as ATF3 and DDIT3 (CHOP). Similar GPR4-mediated acidosis induction of the inflammatory genes was also noted in other types of endothelial cells including human lung microvascular endothelial cells and pulmonary artery endothelial cells. Further analyses indicated that the NF-κB pathway was important for the acidosis/GPR4-induced inflammatory gene expression. Moreover, acidosis activation of GPR4 increased the adhesion of HUVEC to U937 monocytic cells under a flow condition. Importantly, treatment with a recently identified GPR4 antagonist significantly reduced the acidosis/GPR4-mediated endothelial cell inflammatory response. Taken together, these results show that activation of GPR4 by acidosis stimulates the expression of a wide range of inflammatory genes in endothelial cells. Such inflammatory response can be suppressed by GPR4 small molecule inhibitors and hold potential therapeutic value. PMID:23613998

Pro-apoptotic BIM is an essential initiator of physiological endothelial cell death independent of regulation by FOXO3

PubMed Central

Koenig, M N; Naik, E; Rohrbeck, L; Herold, M J; Trounson, E; Bouillet, P; Thomas, T; Voss, A K; Strasser, A; Coultas, L

2014-01-01

The growth of new blood vessels by angiogenesis is essential for normal development, but can also cause or contribute to the pathology of numerous diseases. Recent studies have shown that BIM, a pro-apoptotic BCL2-family protein, is required for endothelial cell apoptosis in vivo, and can contribute to the anti-angiogenic effect of VEGF-A inhibitors in certain tumor models. Despite its importance, the extent to which BIM is autonomously required for physiological endothelial apoptosis remains unknown and its regulation under such conditions is poorly defined. While the transcription factor FOXO3 has been proposed to induce Bim in response to growth factor withdrawal, evidence for this function is circumstantial. We report that apoptosis was reduced in Bim−/− primary endothelial cells, demonstrating a cell-autonomous role for BIM in endothelial death following serum and growth factor withdrawal. In conflict with in vitro studies, BIM-dependent endothelial death in vivo did not require FOXO3. Moreover, endothelial apoptosis proceeded normally in mice lacking FOXO-binding sites in the Bim promoter. Bim mRNA was upregulated in endothelial cells starved of serum and growth factors and this was accompanied by the downregulation of miRNAs of the miR-17∼92 cluster. Bim mRNA levels were also elevated in miR-17∼92+/− endothelial cells cultured under steady-state conditions, suggesting that miR-17∼92 cluster miRNAs may contribute to regulating overall Bim mRNA levels in endothelial cells. PMID:24971484

Engineered living blood vessels: functional endothelia generated from human umbilical cord-derived progenitors.

PubMed

Schmidt, Dörthe; Asmis, Lars M; Odermatt, Bernhard; Kelm, Jens; Breymann, Christian; Gössi, Matthias; Genoni, Michele; Zund, Gregor; Hoerstrup, Simon P

2006-10-01

Tissue-engineered living blood vessels (TEBV) with growth capacity represent a promising new option for the repair of congenital malformations. We investigate the functionality of TEBV with endothelia generated from human umbilical cord blood-derived endothelial progenitor cells. Tissue-engineered living blood vessels were generated from human umbilical cord-derived myofibroblasts seeded on biodegradable vascular scaffolds, followed by endothelialization with differentiated cord blood-derived endothelial progenitor cells. During in vitro maturation the TEBV were exposed to physiologic conditioning in a flow bioreactor. For functional assessment, a subgroup of TEBV was stimulated with tumor necrosis factor-alpha. Control vessels endothelialized with standard vascular endothelial cells were treated in parallel. Analysis of the TEBV included histology, immunohistochemistry, biochemistry (extracellular matrix analysis, DNA), and biomechanical testing. Endothelia were analyzed by flow cytometry and immunohistochemistry (CD31, von Willebrand factor, thrombomodulin, tissue factor, endothelial nitric oxide synthase). Histologically, a three-layered tissue organization of the TEBV analogous to native vessels was observed, and biochemistry revealed the major matrix constituents (collagen, proteoglycans) of blood vessels. Biomechanical properties (Young's modulus, 2.03 +/- 0.65 MPa) showed profiles resembling those of native tissue. Endothelial progenitor cells expressed typical endothelial cell markers CD31, von Willebrand factor, and endothelial nitric oxide synthase comparable to standard vascular endothelial cells. Stimulation with tumor necrosis factor-alpha resulted in physiologic upregulation of tissue factor and downregulation of thrombomodulin expression. These results indicate that TEBV with tissue architecture and functional endothelia similar to native blood vessels can be successfully generated from human umbilical cord progenitor cells. Thus, blood-derived progenitor cells obtained before or at birth may enable the clinical realization of tissue engineering constructs for pediatric applications.

Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

PubMed

Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

2008-08-01

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

Potential proinflammatory effects of hydroxyapatite nanoparticles on endothelial cells in a monocyte–endothelial cell coculture model

PubMed Central

Liu, Xin; Sun, Jiao

2014-01-01

Currently, synthetic hydroxyapatite nanoparticles (HANPs) are used in nanomedicine fields. The delivery of nanomedicine to the bloodstream exposes the cardiovascular system to a potential threat. However, the possible adverse cardiovascular effects of HANPs remain unclear. Current observations using coculture models of endothelial cells and monocytes with HANPs to mimic the complex physiological functionality of the vascular system demonstrate that monocytes could play an important role in the mechanisms of endothelium dysfunction induced by the exposure to HANPs. Our transmission electron microscopy analysis revealed that both monocytes and endothelial cells could take up HANPs. Moreover, our findings demonstrated that at a subcytotoxic dose, HANPs alone did not cause direct endothelial cell injury, but they did induce an indirect activation of endothelial cells, resulting in increased interleukin-6 production and elevated adhesion molecule expression after coculture with monocytes. The potential proinflammatory effect of HANPs is largely mediated by the release of soluble factors from the activated monocytes, leading to an inflammatory response of the endothelium, which is possibly dependent on p38/c-Jun N-terminal kinase, and nuclear factor-kappa B signaling activation. The use of in vitro monocyte–endothelial cell coculture models for the biocompatibility assessment of HANPs could reveal their potential proinflammatory effects on endothelial cells, suggesting that exposure to HANPs possibly increases the risk of cardiovascular disease. PMID:24648726

