An endothelial cell niche induces hepatic specification through dual repression of Wnt and Notch signaling

PubMed Central

Han, Songyan; Dziedzic, Noelle; Gadue, Paul; Keller, Gordon M.; Gouon-Evans, Valerie

2012-01-01

Complex cross-talk between endoderm and the microenvironment is an absolute requirement to orchestrate hepatic specification and expansion. In the mouse, the septum transversum and cardiac mesoderm, through secreted BMPs and FGFs, respectively, instruct the adjacent ventral endoderm to become hepatic endoderm. Consecutively, endothelial cells promote expansion of the specified hepatic endoderm. Using a mouse reporter embryonic stem (ES) cell line in which hCD4 and hCD25 were targeted to the Foxa2 and Foxa3 loci, we reconstituted an in vitro culture system in which committed endoderm cells co-expressing hCD4-Foxa2 and hCD25-Foxa3 were isolated, and co-cultured with endothelial cells in the presence of BMP4 and bFGF. In this culture setting, we provide mechanistic evidence that endothelial cells function not only to promote hepatic endoderm expansion, but are also required at an earlier step for hepatic specification, at least in part through regulation of the Wnt and Notch pathways. Activation of Wnt and Notch by chemical or genetic approaches increases endoderm cell numbers but inhibits hepatic specification, and conversely, chemical inhibition of both pathways enhances hepatic specification and reduces proliferation. Using identical co-culture conditions, we defined a similar dependence of endoderm harvested from embryos on endothelial cells to support their growth and hepatic specification. Our findings (1) confirm a conserved role of Wnt repression for mouse hepatic specification, (2) uncover a novel role for Notch repression in the hepatic fate decision, and (3) demonstrate that repression of Wnt and Notch signaling in hepatic endoderm is controlled by the endothelial cell niche. PMID:21732480

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.

Variable promoter methylation contributes to differential expression of key genes in human placenta-derived venous and arterial endothelial cells.

PubMed

Joo, Jihoon E; Hiden, Ursula; Lassance, Luciana; Gordon, Lavinia; Martino, David J; Desoye, Gernot; Saffery, Richard

2013-07-15

The endothelial compartment, comprising arterial, venous and lymphatic cell types, is established prenatally in association with rapid phenotypic and functional changes. The molecular mechanisms underpinning this process in utero have yet to be fully elucidated. The aim of this study was to investigate the potential for DNA methylation to act as a driver of the specific gene expression profiles of arterial and venous endothelial cells. Placenta-derived venous and arterial endothelial cells were collected at birth prior to culturing. DNA methylation was measured at >450,000 CpG sites in parallel with expression measurements taken from 25,000 annotated genes. A consistent set of genomic loci was found to show coordinate differential methylation between the arterial and venous cell types. This included many loci previously not investigated in relation to endothelial function. An inverse relationship was observed between gene expression and promoter methylation levels for a limited subset of genes implicated in endothelial function, including NOS3, encoding endothelial Nitric Oxide Synthase. Endothelial cells derived from the placental vasculature at birth contain widespread methylation of key regulatory genes. These are candidates involved in the specification of different endothelial cell types and represent potential target genes for environmentally mediated epigenetic disruption in utero in association with cardiovascular disease risk later in life.

Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

PubMed Central

Tracy, Russell P.; Parikh, Megha A.; Hoffman, Eric A.; Shimbo, Daichi; Austin, John H. M.; Smith, Benjamin M.; Hueper, Katja; Vogel-Claussen, Jens; Lima, Joao; Gomes, Antoinette; Watson, Karol; Kawut, Steven; Barr, R. Graham

2017-01-01

Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50–79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema. PMID:28291826

Hemogenic endothelial cell specification requires c-kit, notch signaling, and p27-mediated cell-cycle control

USDA-ARS?s Scientific Manuscript database

Delineating the mechanism or mechanisms that regulate the specification of hemogenic endothelial cells from primordial endothelium is critical for optimizing their derivation from human stem cells for clinical therapies. We previously determined that retinoic acid (RA) is required for hemogenic spec...

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

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.

NgBR is essential for endothelial cell glycosylation and vascular development.

PubMed

Park, Eon Joo; Grabińska, Kariona A; Guan, Ziqiang; Sessa, William C

2016-02-01

NgBR is a transmembrane protein identified as a Nogo-B-interacting protein and recently has been shown to be a subunit required for cis-prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial-specific NgBR knockout embryos. Here, we show that endothelial-specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE-cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo-B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development. © 2016 The Authors.

Endothelial Galectin-1 Binds to Specific Glycans on Nipah Virus Fusion Protein and Inhibits Maturation, Mobility, and Function to Block Syncytia Formation

PubMed Central

Garner, Omai B.; Aguilar, Hector C.; Fulcher, Jennifer A.; Levroney, Ernest L.; Harrison, Rebecca; Wright, Lacey; Robinson, Lindsey R.; Aspericueta, Vanessa; Panico, Maria; Haslam, Stuart M.; Morris, Howard R.; Dell, Anne

2010-01-01

Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell. PMID:20657665

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

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 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.

Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-Thrombospondin-1 axis

PubMed Central

Lee, Joo-Hyeon; Bhang, Dong Ha; Beede, Alexander; Huang, Tian Lian; Stripp, Barry R.; Bloch, Kenneth D.; Wagers, Amy J.; Tseng, Yu-Hua; Ryeom, Sandra; Kim, Carla F.

2014-01-01

SUMMARY Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal three-dimensional (3D) co-cultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell co-cultures. Gain and loss of function experiments showed BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of Thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1-null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide new tools to understand the mechanisms of respiratory diseases at the single cell level. PMID:24485453

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.

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

Immunization of Cattle by Infection with Cowdria ruminantium Elicits T Lymphocytes That Recognize Autologous, Infected Endothelial Cells and Monocytes†

PubMed Central

Mwangi, Duncan M.; Mahan, Suman M.; Nyanjui, John K.; Taracha, Evans L. N.; McKeever, Declan J.

1998-01-01

Peripheral blood mononuclear cells (PBMC) from immune cattle proliferate in the presence of autologous Cowdria ruminantium-infected endothelial cells and monocytes. Endothelial cells required treatment with T-cell growth factors to induce class II major histocompatibility complex expression prior to infection and use as stimulators. Proliferative responses to both infected autologous endothelial cells and monocytes were characterized by expansion of a mixture of CD4+, CD8+, and γδ T cells. However, γδ T cells dominated following several restimulations. Reverse transcription-PCR analysis of cytokine expression by C. ruminantium-specific T-cell lines and immune PBMC revealed weak interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-γ) transcripts at 3 to 24 h after stimulation. Strong expression of IFN-γ, tumor necrosis factor alpha (TNF-α), TNF-β, and IL-2 receptor α-chain mRNA was detected in T-cell lines 48 h after antigen stimulation. Supernatants from these T-cell cultures contained IFN-γ protein. Our findings suggest that in immune cattle a C. ruminantium-specific T-cell response is induced and that infected endothelial cells and monocytes may present C. ruminantium antigens to specific T lymphocytes in vivo during infection and thereby play a role in induction of protective immune responses to the pathogen. PMID:9573061

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

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.

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.

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.

A sensitive ELISA for measuring the adhesion of leukocytic cells to human endothelial cells.

PubMed

Krakauer, T

1994-12-28

A new, sensitive ELISA using monoclonal antibodies reactive with surface molecules specific for various leukocytes was devised to measure the attachment of these cells to cultured monolayers of human umbilical vein endothelial cells. Preparations of peripheral blood mononuclear cells, a human monocytic cell line (THP-1) and a human lymphoblastic T cell line (MOLT-4) were used to test the sensitivity of this method and compare it with the conventional 51Cr-radiolabeled cell assay. The extent of adhesion to endothelial cells was assayed by measuring the optical density produced by a complex of peroxidase-labeled streptavidin, biotin-conjugated F(ab')2 anti-mouse Ig and monoclonal antibody on fixed leukocytic cells that had adhered to endothelial cells. This method is fast and sensitive, eliminates the use of radioisotopes, and, because the detection uses a specific marker on the cell of interest, can be used in preparations of unseparated mixtures of cells. As this is a microassay, using relatively small number of cells and reagents, the methodology can be applied to screen a large number of therapeutic agents that may regulate adhesion. Using this method, the anti-inflammatory corticosteroid, dexamethasone, was found to inhibit the adhesion of THP-1 and MOLT-4 cells to cytokine-activated endothelial cells.

Notch1 acts via Foxc2 to promote definitive hematopoiesis via effects on hemogenic endothelium

PubMed Central

Jang, Il Ho; Lu, Yi-Fen; Zhao, Long; Wenzel, Pamela L.; Kume, Tsutomu; Datta, Sumon M.; Arora, Natasha; Guiu, Jordi; Lagha, Mounia; Kim, Peter G.; Do, Eun Kyoung; Kim, Jae Ho; Schlaeger, Thorsten M.; Zon, Leonard I.; Bigas, Anna; Burns, Caroline E.