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

PubMed

Rops, Angelique L; van der Vlag, Johan; Jacobs, Cor W; Dijkman, Henry B; Lensen, Joost F; Wijnhoven, Tessa J; van den Heuvel, Lambert P; van Kuppevelt, Toin H; Berden, Jo H

2004-12-01

The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

PubMed Central

Lopes, Flavia C. M.; Traina, Fabiola; Almeida, Camila B.; Leonardo, Flavia C.; Franco-Penteado, Carla F.; Garrido, Vanessa T.; Colella, Marina P.; Soares, Raquel; Olalla-Saad, Sara T.; Costa, Fernando F.; Conran, Nicola

2015-01-01

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified. PMID:25769545

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells

PubMed Central

Pipparelli, Aurélien; Arsenijevic, Yvan; Thuret, Gilles; Gain, Philippe

2013-01-01

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy. PMID:23626771

P-selectin mediates neutrophil adhesion to endothelial cell borders.

PubMed

Burns, A R; Bowden, R A; Abe, Y; Walker, D C; Simon, S I; Entman, M L; Smith, C W

1999-03-01

During an acute inflammatory response, endothelial P-selectin (CD62P) can mediate the initial capture of neutrophils from the free flowing bloodstream. P-selectin is stored in secretory granules (Weibel-Palade bodies) and is rapidly expressed on the endothelial surface after stimulation with histamine or thrombin. Because neutrophil transmigration occurs preferentially at endothelial borders, we wished to determine whether P-selectin-dependent neutrophil capture (adhesion) occurs at endothelial cell borders. Under static or hydrodynamic flow (2 dyn/cm2) conditions, histamine (10(-4) M) or thrombin (0.2 U/mL) treatment induced preferential (> or = 75%) neutrophil adhesion to the cell borders of endothelial monolayers. Blocking antibody studies established that neutrophil adhesion was completely P-selectin dependent. P-selectin surface expression increased significantly after histamine treatment and P-selectin immunostaining was concentrated along endothelial borders. We conclude that preferential P-selectin expression along endothelial borders may be an important mechanism for targeting neutrophil migration at endothelial borders.

Inhibition of TGF-β Signaling in SHED Enhances Endothelial Differentiation.

PubMed

Xu, J G; Gong, T; Wang, Y Y; Zou, T; Heng, B C; Yang, Y Q; Zhang, C F

2018-02-01

Low efficiency of deriving endothelial cells (ECs) from adult stem cells hampers their utilization in tissue engineering studies. The purpose of this study was to investigate whether suppression of transforming growth factor beta (TGF-β) signaling could enhance the differentiation efficiency of dental pulp-derived stem cells into ECs. We initially used vascular endothelial growth factor A (VEGF-A) to stimulate 2 dental pulp-derived stem cells (dental pulp stem cells and stem cells from human exfoliated deciduous teeth [SHED]) and compared their differentiation capacity into ECs. We further evaluated whether the vascular endothelial growth factor receptor I (VEGF-RI)-specific ligand placental growth factor-1 (PlGF-1) could mediate endothelial differentiation. Finally, we investigated whether the TGF-β signaling inhibitor SB-431542 could enhance the inductive effect of VEGF-A on endothelial differentiation, as well as the underlying mechanisms involved. ECs differentiated from dental pulp-derived stem cells exhibited the typical phenotypes of primary ECs, with SHED possessing a higher endothelial differentiation potential than dental pulp stem cells. VEGFR1-specific ligand-PLGF exerted a negligible effect on SHED-ECs differentiation. Compared with VEGF-A alone, the combination of VEGF-A and SB-431542 significantly enhanced the endothelial differentiation of SHED. The presence of SB-431542 inhibited the phosphorylation of Suppressor of Mothers Against Decapentaplegic 2/3 (SMAD2/3), allowing for VEGF-A-dependent phosphorylation and upregulation of VEGFR2. Our results indicate that the combination of VEGF-A and SB-431542 could enhance the differentiation of dental pulp-derived stem cells into endothelial cells, and this process is mediated through enhancement of VEGF-A-VEGFR2 signaling and concomitant inhibition of TGF-β-SMAD2/3 signaling.

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

PubMed Central

van den Biggelaar, Maartje; Bouwens, Eveline A.M.; Kootstra, Neeltje A.; Hebbel, Robert P.; Voorberg, Jan; Mertens, Koen

2009-01-01

Background Gene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this study, we explored the feasibility of blood outgrowth endothelial cells as a cellular FVIII delivery device with particular reference to long-term production levels, intracellular storage in Weibel-Palade bodies and agonist-induced regulated secretion. Design and Methods Human blood outgrowth endothelial cells were isolated from peripheral blood collected from healthy donors, transduced at passage 5 using a lentiviral vector encoding human B-domain deleted FVIII-GFP and characterized by flow cytometry and confocal microscopy. Results Blood outgrowth endothelial cells displayed typical endothelial morphology and expressed the endothelial-specific marker VWF. Following transduction with a lentivirus encoding FVIII-GFP, 80% of transduced blood outgrowth endothelial cells expressed FVIII-GFP. Levels of FVIII-GFP positive cells declined slowly upon prolonged culturing. Transduced blood outgrowth endothelial cells expressed 1.6±1.0 pmol/1×106 cells/24h FVIII. Morphological analysis demonstrated that FVIII-GFP was stored in Weibel-Palade bodies together with VWF and P-selectin. FVIII levels were only slightly increased following agonist-induced stimulation, whereas a 6- to 8-fold increase of VWF levels was observed. Subcellular fractionation revealed that 15–22% of FVIII antigen was present within the dense fraction containing Weibel-Palade bodies. Conclusions We conclude that blood outgrowth endothelial cells, by virtue of their ability to store a significant portion of synthesized FVIII-GFP in Weibel-Palade bodies, provide an attractive cellular on-demand delivery device for gene therapy of hemophilia A. PMID:19336741

Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels

PubMed Central

Yu, Jun; Bergaya, Sonia; Murata, Takahisa; Alp, Ilkay F.; Bauer, Michael P.; Lin, Michelle I.; Drab, Marek; Kurzchalia, Teymuras V.; Stan, Radu V.; Sessa, William C.

2006-01-01

Caveolae in endothelial cells have been implicated as plasma membrane microdomains that sense or transduce hemodynamic changes into biochemical signals that regulate vascular function. Therefore we compared long- and short-term flow-mediated mechanotransduction in vessels from WT mice, caveolin-1 knockout (Cav-1 KO) mice, and Cav-1 KO mice reconstituted with a transgene expressing Cav-1 specifically in endothelial cells (Cav-1 RC mice). Arterial remodeling during chronic changes in flow and shear stress were initially examined in these mice. Ligation of the left external carotid for 14 days to lower blood flow in the common carotid artery reduced the lumen diameter of carotid arteries from WT and Cav-1 RC mice. In Cav-1 KO mice, the decrease in blood flow did not reduce the lumen diameter but paradoxically increased wall thickness and cellular proliferation. In addition, in isolated pressurized carotid arteries, flow-mediated dilation was markedly reduced in Cav-1 KO arteries compared with those of WT mice. This impairment in response to flow was rescued by reconstituting Cav-1 into the endothelium. In conclusion, these results showed that endothelial Cav-1 and caveolae are necessary for both rapid and long-term mechanotransduction in intact blood vessels. PMID:16670769

Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system.

PubMed

Yu, J Q; Liu, X F; Chin, L K; Liu, A Q; Luo, K Q

2013-07-21

To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection method, which combines the microfluidic chip system and FRET biosensor. This finding may provide new insight into how glucose causes endothelial cell dysfunction, which is the major cause of diabetes-derived complications.

Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients.

PubMed

Haller, H; Ziegler, E M; Homuth, V; Drab, M; Eichhorn, J; Nagy, Z; Busjahn, A; Vetter, K; Luft, F C

1997-01-01

Endothelial cell activation is important in the pathogenesis of preeclampsia; however, the nature of the activation is unknown. We investigated 22 patients with preeclampsia. 29 normotensive pregnancies, and 18 nonpregnant women to test the hypothesis that serum from preeclamptic patients induces expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) and stimulates intracellular free calcium concentrations [Ca2+]i in cultured endothelial cells. We then asked whether the corresponding integrin adhesive counter receptors lymphocyte function-associated antigen-1 (CD11a/CD18), macrophage-1 antigen (CD11b/CD18), p150,95 (CD11c/CD18), and very late activation antigen-4 (CD49/CD29) are increased in patients with preeclampsia. In the pregnant women, the measurements were conducted both before and after delivery. Integrin expression was measured by fluorescent antibody cell sorting analysis using monoclonal antibodies. ICAM-1 and VCAM-1 were analyzed on endothelial cells by enzyme-linked immunosorbent assay. [Ca2+]i was measured with fura 2. Serum from preeclamptic patients increased endothelial cell ICAM-1 expression but not VCAM-1 expression. Preeclamptic patients' serum also increased [Ca2+]i in endothelial cells compared with serum from normal nonpregnant or normal pregnant women. Endothelial cell [Ca2+]i concentrations were correlated with the ICAM-1 expression in preeclamptic patients (r = .80, P < .001) before but not after delivery. Expression of the integrin counter receptors on leukocytes was similarly increased in preclampsia and normal pregnancy compared with the nonpregnant state. The expression decreased significantly after delivery in both groups. Our results demonstrate that serum from preeclamptic women induces increased ICAM-1 surface expression on endothelial cells, while the expression of the integrin counterreceptors was not different. The effect on endothelial cells may be related to an increase in [Ca2+]i. The effect on cultured endothelial cells and the rapid decrease after delivery suggests the presence of a circulating serum factor which increases endothelial cell [Ca2+]i and enhances adhesion molecule expression.

Multiple protocadherins are expressed in brain microvascular endothelial cells and might play a role in tight junction protein regulation.

PubMed

Dilling, Christina; Roewer, Norbert; Förster, Carola Y; Burek, Malgorzata

2017-10-01

Protocadherins (Pcdhs) are a large family of cadherin-related molecules. They play a role in cell adhesion, cellular interactions, and development of the central nervous system. However, their expression and role in endothelial cells has not yet been characterized. Here, we examined the expression of selected clustered Pcdhs in endothelial cells from several vascular beds. We analyzed human and mouse brain microvascular endothelial cell (BMEC) lines and primary cells, mouse myocardial microvascular endothelial cell line, and human umbilical vein endothelial cells. We examined the mRNA and protein expression of selected Pcdhs using RT-PCR, Western blot, and immunostaining. A strong mRNA expression of Pcdhs was observed in all endothelial cells tested. At the protein level, Pcdhs-gamma were detected using an antibody against the conserved C-terminal domain of Pcdhs-gamma or an antibody against PcdhgC3. Deletion of highly expressed PcdhgC3 led to differences in the tight junction protein expression and mRNA expression of Wnt/mTOR (mechanistic target of rapamycin) pathway genes as well as lower transendothelial electrical resistance. Staining of PcdhgC3 showed diffused cytoplasmic localization in mouse BMEC. Our results suggest that Pcdhs may play a critical role in the barrier-stabilizing pathways at the blood-brain barrier.