2015-01-01

Hematopoietic and vascular development share many common features, including cell surface markers and sites of origin. Recent lineage-tracing studies have established that definitive hematopoietic stem and progenitor cells arise from vascular endothelial–cadherin+ hemogenic endothelial cells of the aorta-gonad-mesonephros region, but the genetic programs underlying the specification of hemogenic endothelial cells remain poorly defined. Here, we discovered that Notch induction enhances hematopoietic potential and promotes the specification of hemogenic endothelium in differentiating cultures of mouse embryonic stem cells, and we identified Foxc2 as a highly upregulated transcript in the hemogenic endothelial population. Studies in zebrafish and mouse embryos revealed that Foxc2 and its orthologs are required for the proper development of definitive hematopoiesis and function downstream of Notch signaling in the hemogenic endothelium. These data establish a pathway linking Notch signaling to Foxc2 in hemogenic endothelial cells to promote definitive hematopoiesis. PMID:25587036

Targeting of phage particles towards endothelial cells by antibodies selected through a multi-parameter selection strategy.

PubMed

Mandrup, Ole A; Lykkemark, Simon; Kristensen, Peter

2017-02-10

One of the hallmarks of cancer is sustained angiogenesis. Here, normal endothelial cells are activated, and their formation of new blood vessels leads to continued tumour growth. An improved patient condition is often observed when angiogenesis is prevented or normalized through targeting of these genomically stable endothelial cells. However, intracellular targets constitute a challenge in therapy, as the agents modulating these targets have to be delivered and internalized specifically to the endothelial cells. Selection of antibodies binding specifically to certain cell types is well established. It is nonetheless a challenge to ensure that the binding of antibodies to the target cell will mediate internalization. Previously selection of such antibodies has been performed targeting cancer cell lines; most often using either monovalent display or polyvalent display. In this article, we describe selections that isolate internalizing antibodies by sequential combining monovalent and polyvalent display using two types of helper phages, one which increases display valence and one which reduces background. One of the selected antibodies was found to mediate internalization into human endothelial cells, although our results confirms that the single stranded nature of the DNA packaged into phage particles may limit applications aimed at targeting nucleic acids in mammalian cells.

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.

Impairment of endothelial cell differentiation from bone marrow-derived mesenchymal stem cells: new insight into the pathogenesis of systemic sclerosis.

PubMed

Cipriani, P; Guiducci, S; Miniati, I; Cinelli, M; Urbani, S; Marrelli, A; Dolo, V; Pavan, A; Saccardi, R; Tyndall, A; Giacomelli, R; Cerinic, M Matucci

2007-06-01

Systemic sclerosis (SSc) is a disorder characterized by vascular damage and fibrosis of the skin and internal organs. Despite marked tissue hypoxia, there is no evidence of compensatory angiogenesis. The ability of mesenchymal stem cells (MSCs) to differentiate into endothelial cells was recently demonstrated. The aim of this study was to determine whether impaired differentiation of MSCs into endothelial cells in SSc might contribute to disease pathogenesis by decreasing endothelial repair. MSCs obtained from 7 SSc patients and 15 healthy controls were characterized. The number of colony-forming unit-fibroblastoid colonies was determined. After culture in endothelial-specific medium, the endothelial-like MSC (EL-MSC) phenotype was assessed according to the surface expression of vascular endothelial growth factor receptors (VEGFRs). Senescence, chemoinvasion, and capillary morphogenesis studies were also performed. MSCs from SSc patients displayed the same phenotype and clonogenic activity as those from controls. In SSc MSCs, a decreased percentage of VEGFR-2+, CXCR4+, VEGFR-2+/CXCR4+ cells and early senescence was detected. After culturing, SSc EL-MSCs showed increased expression of VEGFR-1, VEGFR-2, and CXCR4, did not express CD31 or annexin V, and showed significantly decreased migration after specific stimuli. Moreover, the addition of VEGF and stromal cell-derived factor 1 to cultured SSc EL-MSCs increased their angiogenic potential less than that in controls. Our data strongly suggest that endothelial repair may be affected in SSc. The possibility that endothelial progenitor cells could be used to increase vessel growth in chronic ischemic tissues may open up new avenues in the treatment of vascular damage caused by SSc.

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

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.

Endothelial Notch activity promotes angiogenesis and osteogenesis in bone

NASA Astrophysics Data System (ADS)

Ramasamy, Saravana K.; Kusumbe, Anjali P.; Wang, Lin; Adams, Ralf H.

2014-03-01

Blood vessel growth in the skeletal system and osteogenesis seem to be coupled, suggesting the existence of molecular crosstalk between endothelial and osteoblastic cells. Understanding the nature of the mechanisms linking angiogenesis and bone formation should be of great relevance for improved fracture healing or prevention of bone mass loss. Here we show that vascular growth in bone involves a specialized, tissue-specific form of angiogenesis. Notch signalling promotes endothelial cell proliferation and vessel growth in postnatal long bone, which is the opposite of the well-established function of Notch and its ligand Dll4 in the endothelium of other organs and tumours. Endothelial-cell-specific and inducible genetic disruption of Notch signalling in mice not only impaired bone vessel morphology and growth, but also led to reduced osteogenesis, shortening of long bones, chondrocyte defects, loss of trabeculae and decreased bone mass. On the basis of a series of genetic experiments, we conclude that skeletal defects in these mutants involved defective angiocrine release of Noggin from endothelial cells, which is positively regulated by Notch. Administration of recombinant Noggin, a secreted antagonist of bone morphogenetic proteins, restored bone growth and mineralization, chondrocyte maturation, the formation of trabeculae and osteoprogenitor numbers in endothelial-cell-specific Notch pathway mutants. These findings establish a molecular framework coupling angiogenesis, angiocrine signals and osteogenesis, which may prove significant for the development of future therapeutic applications.

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.

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.

Endothelial Cell Autonomous Role of Akt1: Regulation of Vascular Tone and Ischemia-Induced Arteriogenesis.

PubMed

Lee, Monica Y; Gamez-Mendez, Ana; Zhang, Jiasheng; Zhuang, Zhenwu; Vinyard, David J; Kraehling, Jan; Velazquez, Heino; Brudvig, Gary W; Kyriakides, Themis R; Simons, Michael; Sessa, William C

2018-04-01

The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ubiquitous expression of Akt1, however, raises the possibility of cell-type-dependent Akt1-driven actions, thereby necessitating tissue-specific characterization. Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis. Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair. © 2018 American Heart Association, Inc.

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

PubMed

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

2013-05-01

Endothelial cells from microvasculature are directly involved in a large number of vascular diseases; however, culture of these cells is problematic, since most methodologies employ proteolytic enzymes or mechanical techniques, leading to cell damage and contamination of endothelial cultures with other cellular types. Besides, primary cultured cells have a short life span in vitro and undergo replicative senescence after 3-4 passages, limiting long-term studies. In the present work we report the generation of a spontaneously immortalized endothelial culture obtained from mice pulmonary capillaries. Firstly, primary (third passage) and immortalized (100th) cultures were established. Further, monoclonal populations were obtained by serial dilutions from immortalized cultures. Cells were analyzed according to: (1) morphological appearance, (2) expression of specific endothelial markers by fluorescent staining [von Willebrand Factor (vWF), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE) and Ulex europaeus (UEA-1)] and by flow cytometry (endoglin, VE-cadherin and VCAM-1), and (3) release of nitric oxide (NO), assessed by the specific fluorescent dye DAF-2 DA, and prostacyclin (PGI2), quantified by enzyme immune assay. In both cultures cells grew in monolayers and presented cobblestone appearance at confluence. Positive staining for vWF, eNOS, ACE and UEA-1 was detected in cloned as well as in early-passage cultured cells. Similarly, cultures presented equal expressions of endoglin, VE-cadherin and VCAM-1. Values of NO and PGI2 levels did not differ between cultures. From these results we confirm that the described spontaneously immortalized endothelial cell line is capable of unlimited growth and retains typical morphological and functional properties exhibited by primary cultured cells. Therefore, the endothelial cell line described in the present study can become a suitable tool in the field of endothelium research and can be useful for the investigation of production of endothelial mediators, angiogenesis and inflammation. Copyright © 2013 Elsevier Inc. All rights reserved.

Targeted modulation of reactive oxygen species in the vascular endothelium.