Inhibitive Effects of Quercetin on Myeloperoxidase-Dependent Hypochlorous Acid Formation and Vascular Endothelial Injury.

PubMed

Lu, Naihao; Sui, Yinhua; Tian, Rong; Peng, Yi-Yuan

2018-05-16

Myeloperoxidase (MPO) from activated neutrophils plays important roles in multiple human inflammatory diseases by catalyzing the formation of powerful oxidant hypochlorous acid (HOCl). As a major flavonoid in the human diet, quercetin has been suggested to act as antioxidant and anti-inflammatory agent in vitro and in vivo. In this study, we showed that quercetin inhibited MPO-mediated HOCl formation (75.0 ± 6.2% for 10 μM quercetin versus 100 ± 5.2% for control group, P < 0.01) and cytotoxicity to endothelial cells in vitro, while this flavonoid was nontoxic to endothelial cell cultures ( P > 0.05, all cases). Moreover, quercetin inhibited HOCl generation by stimulated neutrophils (a rich source of MPO) and protected endothelial cells from neutrophils-induced injury. Furthermore, quercetin could inhibit HOCl-induced endothelial dysfunction such as loss of cell viability, and decrease of nitric oxide formation in endothelial cells ( P < 0.05, all cases). Consistent with these in vitro data, quercetin attenuated lipopolysaccharide-induced endothelial dysfunction and increase of MPO activity in mouse aortas, while this flavonoid could protect against HOCl-mediated endothelial dysfunction in isolated aortas ( P < 0.05). Therefore, it was proposed that quercetin attenuated endothelial injury in inflammatory vasculature via inhibition of vascular-bound MPO-mediated HOCl formation or scavenging of HOCl. These data indicate that quercetin is a nontoxic inhibitor of MPO activity and MPO/neutrophils-induced cytotoxicity in endothelial cells and may be useful for targeting MPO-dependent vascular disease and inflammation.

Catheter-directed Intraportal Delivery of Endothelial Cell Therapy for Liver Regeneration: A Feasibility Study in a Large-Animal Model of Cirrhosis.

PubMed

Lee, Kyungmouk Steve; Santagostino, Sara F; Li, David; Ramjit, Amit; Serrano, Kenneth; Ginsberg, Michael D; Ding, Bi-Sen; Rafii, Shahin; Madoff, David C

2017-10-01

Purpose To demonstrate the feasibility of imaging-guided catheter-directed delivery of endothelial cell therapy in a porcine model of cirrhosis for liver regeneration. Materials and Methods After approval from the institutional animal care and use committee, autologous liver endothelial cells were grown from core hepatic specimens from swine. Cirrhosis was induced in swine by means of transcatheter infusion of ethanol and iodized oil into the hepatic artery. Three weeks after induction of cirrhosis, the swine were randomly assigned to receive autologous cell therapy (endothelial cells, n = 4) or control treatment (phosphate-buffered saline, n = 4) by means of imaging-guided transhepatic intraportal catheterization. Fluorescence-activated cell sorting analysis was performed on biopsy samples 1 hour after therapy. Three weeks after intraportal delivery of endothelial cells, the swine were euthanized and the explanted liver underwent quantitative pathologic examination. Statistical analysis was performed with an unpaired t test by using unequal variance. Results Liver endothelial cells were successfully isolated, cultured, and expanded from eight 20-mm, 18-gauge hepatic core samples to 50 × 10 6 autologous cells per pig. Intraportal delivery of endothelial cell therapy or saline was technically successful in all eight swine, with no complications. Endothelial cells were present in the liver for a minimum of 1 hour after intraportal infusion. Swine treated with endothelial cell therapy showed mean levels of surrogate markers of hepatobiliary injury that were consistent with decreases in hepatic fibrosis and biliary ductal damage relative to the control animals, although statistical significance was not met in this pilot study: The mean percentage of positive pixels at Masson trichrome staining was 7.28% vs 5.57%, respectively (P = .20), the mean proliferation index with cytokeratin wide-spectrum was 2.55 vs 1.13 (P = .06), and the mean proliferation index with Ki67 was 7.08 vs 4.96 (P = .14). Conclusion The results confirm the feasibility of imaging-guided catheter-directed endothelial cell therapy with an intraportal technique for the treatment of cirrhosis in a porcine model. A trend toward decreased liver fibrosis with endothelial cell therapy was observed. Larger animal studies and human studies are necessary to confirm significance. © RSNA, 2017.

Signaling hierarchy regulating human endothelial cell development

USDA-ARS?s Scientific Manuscript database

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

The role of corneal endothelial morphology in graft assessment and prediction of endothelial cell loss during organ culture of human donor corneas.

PubMed

Hermel, Martin; Salla, Sabine; Fuest, Matthias; Walter, Peter

2017-03-01

Endothelial assessment is crucial in the release of corneas for grafting. We retrospectively analysed the role of endothelial morphology parameters in predicting endothelial cell loss during organ culture. Human donor corneas were cultured in minimal essential medium with 2% fetal calf serum and antibiotics. Initial endothelial morphology was assessed microscopically using score parameters polymegethism (POL), pleomorphism (PLE), granulation (GRA), vacuolization (VAC), segmentation of cell membranes (SEG), Descemet's folds (DF), trypan blue-positive cells (TBPC) and endothelial cell-free areas (ECFA). Some corneas were primarily rejected based on endothelial assessment. Endothelial cell density (ECD) was assessed at the beginning (I-ECD) and end of culture. Corneas were then placed in dehydration medium (as above + 5% dextran 500). In a subgroup, ECD was reassessed after dehydration. Endothelial cell loss during culture (ECL@Culture) and culture+dehydration (ECL-Culture&Dehydration) were calculated. Data were given as mean ± SD and analysed using multiple linear and logistic regression. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. I-ECD was 2812 ± 360/mm 2 (n = 2356). The decision to reject a cornea due to endothelial assessment was associated negatively with I-ECD (OR = 0.77/100 cells, CI 0.7-0.82) and positively with ECFA (OR = 2.7, CI 1.69-4.35), SEG (OR =1.3, CI 1.01-1.68) and donor age (OR = 1.26/decade, CI 1.33-1.41). ECL@Culture was 153 ± 201/mm 2 (n = 1277), ECL@Culture&Dehydration was 169 ± 183/mm 2 (n = 918). ECL@Culture was associated positively with donor age, I-ECD, GRA and TBPC, and negatively with PLE, and DF. ECL@Culture&Dehydration was associated positively with age, sex, initial ECD, POL, PLE, VAC and TBPC. Morphological parameters displayed associations with the exclusion of corneas from culture and with endothelial cell loss. Appropriate parameter selection for screening purposes may help improve graft quality. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

PubMed

Asgeirsdóttir, Sigridur A; Talman, Eduard G; de Graaf, Inge A; Kamps, Jan A A M; Satchell, Simon C; Mathieson, Peter W; Ruiters, Marcel H J; Molema, Grietje

2010-01-25

Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. Copyright 2009 Elsevier B.V. All rights reserved.

NaK-ATPase pump sites in cultured bovine corneal endothelium of varying cell density at confluence.

PubMed

Crawford, K M; Ernst, S A; Meyer, R F; MacCallum, D K

1995-06-01

The driving force for ion and water flow necessary for efficient deturgesence of the corneal stroma resides in the ouabain-sensitive sodium (Na) pump of corneal endothelial cells. Using a cell culture model of corneal endothelial cell hypertrophy, the authors examined the expression of Na pumps at the cell surface to see how this central element of the endothelial pump changed as corneal endothelial cell density decreased to a level associated with corneal decompensation in vivo. 3H-ouabain binding to NaK-ATPase at saturating conditions was used to quantitate the number of Na pump sites on cultured bovine corneal endothelial cells as the confluent density decreased from approximately 2750 cells/mm2 to approximately 275 cells/mm2. The mean number of Na pump sites per cell at confluence (1.92 +/- 0.07 x 10(6)) did not change as the cell density decreased 2.7-fold from 2763 cells/mm2 to 1000 cells/mm2. However, pump site expression doubled to approximately 4 x 10(6) sites/cell as the cell density decreased from 1000 cells/mm2 to 275 cells/mm2. Despite the incremental increase in Na pump site expression that occurred as the cells hypertrophied below a density of 1000/mm2 to achieve confluence, this increase was insufficient to prevent a decrease in Na pump site density of the intact monolayer, expressed as pump sites/mm2. The confluent cell density of cultured bovine corneal endothelial cells can be varied from that found in the normal native cornea to that associated with corneal decompensation. In confluent cultures with cell densities ranging from 2750 cells/mm2 to 1000 cells/mm2, the number of pump sites per cell remains relatively unchanged. Below cell densities of 1000 cells/mm2, the number of pump sites per cell progressively increases. The increased Na pump site abundance in markedly hypertrophied endothelial cells cannot adequately compensate for the progressive reduction in the number of transporting cells per unit area within the intact monolayer. Even when considered with the decrease in the size of the paracellular ion conductive pathway that is a consequence of progressive endothelial hypertrophy, the overall pumping capacity of the intact endothelial monolayer declines.

Cellular proliferation after experimental glaucoma filtration surgery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jampel, H.D.; McGuigan, L.J.; Dunkelberger, G.R.

1988-01-01

We used light microscopic autoradiography to determine the time course of cellular incorporation of tritiated thymidine (a correlate of cell division) following glaucoma filtration surgery in seven eyes of four cynomolgus monkeys with experimental glaucoma. Incorporation of tritiated thymidine was detected as early as 24 hours postoperatively. Peak incorporation occurred five days postoperatively and had returned to baseline levels by day 11. Cells incorporating tritiated thymidine included keratocytes, episcleral cells, corneal and capillary endothelial cells, and conjunctival and corneal epithelial cells. Transmission electron microscopy was correlated with the autoradiographic results to demonstrate that fibroblasts were dividing on the corneoscleral margin.more » These findings have potential clinical implications for the use of antiproliferative agents after filtration surgery.« less

Implantation of Vascular Grafts Lined with Genetically Modified Endothelial Cells

NASA Astrophysics Data System (ADS)

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

1989-06-01

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

Timing of galectin-1 exposure differentially modulates Nipah virus entry and syncytium formation in endothelial cells.

PubMed

Garner, Omai B; Yun, Tatyana; Pernet, Olivier; Aguilar, Hector C; Park, Arnold; Bowden, Thomas A; Freiberg, Alexander N; Lee, Benhur; Baum, Linda G

2015-03-01

Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by "bridging" the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Circulating endothelial progenitor cells and cardiovascular outcomes.