PubMed

Shuvaev, Vladimir V; Muzykantov, Vladimir R

2011-07-15

'Endothelial cells lining vascular luminal surface represent an important site of signaling and injurious effects of reactive oxygen species (ROS) produced by other cells and endothelium itself in ischemia, inflammation and other pathological conditions. Targeted delivery of ROS modulating enzymes conjugated with antibodies to endothelial surface molecules (vascular immunotargeting) provides site-specific interventions in the endothelial ROS, unattainable by other formulations including PEG-modified enzymes. Targeting of ROS generating enzymes (e.g., glucose oxidase) provides ROS- and site-specific models of endothelial oxidative stress, whereas targeting of antioxidant enzymes SOD and catalase offers site-specific quenching of superoxide anion and H(2)O(2). These targeted antioxidant interventions help to clarify specific role of endothelial ROS in vascular and pulmonary pathologies and provide basis for design of targeted therapeutics for treatment of these pathologies. In particular, antibody/catalase conjugates alleviate acute lung ischemia/reperfusion injury, whereas antibody/SOD conjugates inhibit ROS-mediated vasoconstriction and inflammatory endothelial signaling. Encapsulation in protease-resistant, ROS-permeable carriers targeted to endothelium prolongs protective effects of antioxidant enzymes, further diversifying the means for targeted modulation of endothelial ROS. Copyright © 2011 Elsevier B.V. All rights reserved.

Primitive erythrocytes are generated from hemogenic endothelial cells.

PubMed

Stefanska, Monika; Batta, Kiran; Patel, Rahima; Florkowska, Magdalena; Kouskoff, Valerie; Lacaud, Georges

2017-07-25

Primitive erythroblasts are the first blood cells generated during embryonic hematopoiesis. Tracking their emergence both in vivo and in vitro has remained challenging due to the lack of specific cell surface markers. To selectively investigate primitive erythropoiesis, we have engineered a new transgenic embryonic stem (ES) cell line, where eGFP expression is driven by the regulatory sequences of the embryonic βH1 hemoglobin gene expressed specifically in primitive erythroid cells. Using this ES cell line, we observed that the first primitive erythroblasts are detected in vitro around day 1.5 of blast colony differentiation, within the cell population positive for the early hematopoietic progenitor marker CD41. Moreover, we establish that these eGFP + cells emerge from a hemogenic endothelial cell population similarly to their definitive hematopoietic counterparts. We further generated a corresponding βH1-eGFP transgenic mouse model and demonstrated the presence of a primitive erythroid primed hemogenic endothelial cell population in the developing embryo. Taken together, our findings demonstrate that both in vivo and in vitro primitive erythrocytes are generated from hemogenic endothelial cells.

Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

PubMed Central

Sahara, Makoto; Hansson, Emil M; Wernet, Oliver; Lui, Kathy O; Später, Daniela; Chien, Kenneth R

2014-01-01

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC+CD31+CD34+CD14−KDRhigh endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR+ mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs. PMID:24810299

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

PubMed

Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

2017-10-01

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Endothelial Response to Glucocorticoids in Inflammatory Diseases

PubMed Central

Zielińska, Karolina A.; Van Moortel, Laura; Opdenakker, Ghislain; De Bosscher, Karolien; Van den Steen, Philippe E.

2016-01-01

The endothelium plays a crucial role in inflammation. A balanced control of inflammation requires the action of glucocorticoids (GCs), steroidal hormones with potent cell-specific anti-inflammatory properties. Besides the classic anti-inflammatory effects of GCs on leukocytes, recent studies confirm that endothelial cells also represent an important target for GCs. GCs regulate different aspects of endothelial physiology including expression of adhesion molecules, production of pro-inflammatory cytokines and chemokines, and maintenance of endothelial barrier integrity. However, the regulation of endothelial GC sensitivity remains incompletely understood. In this review, we specifically examine the endothelial response to GCs in various inflammatory diseases ranging from multiple sclerosis, stroke, sepsis, and vasculitis to atherosclerosis. Shedding more light on the cross talk between GCs and endothelium will help to improve existing therapeutic strategies and develop new therapies better tailored to the needs of patients. PMID:28018358

Defective angiogenesis delays thrombus resolution: a potential pathogenetic mechanism underlying chronic thromboembolic pulmonary hypertension

PubMed Central

Panzenboeck, Adelheid; Winter, Max P; Schubert, Uwe; Voswinckel, Robert; Frey, Maria K; Jakowitsch, Johannes; Alimohammadi, Arman; Hobohm, Lukas; Mangold, Andreas; Bergmeister, Helga; Sibilia, Maria; Wagner, Erwin F; Mayer, Eckhard; Klepetko, Walter; Hoelzenbein, Thomas J; Preissner, Klaus T; Lang, Irene M

2015-01-01

Objective Restoration of patency is a natural target of vascular remodeling following venous thrombosis that involves vascular endothelial cells and smooth muscle cells as well as leukocytes. Acute pulmonary emboli usually resolve within six months. However, in some instances, thrombi transform into fibrous vascular obstructions, resulting in occlusion of the deep veins, or in chronic thromboembolic pulmonary hypertension (CTEPH). We proposed that dysregulated thrombus angiogenesis may contribute to thrombus persistence. Approach and Results Mice with an endothelial-cell-specific conditional deletion of vascular endothelial growth factor receptor 2/kinase insert domain protein receptor (VEGF-R2/Kdr) were utilized in a model of stagnant flow venous thrombosis closely resembling human deep vein thrombosis. Biochemical and functional analyses were performed on pulmonary endarterectomy specimens from patients with CTEPH, a human model of non-resolving venous thromboembolism. Endothelial cell-specific deletion of Kdr and subsequent ablation of thrombus vascularization delayed thrombus resolution. In accordance with these findings, organized human CTEPH thrombi were largely devoid of vascular structures. Several vessel-specific genes such as KDR, vascular endothelial cadherin and podoplanin were expressed at lower levels in white CTEPH thrombi than in organizing deep vein thrombi and organizing thrombi from aortic aneurysms. In addition, red CTEPH thrombi attenuated the angiogenic response induced by VEGF. Conclusions In the present work, we propose a mechanism of thrombus non-resolution demonstrating that endothelial cell-specific deletion of Kdr abates thrombus vessel formation, misguiding thrombus resolution. Medical conditions associated with the development of CTEPH may be compromising early thrombus angiogenesis. PMID:24526692

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

Specific Accumulation of Tumor-Derived Adhesion Factor in Tumor Blood Vessels and in Capillary Tube-Like Structures of Cultured Vascular Endothelial Cells

NASA Astrophysics Data System (ADS)

Akaogi, Kotaro; Okabe, Yukie; Sato, Junji; Nagashima, Yoji; Yasumitsu, Hidetaro; Sugahara, Kazuyuki; Miyazaki, Kaoru

1996-08-01

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

Analysis of proteome response to the mobile phone radiation in two types of human primary endothelial cells.

PubMed

Nylund, Reetta; Kuster, Niels; Leszczynski, Dariusz

2010-10-18

Use of mobile phones has widely increased over the past decade. However, in spite of the extensive research, the question of potential health effects of the mobile phone radiation remains unanswered. We have earlier proposed, and applied, proteomics as a tool to study biological effects of the mobile phone radiation, using as a model human endothelial cell line EA.hy926. Exposure of EA.hy926 cells to 900 MHz GSM radiation has caused statistically significant changes in expression of numerous proteins. However, exposure of EA.hy926 cells to 1800 MHz GSM signal had only very small effect on cell proteome, as compared with 900 MHz GSM exposure. In the present study, using as model human primary endothelial cells, we have examined whether exposure to 1800 MHz GSM mobile phone radiation can affect cell proteome. Primary human umbilical vein endothelial cells and primary human brain microvascular endothelial cells were exposed for 1 hour to 1800 MHz GSM mobile phone radiation at an average specific absorption rate of 2.0 W/kg. The cells were harvested immediately after the exposure and the protein expression patterns of the sham-exposed and radiation-exposed cells were examined using two dimensional difference gel electrophoresis-based proteomics (2DE-DIGE). There were observed numerous differences between the proteomes of human umbilical vein endothelial cells and human brain microvascular endothelial cells (both sham-exposed). These differences are most likely representing physiological differences between endothelia in different vascular beds. However, the exposure of both types of primary endothelial cells to mobile phone radiation did not cause any statistically significant changes in protein expression. Exposure of primary human endothelial cells to the mobile phone radiation, 1800 MHz GSM signal for 1 hour at an average specific absorption rate of 2.0 W/kg, does not affect protein expression, when the proteomes were examined immediately after the end of the exposure and when the false discovery rate correction was applied to analysis. This observation agrees with our earlier study showing that the 1800 MHz GSM radiation exposure had only very limited effect on the proteome of human endothelial cell line EA.hy926, as compared with the effect of 900 MHz GSM radiation.

PAR-2 triggers placenta-derived protease-induced altered VE-cadherin reorganization at endothelial junctions in preeclampsia.