PubMed

Werner, Nikos; Kosiol, Sonja; Schiegl, Tobias; Ahlers, Patrick; Walenta, Katrin; Link, Andreas; Böhm, Michael; Nickenig, Georg

2005-09-08

Endothelial progenitor cells derived from bone marrow are believed to support the integrity of the vascular endothelium. The number and function of endothelial progenitor cells correlate inversely with cardiovascular risk factors, but the prognostic value associated with circulating endothelial progenitor cells has not been defined. The number of endothelial progenitor cells positive for CD34 and kinase insert domain receptor (KDR) was determined with the use of flow cytometry in 519 patients with coronary artery disease as confirmed on angiography. After 12 months, we evaluated the association between baseline levels of endothelial progenitor cells and death from cardiovascular causes, the occurrence of a first major cardiovascular event (myocardial infarction, hospitalization, revascularization, or death from cardiovascular causes), revascularization, hospitalization, and death from all causes. A total of 43 participants died, 23 from cardiovascular causes. A first major cardiovascular event occurred in 214 patients. The cumulative event-free survival rate increased stepwise across three increasing baseline levels of endothelial progenitor cells in an analysis of death from cardiovascular causes, a first major cardiovascular event, revascularization, and hospitalization. After adjustment for age, sex, vascular risk factors, and other relevant variables, increased levels of endothelial progenitor cells were associated with a reduced risk of death from cardiovascular causes (hazard ratio, 0.31; 95 percent confidence interval, 0.16 to 0.63; P=0.001), a first major cardiovascular event (hazard ratio, 0.74; 95 percent confidence interval, 0.62 to 0.89; P=0.002), revascularization (hazard ratio, 0.77; 95 percent confidence interval, 0.62 to 0.95; P=0.02), and hospitalization (hazard ratio, 0.76; 95 percent confidence interval, 0.63 to 0.94; P=0.01). Endothelial progenitor-cell levels were not predictive of myocardial infarction or of death from all causes. The level of circulating CD34+KDR+ endothelial progenitor cells predicts the occurrence of cardiovascular events and death from cardiovascular causes and may help to identify patients at increased cardiovascular risk. Copyright 2005 Massachusetts Medical Society.

Factor X/Xa elicits protective signaling responses in endothelial cells directly via PAR-2 and indirectly via endothelial protein C receptor-dependent recruitment of PAR-1.

PubMed

Bae, Jong-Sup; Yang, Likui; Rezaie, Alireza R

2010-11-05

We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.

Phloretin attenuates hyperuricemia-induced endothelial dysfunction through co-inhibiting inflammation and GLUT9-mediated uric acid uptake.

PubMed

Liu, Shuyun; Yuan, Yujia; Zhou, Yijie; Zhao, Meng; Chen, Younan; Cheng, Jingqiu; Lu, Yanrong; Liu, Jingping

2017-10-01

Hyperuricemia is an important risk factor for cardiovascular and renal diseases. Phloretin had shown antioxidant and anti-inflammatory properties, but its role in endothelial injury is rarely reported. In this study, we aimed to investigate the protective effect of phloretin on UA-induced injury in human umbilical vein endothelial cells. The effects of UA and phloretin on cell viability, inflammation, THP-1 monocyte adhesion, endothelial cell tube formation, GLUT9 expression and UA uptake in human umbilical vein endothelial cells were evaluated. The changes of nuclear factor-kappa B/extracellular regulated protein kinases signalling were also analysed. Our results showed that UA reduced cell viability and tube formation, and increased inflammation and monocytes adhesion in human umbilical vein endothelial cells in a dose-dependent manner. In contrast, phloretin significantly attenuated pro-inflammatory factors expression and endothelial injury induced by UA. Phloretin inhibited the activation of extracellular regulated protein kinases/nuclear factor-kappa B pathway, and reduced GLUT9 and it mediated UA uptake in human umbilical vein endothelial cells. These results indicated that phloretin attenuated UA-induced endothelial injury via a synergic mechanism including direct anti-inflammatory effect and lowering cellular UA uptake. Our study suggested that phloretin might be a promising therapy for hyperuricemia-related cardiovascular diseases. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Expansion and cryopreservation of porcine and human corneal endothelial cells.

PubMed

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

2017-08-01

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

Vascular endothelial cells express isoforms of protein kinase A inhibitor.

PubMed

Lum, Hazel; Hao, Zengping; Gayle, Dave; Kumar, Priyadarsini; Patterson, Carolyn E; Uhler, Michael D

2002-01-01

The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKI alpha, human PKI gamma, and mouse PKI beta sequences detected PKI alpha and PKI gamma mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKI alpha and PKI gamma were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKI alpha mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKI gamma mRNA. Western blot analysis indicated that PKI gamma protein was detected in all three cell types, whereas PKI alpha was found in HBMECs. In summary, endothelial cells from three different vascular beds express PKI alpha and PKI gamma, which may be physiologically important in endothelial barrier function.

Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function.

PubMed

Li, Xiaocong; Jiang, Chunyu; Zhao, Jungong

2016-08-01

Wound healing is deeply dependent on neovascularization to restore blood flow. The neovascularization of endothelial progenitor cells (EPCs) through paracrine secretion has been reported in various tissue repair models. Exosomes, key components of cell paracrine mechanism, have been rarely reported in wound healing. Exosomes were isolated from the media of EPCs obtained from human umbilical cord blood. Diabetic rats wound model was established and treated with exosomes. The in vitro effects of exosomes on the proliferation, migration and angiogenic tubule formation of endothelial cells were investigated. We revealed that human umbilical cord blood EPCs derived exosomes transplantation could accelerate cutaneous wound healing in diabetic rats. We also showed that exosomes enhanced the proliferation, migration and tube formation of vascular endothelial cells in vitro. Furthermore, we found that endothelial cells stimulated with these exosomes would increase expression of angiogenesis-related molecules, including FGF-1, VEGFA, VEGFR-2, ANG-1, E-selectin, CXCL-16, eNOS and IL-8. Taken together, our findings indicated that EPCs-derived exosomes facilitate wound healing by positively modulating vascular endothelial cells function. Copyright © 2016 Elsevier Inc. All rights reserved.