PubMed

Gu, Y; Groome, L J; Alexander, J S; Wang, Y

2012-10-01

PAR-2 is a G-protein coupled protease receptor whose activation in endothelial cells (ECs) is associated with increased solute permeability. VE-cadherin is an endothelial-specific junction protein, which exhibits a disorganized distribution at cell junction during inflammation and is a useful indicator of endothelial barrier dysfunction. In the present study, we tested the hypothesis that PAR-2 activation mediates placenta-derived chymotrypsin-like protease (CLP)-induced endothelial junction disturbance and permeability in preeclampsia (PE). PAR-2 and VE-cadherin were examined by immunofluorescent staining. Specific CLP induced PAR-2 activation and altered VE-cadherin distribution was assessed following depletion of protease chymotrypsin in the placental conditioned medium and after PAR-2 siRNA. VE-cadherin assembly was determined by treating cells with protease chymotrypsin and/or the specific PAR-2 agonist SLIGKV-NH2. Our results showed: 1) placental conditioned medium not only disturbed VE-cadherin distribution at cell junctions but also activated PAR-2 in ECs; 2) PAR-2 siRNA blocked the placental conditioned medium induced PAR-2 upregulation and disorganization of VE-cadherin at cell junctions; 3) PAR-2 agonist induced PAR-2 activation and VE-cadherin reorganization were dose-dependent; and 4) PAR-2 agonist could stimulate ERK1/2 activation. These results strongly suggest that proteases produced by the placenta elicit endothelial barrier dysfunction via a PAR-2 signaling regulatory mechanism in PE. Copyright © 2012 Elsevier Ltd. All rights reserved.

PAR-2 triggers placenta-derived protease-induced altered VE-cadherin reorganization at endothelial junctions in preeclampsia

PubMed Central

Gu, Yang; Groome, Lynn J.; Alexander, J. Steven; Wang, Yuping

2014-01-01

PAR-2 is a G-protein coupled protease receptor whose activation in endothelial cells (ECs) is associated with increased solute permeability. VE-cadherin is an endothelial specific junction protein, which exhibits a disorganized distribution at cell junction during inflammation and is a useful indicator of endothelial barrier dysfunction. In the present study, we tested the hypothesis that PAR-2 activation mediates placenta-derived chymotrypsin-like protease (CLP)-induced endothelial junction disturbance and permeability in preeclampsia (PE). PAR-2 and VE-cadherin were examined by immunofluorescent staining. Specific CLP-induced PAR-2 activation and altered VE-cadherin distribution was assessed following depletion of protease chymotrypsin in the placental conditioned medium and after PAR-2 siRNA. VE-cadherin assembly was determined by treating cells with protease chymotrypsin and/or the specific PAR-2 agonist SLIGKV-NH2. Our results showed: 1) placental conditioned medium not only disturbed VE-cadherin distribution at cell junctions but also activated PAR-2 in ECs; 2) PAR-2 siRNA blocked the placental conditioned medium induced PAR-2 upregulation and disorganization of VE-cadherin at cell junctions; 3) PAR-2 agonist induced PAR-2 activation and VE-cadherin reorganization were dose-dependent; and 4) PAR-2 agonist could stimulate ERK1/2 activation. These results strongly suggest that proteases produced by the placenta elicit endothelial barrier dysfunction via a PAR-2 signaling regulatory mechanism in PE. PMID:22840244

Polymerisation of fibrin αC-domains promotes endothelial cell migration and proliferation.

PubMed

Yakovlev, S; Mikhailenko, I; Tsurupa, G; Belkin, A M; Medved, L

2014-12-01

Upon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerisation of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signalling pathways. The major goal of this study was to test the impact of αC-domain polymerisation on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells. In an in vitro wound healing assay, confluent endothelial cell monolayers on tissue culture plates coated with the αC monomer or αC polymers were wounded by scratching and wound closure was monitored by time-lapse videomicroscopy. Although the plates were coated with equal amounts of αC species, as confirmed by ELISA, wound closure by the cells occurred much faster on αC polymers, indicating that αC-domain polymerisation promotes cell migration and proliferation. In agreement, endothelial cell proliferation was also more efficient on αC polymers, as revealed by cell proliferation assay. Wound closure on both types of substrates was equally inhibited by the integrin-blocking GRGDSP peptide and a specific antagonist of the ERK1/2 signalling pathway. In contrast, blocking the FAK signaling pathway by a specific antagonist decreased wound closure only on αC polymers. These results indicate that polymerisation of the αC-domains enhances integrin-dependent endothelial cell migration and proliferation mainly through the FAK signalling pathway. Furthermore, clustering of integrin-binding RGD motifs in αC polymers is the major mechanism triggering these events.

Endothelial cell-derived GABA signaling modulates neuronal migration and postnatal behavior

PubMed Central

Li, Suyan; Kumar T, Peeyush; Joshee, Sampada; Kirschstein, Timo; Subburaju, Sivan; Khalili, Jahan S; Kloepper, Jonas; Du, Chuang; Elkhal, Abdallah; Szabó, Gábor; Jain, Rakesh K; Köhling, Rüdiger; Vasudevan, Anju

2018-01-01

The cerebral cortex is essential for integration and processing of information that is required for most behaviors. The exquisitely precise laminar organization of the cerebral cortex arises during embryonic development when neurons migrate successively from ventricular zones to coalesce into specific cortical layers. While radial glia act as guide rails for projection neuron migration, pre-formed vascular networks provide support and guidance cues for GABAergic interneuron migration. This study provides novel conceptual and mechanistic insights into this paradigm of vascular-neuronal interactions, revealing new mechanisms of GABA and its receptor-mediated signaling via embryonic forebrain endothelial cells. With the use of two new endothelial cell specific conditional mouse models of the GABA pathway (Gabrb3ΔTie2-Cre and VgatΔTie2-Cre), we show that partial or complete loss of GABA release from endothelial cells during embryogenesis results in vascular defects and impairs long-distance migration and positioning of cortical interneurons. The downstream effects of perturbed endothelial cell-derived GABA signaling are critical, leading to lasting changes to cortical circuits and persistent behavioral deficits. Furthermore, we illustrate new mechanisms of activation of GABA signaling in forebrain endothelial cells that promotes their migration, angiogenesis and acquisition of blood-brain barrier properties. Our findings uncover and elucidate a novel endothelial GABA signaling pathway in the CNS that is distinct from the classical neuronal GABA signaling pathway and shed new light on the etiology and pathophysiology of neuropsychiatric diseases, such as autism spectrum disorders, epilepsy, anxiety, depression and schizophrenia. PMID:29086765

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.

Histone Acetylation Regulates the Cell-Specific and Interferon-γ–Inducible Expression of Extracellular Superoxide Dismutase in Human Pulmonary Arteries

PubMed Central

Stepp, Marcus W.; Vorst, Alan L.; Folz, Rodney J.

2011-01-01

Extracellular superoxide dismutase (EC-SOD) is the major antioxidant enzyme present in the vascular wall, and is responsible for both the protection of vessels from oxidative stress and for the modulation of vascular tone. Concentrations of EC-SOD in human pulmonary arteries are very high relative to other tissues, and the expression of EC-SOD appears highly restricted to smooth muscle. The molecular basis for this smooth muscle–specific expression of EC-SOD is not known. Here we assessed the role of epigenetic factors in regulating the cell-specific and IFN-γ–inducible expression of EC-SOD in human pulmonary artery cells. The analysis of CpG site methylation within the promoter and coding regions of the EC-SOD gene demonstrated higher levels of DNA methylation within the distal promoter region in endothelial cells compared with smooth muscle cells. Exposure of both cell types to DNA demethylation agents reactivated the transcription of EC-SOD in endothelial cells alone. However, exposure to the histone deacetylase inhibitor trichostatin A (TSA) significantly induced EC-SOD gene expression in both endothelial cells and smooth muscle cells. Concentrations of EC-SOD mRNA were also induced up to 45-fold by IFN-γ in smooth muscle cells, but not in endothelial cells. The IFN-γ–dependent expression of EC-SOD was regulated by the Janus tyrosine kinase/signal transducers and activators of transcription proteins signaling pathway. Simultaneous exposure to TSA and IFN-γ produced a synergistic effect on the induction of EC-SOD gene expression, but only in endothelial cells. These findings provide strong evidence that EC-SOD cell-specific and IFN-γ–inducible expression in pulmonary artery cells is regulated, to a major degree, by epigenetic mechanisms that include histone acetylation and DNA methylation. PMID:21493784

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.

Specialized mouse embryonic stem cells for studying vascular development.