Lymphatic endothelial cell line (CH3) from a recurrent retroperitoneal lymphangioma.

PubMed

Way, D; Hendrix, M; Witte, M; Witte, C; Nagle, R; Davis, J

1987-09-01

An endothelial cell line derived from a massive recurrent chyle-containing retroperitoneal lymphangioma was isolated in monolayer culture. Scanning and transmission electron microscopy and immunohistochemistry confirmed a close resemblance to blood vascular endothelium with typical cobblestone morphology, positive immunofluorescence staining for endothelial marker Factor VIII-associated antigen and fibronectin, and prominent Weibel-Palade bodies. The endothelial cells also exhibited other ultrastructural features characteristic of lymphatic endothelium, including sparse microvillous surface projections, overlapping intercellular junctions, and abundant intermediate filaments. This endothelial cell line represents a new source of proliferating lymphatic endothelium for future study, including structural and functional comparison to blood vascular endothelium.

Static mechanical strain induces capillary endothelial cell cycle re-entry and sprouting.

PubMed

Zeiger, A S; Liu, F D; Durham, J T; Jagielska, A; Mahmoodian, R; Van Vliet, K J; Herman, I M

2016-08-16

Vascular endothelial cells are known to respond to a range of biochemical and time-varying mechanical cues that can promote blood vessel sprouting termed angiogenesis. It is less understood how these cells respond to sustained (i.e., static) mechanical cues such as the deformation generated by other contractile vascular cells, cues which can change with age and disease state. Here we demonstrate that static tensile strain of 10%, consistent with that exerted by contractile microvascular pericytes, can directly and rapidly induce cell cycle re-entry in growth-arrested microvascular endothelial cell monolayers. S-phase entry in response to this strain correlates with absence of nuclear p27, a cyclin-dependent kinase inhibitor. Furthermore, this modest strain promotes sprouting of endothelial cells, suggesting a novel mechanical 'angiogenic switch'. These findings suggest that static tensile strain can directly stimulate pathological angiogenesis, implying that pericyte absence or death is not necessarily required of endothelial cell re-activation.

The Association Between PD-L1 Expression and the Clinical Outcomes to Vascular Endothelial Growth Factor-Targeted Therapy in Patients With Metastatic Clear Cell Renal Cell Carcinoma

PubMed Central

Shin, Su-Jin; Jeon, Yoon Kyung; Cho, Yong Mee; Lee, Jae-Lyun; Chung, Doo Hyun; Park, Ji Young

2015-01-01

Background. Vascular endothelial growth factor pathway (VEGF)-tyrosine kinase inhibitors (TKIs) are used as the first-line treatment for patients with metastatic clear cell renal cell carcinoma (mCCRCC). Recently, programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) blockade emerged as promising therapy for renal cell carcinoma. However, the expression pattern and prognostic implication of programmed death-ligands (PD-Ls) in mCCRCC patients receiving VEGF-TKI remain unclear. Patients and Methods. PD-L1 and PD-L2 expression in tumor cells and the quantities of PD-1+ tumor-infiltrating lymphocytes were immunohistochemically evaluated in 91 mCCRCC patients treated with VEGF-TKI, and their associations with VEGF-TKI responsiveness and clinical outcome were analyzed. Results. PD-L1 immunopositivity was observed in 17.6% and significantly associated with a high International Society of Urological Pathology grade (p = .031) and sarcomatoid features (p = .014). PD-L2 immunopositivity was observed in 39.6% and was not associated with any of the assessed clinicopathological variables. PD-L1-positive cases showed poor VEGF-TKI responsiveness (p = .012) compared with PD-L1-negative cases. In univariate survival analysis, PD-L1 immunopositivity was significantly associated with shorter overall survival (OS) (p = .037) and progression-free survival (PFS) (p = .043). Multivariate survival analysis revealed that PD-L1 expression was independently associated with poor OS (p = .038) and PFS (p = .013) in addition to tumor necrosis (p = .006; p = .029, respectively) and Memorial Sloan Kettering Cancer Center score (p = .018; p = .032, respectively). PD-L2 expression was neither associated with VEGF-TKI responsiveness nor patients’ outcome. Conclusion. PD-L1 expression was significantly related to lack of VEGF-TKI responsiveness and independently associated with shorter survival in mCCRCC patients after VEGF-TKI treatment. PD-L1 may have a predictive and prognostic value for determining the value of VEGF-TKI treatment in patients with mCCRCC. Implications for Practice: Vascular endothelial growth factor pathway (VEGF)-tyrosine kinase inhibitors (TKIs) are essential for the treatment of metastatic renal cell carcinoma patients, but the treatment suffers from a lack of predictive markers. This study demonstrates that PD-L1 expression is a predictor for unfavorable response to VEGF-TKI and a prognostic indicator for poor overall survival and progression-free survival in patients with metastatic clear cell renal cell carcinoma receiving VEGF-TKI. PMID:26424759

Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule Are Induced by Ionizing Radiation on Lymphatic Endothelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez-Ruiz, María E., E-mail: mrruiz@unav.es; Radiation Oncology, University Clinic, University of Navarra, Pamplona; Garasa, Saray