PubMed

Glaser, Drew E; Burns, Andrew B; Hatano, Rachel; Medrzycki, Magdalena; Fan, Yuhong; McCloskey, Kara E

2014-01-01

Vascular progenitor cells are desirable in a variety of therapeutic strategies; however, the lineage commitment of endothelial and smooth muscle cell from a common progenitor is not well-understood. Here, we report the generation of the first dual reporter mouse embryonic stem cell (mESC) lines designed to facilitate the study of vascular endothelial and smooth muscle development in vitro. These mESC lines express green fluorescent protein (GFP) under the endothelial promoter, Tie-2, and Discomsoma sp. red fluorescent protein (RFP) under the promoter for alpha-smooth muscle actin (α-SMA). The lines were then characterized for morphology, marker expression, and pluripotency. The mESC colonies were found to exhibit dome-shaped morphology, alkaline phosphotase activity, as well as expression of Oct 3/4 and stage-specific embryonic antigen-1. The mESC colonies were also found to display normal karyotypes and are able to generate cells from all three germ layers, verifying pluripotency. Tissue staining confirmed the coexpression of VE (vascular endothelial)-cadherin with the Tie-2 GFP+ expression on endothelial structures and smooth muscle myosin heavy chain with the α-SMA RFP+ smooth muscle cells. Lastly, it was verified that the developing mESC do express Tie-2 GFP+ and α-SMA RFP+ cells during differentiation and that the GFP+ cells colocalize with the vascular-like structures surrounded by α-SMA-RFP cells. These dual reporter vascular-specific mESC permit visualization and cell tracking of individual endothelial and smooth muscle cells over time and in multiple dimensions, a powerful new tool for studying vascular development in real time.

RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border

PubMed Central

Marcos-Ramiro, Beatriz; García-Weber, Diego; Barroso, Susana; Feito, Jorge; Ortega, María C.; Cernuda-Morollón, Eva; Reglero-Real, Natalia; Fernández-Martín, Laura; Durán, Maria C.; Alonso, Miguel A.; Correas, Isabel; Cox, Susan; Ridley, Anne J.

2016-01-01

Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation. PMID:27138256

Angiocrine functions of organ-specific endothelial cells

PubMed Central

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

2016-01-01

Preface Endothelial cells lining blood vessel capillaries are not just passive conduits for delivering blood. Tissue-specific endothelium establish specialized vascular niches that deploy specific sets of growth factors, known as angiocrine factors, which actively participate in inducing, specifying, patterning, and guiding organ regeneration and maintaining homeostasis and metabolism. Angiocrine factors upregulated in response to injury orchestrates self-renewal and differentiation of tissue-specific repopulating resident stem and progenitor cells into functional organs. Uncovering the precise mechanisms whereby physiological-levels of angiocrine factors are spatially and temporally produced, and distributed by organotypic endothelium to repopulating cells, will lay the foundation for driving organ repair without scarring. PMID:26791722

Cardiac Remodeling: Endothelial Cells Have More to Say Than Just NO

PubMed Central

Segers, Vincent F. M.; Brutsaert, Dirk L.; De Keulenaer, Gilles W.

2018-01-01

The heart is a highly structured organ consisting of different cell types, including myocytes, endothelial cells, fibroblasts, stem cells, and inflammatory cells. This pluricellularity provides the opportunity of intercellular communication within the organ, with subsequent optimization of its function. Intercellular cross-talk is indispensable during cardiac development, but also plays a substantial modulatory role in the normal and failing heart of adults. More specifically, factors secreted by cardiac microvascular endothelial cells modulate cardiac performance and either positively or negatively affect cardiac remodeling. The role of endothelium-derived small molecules and peptides—for instance NO or endothelin-1—has been extensively studied and is relatively well defined. However, endothelial cells also secrete numerous larger proteins. Information on the role of these proteins in the heart is scattered throughout the literature. In this review, we will link specific proteins that modulate cardiac contractility or cardiac remodeling to their expression by cardiac microvascular endothelial cells. The following proteins will be discussed: IL-6, periostin, tenascin-C, thrombospondin, follistatin-like 1, frizzled-related protein 3, IGF-1, CTGF, dickkopf-3, BMP-2 and−4, apelin, IL-1β, placental growth factor, LIF, WISP-1, midkine, and adrenomedullin. In the future, it is likely that some of these proteins can serve as markers of cardiac remodeling and that the concept of endothelial function and dysfunction might have to be redefined as we learn more about other factors secreted by ECs besides NO. PMID:29695980

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.

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

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

Kim, Hyung Gyun; Kim, Ji Young; Hwang, Yong Pil

2006-12-15

Endothelial cells produce adhesion molecules after being stimulated with various inflammatory cytokines. These adhesion molecules play an important role in the development of atherogenesis. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of kahweol, a coffee-specific diterpene. This study examined the effects of kahweol on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. Kahweol inhibited the adhesion of TNF{alpha}-induced monocytes to endothelial cells and suppressed the TNF{alpha}-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNF{alpha}-induced JAK2-PI3K/Akt-NF-{kappa}B activation pathway in these cells. Overall, kahweol hasmore » anti-inflammatory and anti-atherosclerotic activities, which occurs partly by down-regulating the pathway that affects the expression and interaction of the cell adhesion molecules on endothelial cells.« less

Production of platelet-derived endothelial cell growth factor by normal and transformed human cells in culture.

PubMed Central

Usuki, K; Heldin, N E; Miyazono, K; Ishikawa, F; Takaku, F; Westermark, B; Heldin, C H

1989-01-01

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa endothelial cell mitogen which has angiogenic properties in vivo. We report here that human foreskin fibroblasts, a human squamous cell carcinoma cell line, and 2 out of the 3 human thyroid carcinoma cell lines investigated produce PD-ECGF, whereas 21 other cell lines examined do not. The positive cell lines contained a 1.8-kilobase PD-ECGF mRNA, and a 45-kDa protein could be demonstrated in lysates of the cell lines by immunoblotting and immunoprecipitation using a specific antiserum against PD-ECGF. Furthermore, the cell lysates contained mitogenic activity for endothelial cells that was neutralized by the PD-ECGF antiserum. PD-ECGF was found to be secreted only slowly from the producer cells, consistent with the previous finding that the primary translation product lacks a signal sequence. The restricted expression and intracellular sequestration of PD-ECGF imply a strictly controlled function in endothelial cell proliferation and angiogenesis. Aberrant production of PD-ECGF may play a role in tumor angiogenesis. Images PMID:2678104

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.

Morphological changes in vascular and circulating blood cells following exposure to detergent sclerosants.

PubMed

Cooley-Andrade, O; Connor, D E; Ma, D D F; Weisel, J W; Parsi, K

2016-04-01

To investigate morphological changes in vascular and circulating blood cells following exposure to detergent sclerosants sodium tetradecyl sulfate and polidocanol. Samples of whole blood, isolated leukocytes, platelets, endothelial cells, and fibroblasts were incubated with varying concentrations of sclerosants. Whole blood smears were stained with Giemsa and examined by light and bright field microscopy. Phalloidin and Hoechst stains were used to analyze cytoplasmic and nuclear morphology by fluorescence microscopy. Endothelial cell and fibroblasts were analyzed by live cell imaging. Higher concentrations of sclerosants induced cell lysis. Morphological changes in intact cells were observed at sublytic concentrations of detergents. Low concentration sodium tetradecyl sulfate induced erythrocyte acanthocytosis and macrocytosis, while polidocanol induced Rouleaux formation and increased the population of target cells and stomatocytes. Leukocytes showed swelling, blebbing, vacuolation, and nuclear degradation following exposure to sodium tetradecyl sulfate, while polidocanol induced pseudopodia formation, chromatin condensation, and fragmentation. Platelets exhibited pseudopodia with sodium tetradecyl sulfate and a "fried egg" appearance with polidocanol. Exposure to sodium tetradecyl sulfate resulted in size shrinkage in both endothelial cell and fibroblasts, while endothelial cell developed distinct spindle morphology. Polidocanol induced cytoplasmic microfilament bundles in both endothelial cell and fibroblasts. Patchy chromatin condensation was observed following exposure of fibroblasts to either agent. Detergent sclerosants are biologically active at sublytic concentrations. The observed morphological changes are consistent with cell activation, apoptosis, and oncosis. The cellular response is concentration dependent, cell-specific, and sclerosant specific. © The Author(s) 2015.

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.

Subregional localization and characterization of Ly6aGFP-expressing hematopoietic cells in the mouse embryonic head.