Purpose/Objectives: The goal of this study was to assess the effects of ionizing radiation on the expression of the integrin ligands ICAM-1 and VCAM that control leucocyte transit by lymphatic endothelial cells. Materials/Methods: Confluent monolayers of primary human lymphatic endothelial cells (LEC) were irradiated with single dose of 2, 5, 10 or 20 Gy, with 6 MeV-x-rays using a Linear-Accelerator. ICAM-1 and VCAM expression was determined by flow cytometry. Human tissue specimens received a single dose of 20 Gy with 15 MeV-x-rays. MC38, B16-OVA or B16-VEGF-C tumors grown in C57BL/6 mice were irradiated with single dose of 20Gy using amore » Linear-Accelerator fitted with a 10mm Radiosurgery collimator. Clinical samples were obtained from patients previous and 4 weeks after complete standard radiotherapy. ICAM-1 and VCAM expression was detected in all tissue specimens by confocal microscopy. To understand the role of TGFβ in this process anti-TGFβ blocking mAb were injected i.p. 30min before radiotherapy. Cell adhesion to irradiated LEC was analyzed in adhesion experiments performed in the presence or in the absence of anti- TGFβ and /or anti-ICAM1 blocking mAb. Results: We demonstrate that lymphatic endothelial cells in tumor samples experience induction of surface ICAM-1 and VCAM when exposed to ionizing radiation in a dose- and time-dependent manner. These effects can be recapitulated in cultured LEC, and are in part mediated by TGFβ. These data are consistent with increases in ICAM-1 and VCAM expression on LYVE-1+ endothelial cells in freshly explanted human tumor tissue and in mouse transplanted tumors after radiotherapy. Finally, ICAM-1 and VCAM expression accounts for enhanced adherence of human T lymphocytes to irradiated LEC. Conclusion: Our results show induction of ICAM-1 and VCAM on LVs in irradiated lesions and offer a starting point for elucidating the biological and therapeutic implications of targeting leukocyte traffic in combination to immunotherapy.« less

Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule Are Induced by Ionizing Radiation on Lymphatic Endothelium.

PubMed

Rodriguez-Ruiz, María E; Garasa, Saray; Rodriguez, Inmaculada; Solorzano, Jose Luis; Barbes, Benigno; Yanguas, Alba; Teijeira, Alvaro; Etxeberria, Iñaki; Aristu, José Javier; Halin, Cornelia; Melero, Ignacio; Rouzaut, Ana

2017-02-01

The goal of this study was to assess the effects of ionizing radiation on the expression of the integrin ligands ICAM-1 and VCAM that control leucocyte transit by lymphatic endothelial cells. Confluent monolayers of primary human lymphatic endothelial cells (LEC) were irradiated with single dose of 2, 5, 10 or 20 Gy, with 6 MeV-x-rays using a Linear-Accelerator. ICAM-1 and VCAM expression was determined by flow cytometry. Human tissue specimens received a single dose of 20 Gy with 15 MeV-x-rays. MC38, B16-OVA or B16-VEGF-C tumors grown in C57BL/6 mice were irradiated with single dose of 20Gy using a Linear-Accelerator fitted with a 10mm Radiosurgery collimator. Clinical samples were obtained from patients previous and 4 weeks after complete standard radiotherapy. ICAM-1 and VCAM expression was detected in all tissue specimens by confocal microscopy. To understand the role of TGFβ in this process anti-TGFβ blocking mAb were injected i.p. 30min before radiotherapy. Cell adhesion to irradiated LEC was analyzed in adhesion experiments performed in the presence or in the absence of anti- TGFβ and /or anti-ICAM1 blocking mAb. We demonstrate that lymphatic endothelial cells in tumor samples experience induction of surface ICAM-1 and VCAM when exposed to ionizing radiation in a dose- and time-dependent manner. These effects can be recapitulated in cultured LEC, and are in part mediated by TGFβ. These data are consistent with increases in ICAM-1 and VCAM expression on LYVE-1+ endothelial cells in freshly explanted human tumor tissue and in mouse transplanted tumors after radiotherapy. Finally, ICAM-1 and VCAM expression accounts for enhanced adherence of human T lymphocytes to irradiated LEC. Our results show induction of ICAM-1 and VCAM on LVs in irradiated lesions and offer a starting point for elucidating the biological and therapeutic implications of targeting leukocyte traffic in combination to immunotherapy. Copyright © 2016 Elsevier Inc. All rights reserved.

Heparin use in a rat hemorrhagic shock model induces biologic activity in mesenteric lymph separate from shock.

PubMed

Qin, Yong; Prescott, Lauriston M; Deitch, Edwin A; Kaiser, Vicki L

2011-04-01

Experimental data have shown that mesenteric lymph from rats subjected to trauma-hemorrhagic shock (THS) but not trauma-sham shock induces neutrophil activation, cytotoxicity, decreased red blood cell (RBC) deformability, and bone marrow colony growth suppression. These data have led to the hypothesis that gut factors produced from THS enter the systemic circulation via the mesenteric lymphatics and contribute to the progression of multiple organ failure after THS. Ongoing studies designed to identify bioactive lymph agents implicated factors associated with the heparin use in the THS procedure. We investigated if heparin itself was responsible for reported toxicity to human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cell toxicity was not induced by lymph when alternate anticoagulants (citrate and EDTA) were used in THS. Human umbilical vein endothelial cell toxicity was induced by lymph after heparin but not saline or citrate injection into trauma-sham shock and naive animals and was dose dependent. Activities of both heparin-releasable lipases (lipoprotein and hepatic) were detected in the plasma and lymph from THS and naive animals receiving heparin but not citrate or saline. Lymph-induced HUVEC toxicity correlated with lymph lipase activities. Finally, incubation of HUVECs with purified lipoprotein lipase added to naive lymph-induced toxicity in vitro. These data show that heparin, not THS, is responsible for the reported lymph-mediated HUVEC toxicity through its release of lipases into the lymph. These findings can provide alternative explanations for several of the THS effects reported in the literature using heparin models, thus necessitating a review of previous work in this field.