PubMed

Li, Zhuan; Vink, Chris S; Mariani, Samanta A; Dzierzak, Elaine

2016-08-01

Hematopoietic cell generation in the midgestation mouse embryo occurs through the natural transdifferentiation of temporally and spatially restricted set of hemogenic endothelial cells. These cells take on hematopoietic fate in the aorta, vitelline and umbilical arteries and appear as hematopoietic cell clusters that emerge from the vascular wall. Genetic and live imaging data have supported this. Recently, the embryonic head has been shown to contain fully functional hematopoietic stem cells (HSC). By lineage tracing, cerebrovascular specific endothelial cells were shown to contribute to the postnatal mouse hematopoietic system. Since Ly6aGFP is a marker of all HSCs, some hematopoietic cluster cells and hemogenic endothelial cells in the midgestation mouse aorta, we examine here whether embryonic head HSCs and vascular endothelial cells are positive for this marker. Whereas some head vasculature, single hematopoietic cells and all HSCs are Ly6aGFP expressing, we do not find clusters of hematopoietic cells emerging from the cerebrovasculature that are characteristic of endothelial-to-hematopoietic transition. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Treatment with platelet lysate induces endothelial differentation of bone marrow mesenchymal stem cells under fluid shear stress

PubMed Central

Homayouni Moghadam, Farshad; Tayebi, Tahereh; Moradi, Alireza; Nadri, Hamid; Barzegar, Kazem; Eslami, Gilda

2014-01-01

By considering stem cell-based therapies as a new hope for the treatment of some tragic diseases, marrow stromal cells or marrow mesenchymal stem cells (MSCs) were considered as a suitable and safe multipotential cell source for this new therapeutic approach. For this purpose, many investigations have been performed on differentiation of MSCs toward specific cell lines to overcome the demand for providing the organ specific cells for cell therapy or preparation of engineered tissues. In the present study, differentiation of MSCs to endothelial cells (ECs) by mechanical and chemical stimulation was evaluated. Fluid shear stress (FSS) was used as mechanical inducer, while platelet lysate (PL) and estradiol (E) were used as chemical induction factors. MSCs were placed under FSS with different forces (2, 5 and 10dyn/cm2) for different periods (6, 12 and 24 hours). In some groups, PL and E were added to the culture media to evaluate their effect on expression of EC specific markers. This investigation revealed that FSS with low tension (2.5-5 dyn/cm2) for a long time (24 hours) or high tension (10 dyn/cm2) in short time (6 hours) in the presence of PL could differentiate MSCs toward ECs. The presence of PL was necessary for initiation of endothelial differentiation, and in the absence of PL, there was not any expression of CD34 and Cadherin5 (Cdh5) among cells. Adding E to the culture medium did not change the rate of endothelial differentiation under FSS. Generated endothelial progenitors could produce von Willebrand factor (vWF) after two weeks culture and also they formed tubular structures after culture on matrigel. PMID:26417289

Treatment with platelet lysate induces endothelial differentation of bone marrow mesenchymal stem cells under fluid shear stress.

PubMed

Homayouni Moghadam, Farshad; Tayebi, Tahereh; Moradi, Alireza; Nadri, Hamid; Barzegar, Kazem; Eslami, Gilda

2014-01-01

By considering stem cell-based therapies as a new hope for the treatment of some tragic diseases, marrow stromal cells or marrow mesenchymal stem cells (MSCs) were considered as a suitable and safe multipotential cell source for this new therapeutic approach. For this purpose, many investigations have been performed on differentiation of MSCs toward specific cell lines to overcome the demand for providing the organ specific cells for cell therapy or preparation of engineered tissues. In the present study, differentiation of MSCs to endothelial cells (ECs) by mechanical and chemical stimulation was evaluated. Fluid shear stress (FSS) was used as mechanical inducer, while platelet lysate (PL) and estradiol (E) were used as chemical induction factors. MSCs were placed under FSS with different forces (2, 5 and 10dyn/cm(2)) for different periods (6, 12 and 24 hours). In some groups, PL and E were added to the culture media to evaluate their effect on expression of EC specific markers. This investigation revealed that FSS with low tension (2.5-5 dyn/cm(2)) for a long time (24 hours) or high tension (10 dyn/cm(2)) in short time (6 hours) in the presence of PL could differentiate MSCs toward ECs. The presence of PL was necessary for initiation of endothelial differentiation, and in the absence of PL, there was not any expression of CD34 and Cadherin5 (Cdh5) among cells. Adding E to the culture medium did not change the rate of endothelial differentiation under FSS. Generated endothelial progenitors could produce von Willebrand factor (vWF) after two weeks culture and also they formed tubular structures after culture on matrigel.

Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications

PubMed Central

Kumar VR, Santhosh; Darisipudi, Murthy N.; Steiger, Stefanie; Devarapu, Satish Kumar; Tato, Maia; Kukarni, Onkar P.; Mulay, Shrikant R.; Thomasova, Dana; Popper, Bastian; Demleitner, Jana; Zuchtriegel, Gabriele; Reichel, Christoph; Cohen, Clemens D.; Lindenmeyer, Maja T.; Liapis, Helen; Moll, Solange; Reid, Emma; Stitt, Alan W.; Schott, Brigitte; Gruner, Sabine; Haap, Wolfgang; Ebeling, Martin; Hartmann, Guido

2016-01-01

Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia–induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68+ intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage–derived circulating PAR2 agonist and mediator of endothelial dysfunction–related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases. PMID:26567242

Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/β-catenin pathway during brain angiogenesis

PubMed Central

Vanhollebeke, Benoit; Stone, Oliver A; Bostaille, Naguissa; Cho, Chris; Zhou, Yulian; Maquet, Emilie; Gauquier, Anne; Cabochette, Pauline; Fukuhara, Shigetomo; Mochizuki, Naoki; Nathans, Jeremy; Stainier, Didier YR

2015-01-01

Despite the critical role of endothelial Wnt/β-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/β-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/β-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/β-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function. DOI: http://dx.doi.org/10.7554/eLife.06489.001 PMID:26051822

Vascular Endothelial Cell-Specific Connective Tissue Growth Factor (CTGF) Is Necessary for Development of Chronic Hypoxia-Induced Pulmonary Hypertension.

PubMed

Pi, Liya; Fu, Chunhua; Lu, Yuanquing; Zhou, Junmei; Jorgensen, Marda; Shenoy, Vinayak; Lipson, Kenneth E; Scott, Edward W; Bryant, Andrew J

2018-01-01

Chronic hypoxia frequently complicates the care of patients with interstitial lung disease, contributing to the development of pulmonary hypertension (PH), and premature death. Connective tissue growth factor (CTGF), a matricellular protein of the Cyr61/CTGF/Nov (CCN) family, is known to exacerbate vascular remodeling within the lung. We have previously demonstrated that vascular endothelial-cell specific down-regulation of CTGF is associated with protection against the development of PH associated with hypoxia, though the mechanism for this effect is unknown. In this study, we generated a transgenic mouse line in which the Ctgf gene was floxed and deleted in vascular endothelial cells that expressed Cre recombinase under the control of VE-Cadherin promoter (eCTGF KO mice). Lack of vascular endothelial-derived CTGF protected against the development of PH secondary to chronic hypoxia, as well as in another model of bleomycin-induced pulmonary hypertension. Importantly, attenuation of PH was associated with a decrease in infiltrating inflammatory cells expressing CD11b or integrin α M (ITGAM), a known adhesion receptor for CTGF, in the lungs of hypoxia-exposed eCTGF KO mice. Moreover, these pathological changes were associated with activation of-Rho GTPase family member-cell division control protein 42 homolog (Cdc42) signaling, known to be associated with alteration in endothelial barrier function. These data indicate that endothelial-specific deletion of CTGF results in protection against development of chronic-hypoxia induced PH. This protection is conferred by both a decrease in inflammatory cell recruitment to the lung, and a reduction in lung Cdc42 activity. Based on our studies, CTGF inhibitor treatment should be investigated in patients with PH associated with chronic hypoxia secondary to chronic lung disease.

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

Cytostatic inhibition of endothelial cell growth by the angiogenesis inhibitor TNP-470 (AGM-1470).

PubMed Central

Kusaka, M.; Sudo, K.; Matsutani, E.; Kozai, Y.; Marui, S.; Fujita, T.; Ingber, D.; Folkman, J.

1994-01-01

Recently, we reported the anti-angiogenic action along with anti-tumour activity of TNP-470 (AGM-1470). In this study, the effect of TNP-470 on the growth of human umbilical vein endothelial (HUVE) cells was examined. TNP-470 inhibited the growth of HUVE cells in a biphasic manner. The inhibition was cytostatic in the first phase (complete inhibition at 300 pg ml-1 to 3 micrograms ml-1 with an IC50 of 15 pg ml-1) and cytotoxic in the second phase (> or = 30 micrograms ml-1). The cytostatic inhibition of HUVE cell growth by TNP-470 was durable after washing out TNP-470 in culture. Incorporation of thymidine but not uridine and leucine by HUVE cells was inhibited in the first phase, while that of all three compounds was inhibited in the second phase. Human and rat endothelial cells among various types of cells were the most sensitive to the cytostatic inhibition, while differences in the cytotoxic inhibition were minimal. These results suggest that TNP-470 exerts its specific anti-angiogenic action by inhibiting cytostatically growth of endothelial cells in a relatively specific manner. PMID:8297716

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

MIP-2 causes differential activation of RhoA in mouse aortic versus pulmonary artery endothelial cells

PubMed Central

Moldobaeva, Aigul; Baek, Amy; Wagner, Elizabeth M.

2008-01-01

Previously, we have shown that endothelial cell chemotaxis to the proangiogenic chemokine MIP-2 (macrophage inflammatory protein-2), is much greater in mouse aortic endothelial cells (EC) than pulmonary arterial endothelial cells (PA EC). This was true despite the observation that both cell types display comparable levels of the ligand receptor, CXCR2 (8). Since the systemic arterial circulation is proangiogenic in the adult lung and the pulmonary circulation is relatively resistant to neovascularization, we questioned whether the observed functional heterogeneity is related to inherent differences in cell signaling cascades of the two EC subtypes. Specifically, we measured activation of Rac1 and RhoA, both thought to be involved in EC cell migration. Rac1 showed inconsistent and minimal changes in both cell types after MIP-2 treatment (p>0.05). However, activated RhoA was increased upon exposure to MIP-2 only in aortic EC (61% increase; p<0.05). Decreased RhoA activation after treatment of aortic EC with specific siRNA for RhoA resulted in a functional decrease in EC chemotaxis to MIP-2 (17% increase; p<0.05). Additionally, increased RhoA activation in PA EC with adenoviral infection of RhoA caused an increase in PA EC chemotaxis to MIP-2 (46% increase; p<0.05). Inhibition of RhoA activity with the Rho kinase inhibitor, Y27632 blocked aortic EC chemotaxis and stress fiber formation. Thus, RhoA activation is increased after MIP-2 treatment in mouse aortic endothelial cells but not in pulmonary artery endothelial cells. We conclude that RhoA is part of a signaling pathway essential for aortic cell migration after CXCR2 ligation. This result provides one explanation for the difference in chemotaxis observed in these two endothelial subtypes that express similar levels of CXCR2. PMID:17662312

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.

TAK1 in brain endothelial cells mediates fever and lethargy

PubMed Central

Ridder, Dirk A.; Lang, Ming-Fei; Salinin, Sergei; Röderer, Jan-Peter; Struss, Marcel; Maser-Gluth, Christiane

2011-01-01

Systemic inflammation affects the brain, resulting in fever, anorexia, lethargy, and activation of the hypothalamus–pituitary–adrenal axis. How peripheral inflammatory signals reach the brain is still a matter of debate. One possibility is that, in response to inflammatory stimuli, brain endothelial cells in proximity to the thermoregulatory centers produce cyclooxygenase 2 (COX-2) and release prostaglandin E2, causing fever and sickness behavior. We show that expression of the MAP kinase kinase kinase TAK1 in brain endothelial cells is needed for interleukin 1β (IL-1β)–induced COX-2 production. Exploiting the selective expression of the thyroxine transporter Slco1c1 in brain endothelial cells, we generated a mouse line allowing inducible deletion of Tak1 specifically in brain endothelium. Mice lacking the Tak1 gene in brain endothelial cells showed a blunted fever response and reduced lethargy upon intravenous injection of the endogenous pyrogen IL-1β. In conclusion, we demonstrate that TAK1 in brain endothelial cells induces COX-2, most likely by activating p38 MAPK and c-Jun, and is necessary for fever and sickness behavior. PMID:22143887

PI3 K/Akt/mTOR-mediated translational control regulates proliferation and differentiation of lineage-restricted RoSH stem cell lines

PubMed Central

Que, Jianwen; Lian, Qizhou; El Oakley, Reida M; Lim, Bing; Lim, Sai-Kiang

2007-01-01

Background We have previously derived highly similar lineage-restricted stem cell lines, RoSH and E-RoSH cell lines from mouse embryos and CD9hi SSEA-1- differentiated mouse embryonic stem cells, respectively. These cell lines are not pluripotent and differentiate readily into endothelial cells in vitro and in vivo. Results We investigated the signaling pathway that maintains proliferation of these cells in an undifferentiated state, and demonstrate that PI3 K/Akt/mTOR, but not Raf/MEK/Erk, signaling in these cells was active during proliferation and was downregulated during endothelial differentiation. Inhibition of PI3 K/Akt/mTOR signaling, but not Raf/MEK/Erk, reduced proliferation and induced expression of endothelial specific proteins. During differentiation or inhibition of PI3 K/Akt/mTOR signaling, cyclinD2 transcript abundance in ribosome-enriched RNA but not in total RNA was reduced with a corresponding reduction in protein level. In contrast, transcript abundance of endothelial-specific genes e.g. Kdr, Tek and Pdgfrα in ribosome-enriched RNA fraction was not reduced and their protein levels were increased. Together these observations suggested that translational control mediated by PI3K/Akt/mTOR signaling was critical in regulating proliferation and endothelial differentiation of lineage-restricted RoSH-like stem cell lines. Conclusion This study highlights translation regulation as a critical regulatory mechanism during proliferation and differentiation in stem cells. PMID:17892597

Isolated tumor endothelial cells maintain specific character during long-term culture

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

Matsuda, Kohei; Oral Pathology and Biology, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586; Oral and Maxillofacial Surgery, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, wemore » have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.« less

Side-Specific Endothelial-Dependent Regulation of Aortic Valve Calcification

PubMed Central

Richards, Jennifer; El-Hamamsy, Ismail; Chen, Si; Sarang, Zubair; Sarathchandra, Padmini; Yacoub, Magdi H.; Chester, Adrian H.; Butcher, Jonathan T.

2014-01-01

Arterial endothelial cells maintain vascular homeostasis and vessel tone in part through the secretion of nitric oxide (NO). In this study, we determined how aortic valve endothelial cells (VEC) regulate aortic valve interstitial cell (VIC) phenotype and matrix calcification through NO. Using an anchored in vitro collagen hydrogel culture system, we demonstrate that three-dimensionally cultured porcine VIC do not calcify in osteogenic medium unless under mechanical stress. Co-culture with porcine VEC, however, significantly attenuated VIC calcification through inhibition of myofibroblastic activation, osteogenic differentiation, and calcium deposition. Incubation with the NO donor DETA-NO inhibited VIC osteogenic differentiation and matrix calcification, whereas incubation with the NO blocker l-NAME augmented calcification even in 3D VIC–VEC co-culture. Aortic VEC, but not VIC, expressed endothelial NO synthase (eNOS) in both porcine and human valves, which was reduced in osteogenic medium. eNOS expression was reduced in calcified human aortic valves in a side-specific manner. Porcine leaflets exposed to the soluble guanylyl cyclase inhibitor ODQ increased osteocalcin and α-smooth muscle actin expression. Finally, side-specific shear stress applied to porcine aortic valve leaflet endothelial surfaces increased cGMP production in VEC. Valve endothelial-derived NO is a natural inhibitor of the early phases of valve calcification and therefore may be an important regulator of valve homeostasis and pathology. PMID:23499458

Solid tumor therapy by selectively targeting stromal endothelial cells

PubMed Central

Liu, Shihui; Liu, Jie; Ma, Qian; Cao, Liu; Fattah, Rasem J.; Yu, Zuxi; Bugge, Thomas H.; Finkel, Toren; Leppla, Stephen H.

2016-01-01

Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors. PMID:27357689

The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1.

PubMed

Zheng, Xiangrong; Zhang, Shangshang; Yang, Yujia; Wang, Xia; Zhong, Le; Yu, Xiaohe

2008-11-01

The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 mumol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The expression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regulated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (P<0.05). The MTT test showed that the proliferation rate of HUVEC transfected with HPVA under hypoxia exceeded that of the control group. We conclude that the HRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation of endothelial cells. Taking advantage of these facts could greatly improve the efficiency of gene therapy. The vector would be valuable for various gene transfer studies targeting endothelial cells.

RNAi-mediated downregulation of oral cancer overexpressed 1 (ORAOV1) inhibits vascular endothelial cell proliferation, migration, invasion, and tube formation.

PubMed

Zhao, Xin; Liu, Dongjuan; Wang, Lili; Wu, Ruiqing; Zeng, Xin; Dan, Hongxia; Ji, Ning; Jiang, Lu; Zhou, Yu; Chen, Qianming

2016-04-01

Oral squamous cell carcinoma (OSCC) is one of the top ten tumors threatening human health. Oral cancer overexpressed 1 (ORAOV1) identified within chromosomal region 11q13, one of the most frequently amplified regions in OSCC, has been suggested as a novel candidate oncogene in OSCC, regulating cell cycle, apoptosis, and angiogenesis. In this study, we investigated the role of ORAOV1 in OSCC-induced angiogenesis in vitro. EA.hy926 human endothelial cells were co-cultured with OSCC cells (HSC-3 and SCC-25) transfected with ORAOV1-specific shRNA to downregulate ORAOV1 expression, and analyzed for proliferation, migration, invasion, and tube formation by specific assays. EA.hy926 endothelial cells co-cultured with ORAOV1-deficient OSCC cells exhibited significantly lower proliferation, migration, and invasion, as well as the activity in tube formation compared to that in the control cells. Our results show, for the first time, that ORAOV1 expressed by OSCC cells promotes tube formation by endothelial cells, indicating its involvement in OSCC angiogenesis. Considering the importance of neovascularization in tumor development and metastasis, these findings suggest that targeting ORAOV1 may be a potential therapeutic strategy against OSCC. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.

PubMed

Kaminski, Alexander; Ma, Nan; Donndorf, Peter; Lindenblatt, Nicole; Feldmeier, Gregor; Ong, Lee-Lee; Furlani, Dario; Skrabal, Christian A; Liebold, Andreas; Vollmar, Brigitte; Steinhoff, Gustav

2008-01-01

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1alpha)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1alpha/tumor necrosis factor-alpha (TNF-alpha)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1alpha/TNF-alpha stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (-/-) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1alpha enhanced c-kit+-cell migration. In vivo, SDF-1alpha alone triggered endothelial rolling-not firm adherence-of c-kit+ cells in WT mice. While TNF-alpha alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1alpha+TNF-alpha, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (-/-) mice, firm endothelial adhesion of c-kit+ cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1alpha mediates firm adhesion c-kit+ cells only in the presence of TNF-alpha stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit+-cell adhesion to the vascular endothelium.

Endothelial-to-Osteoblast Conversion Generates Osteoblastic Metastasis of Prostate Cancer.

PubMed

Lin, Song-Chang; Lee, Yu-Chen; Yu, Guoyu; Cheng, Chien-Jui; Zhou, Xin; Chu, Khoi; Murshed, Monzur; Le, Nhat-Tu; Baseler, Laura; Abe, Jun-Ichi; Fujiwara, Keigi; deCrombrugghe, Benoit; Logothetis, Christopher J; Gallick, Gary E; Yu-Lee, Li-Yuan; Maity, Sankar N; Lin, Sue-Hwa

2017-06-05

Prostate cancer (PCa) bone metastasis is frequently associated with bone-forming lesions, but the source of the osteoblastic lesions remains unclear. We show that the tumor-induced bone derives partly from tumor-associated endothelial cells that have undergone endothelial-to-osteoblast (EC-to-OSB) conversion. The tumor-associated osteoblasts in PCa bone metastasis specimens and patient-derived xenografts (PDXs) were found to co-express endothelial marker Tie-2. BMP4, identified in PDX-conditioned medium, promoted EC-to-OSB conversion of 2H11 endothelial cells. BMP4 overexpression in non-osteogenic C4-2b PCa cells led to ectopic bone formation under subcutaneous implantation. Tumor-induced bone was reduced in trigenic mice (Tie2 cre /Osx f/f /SCID) with endothelial-specific deletion of osteoblast cell-fate determinant OSX compared with bigenic mice (Osx f/f /SCID). Thus, tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa. Copyright © 2017 Elsevier Inc. All rights reserved.

Heterozygous Deficiency of PHD2 Restores Tumor Oxygenation and Inhibits Metastasis via Endothelial Normalization

PubMed Central

Loges, Sonja; Schmidt, Thomas; Jonckx, Bart; Tian, Ya-Min; Lanahan, Anthony A.; Pollard, Patrick; de Almodovar, Carmen Ruiz; De Smet, Frederik; Vinckier, Stefan; Aragonés, Julián; Debackere, Koen; Luttun, Aernout; Wyns, Sabine; Jordan, Benedicte; Pisacane, Alberto; Gallez, Bernard; Lampugnani, Maria Grazia; Dejana, Elisabetta; Simons, Michael; Ratcliffe, Peter; Maxwell, Patrick; Carmeliet, Peter

2014-01-01

SUMMARY A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2+/− mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy. PMID:19217150

The innate defense antimicrobial peptides hBD3 and RNase7 are induced in human umbilical vein endothelial cells by classical inflammatory cytokines but not Th17 cytokines.

PubMed

Burgey, Christine; Kern, Winfried V; Römer, Winfried; Sakinc, Türkan; Rieg, Siegbert

2015-05-01

Antimicrobial peptides are multifunctional effector molecules of innate immunity. In this study we investigated whether endothelial cells actively contribute to innate defense mechanisms by expression of antimicrobial peptides. We therefore stimulated human umbilical vein endothelial cells (HUVEC) with inflammatory cytokines, Th17 cytokines, heat-inactivated bacteria, bacterial conditioned medium (BCM) of Staphylococcus aureus and Streptococcus sanguinis, and lipoteichoic acid (LTA). Stimulation with single cytokines induced discrete expression of human β-defensin 3 (hBD3) by IFN-γ or IL-1β and of ribonuclease 7 (RNase7) by TNF-α without any effects on LL-37 gene expression. Stronger hBD3 and RNase7 induction was observed after combined stimulation with IL-1β, TNF-α and IFN-γ and was confirmed by high hBD3 and RNase7 peptide levels in cell culture supernatants. In contrast, Th17 cytokines or stimulation with LTA did not result in AMP production. Moreover, only BCM of an invasive S. aureus bacteremia isolate induced hBD3 in HUVEC. We conclude that endothelial cells actively contribute to prevent dissemination of pathogens at the blood-tissue-barrier by production of AMPs that exhibit microbicidal and immunomodulatory functions. Further investigations should focus on tissue-specific AMP induction in different endothelial cell types, on pathogen-specific induction patterns and potentially involved pattern-recognition receptors of endothelial cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

PubMed Central

Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

2017-01-01

Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

VE-Cadherin–Mediated Epigenetic Regulation of Endothelial Gene Expression

PubMed Central

Morini, Marco F.; Giampietro, Costanza; Corada, Monica; Pisati, Federica; Lavarone, Elisa; Cunha, Sara I.; Conze, Lei L.; O’Reilly, Nicola; Joshi, Dhira; Kjaer, Svend; George, Roger; Nye, Emma; Ma, Anqi; Jin, Jian; Mitter, Richard; Lupia, Michela; Cavallaro, Ugo; Pasini, Diego; Calado, Dinis P.

2018-01-01

Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. Conclusions: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system. PMID:29233846

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

PubMed

Morini, Marco F; Giampietro, Costanza; Corada, Monica; Pisati, Federica; Lavarone, Elisa; Cunha, Sara I; Conze, Lei L; O'Reilly, Nicola; Joshi, Dhira; Kjaer, Svend; George, Roger; Nye, Emma; Ma, Anqi; Jin, Jian; Mitter, Richard; Lupia, Michela; Cavallaro, Ugo; Pasini, Diego; Calado, Dinis P; Dejana, Elisabetta; Taddei, Andrea

2018-01-19

The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5 , vascular endothelial-protein tyrosine phosphatase ( VE-PTP ), and von Willebrand factor ( vWf ). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5 , VE-PTP , and vWf . VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5 , VE-PTP , and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system. © 2017 The Authors.

Changes in thrombospondin-1 levels in the endothelial cells of the anterior pituitary during estrogen-induced prolactin-secreting pituitary tumors

PubMed Central

Sarkar, Abby J; Chaturvedi, Kirti; Chen, Cui Ping; Sarkar, Dipak K

2010-01-01

Thrombospondin-1 (TSP-1), a multifunctional matrix glycoprotein, has been shown to control tumor growth by inhibiting angiogenesis in various tissues. However, the role of this glycoprotein in pituitary angiogenesis is not well studied. In this report, we determined the changes in the production and action of TSP-1 on endothelial cells in anterior pituitary following estradiol treatment, which is known to increase prolactin-secreting tumor growth and vascularization in this tissue. We showed that TSP-1 immunoreactive protein is distributed in the anterior pituitary, particularly in the endothelial cells. Estradiol treatment for 2 and 4 weeks decreased the total tissue immunoreactive level of TSP-1 as well as the endothelial cell-specific immunoreactive level of this protein in the anterior pituitary. The steroid treatment also decreased the protein levels of TSP-1 in anterior pituitary tissues and in purified pituitary endothelial cells in primary cultures. Determination of the effects of TSP-1 on proliferation and migration of pituitary-derived endothelial cells in primary cultures elucidated an inhibitory action of TSP-1 on these vascular cell functions. These results suggest that locally produced TSP-1 may regulate estrogen angiogenic action on the pituitary. PMID:17283240

Differential sex-specific effects of oxygen toxicity in human umbilical vein endothelial cells

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

Zhang, Yuhao; Lingappan, Krithika

Despite the well-established sex-specific differences in the incidence of bronchopulmonary dysplasia (BPD), the molecular mechanism(s) behind these are not completely understood. Pulmonary angiogenesis is critical for alveolarization and arrest in vascular development adversely affects lung development. Human neonatal umbilical vein endothelial cells (HUVECs) provide a robust in vitro model for the study of endothelial cell physiology and function. Male and Female HUVECs were exposed to room air (21% O{sub 2}, 5% CO{sub 2}) or hyperoxia (95% O{sub 2}, 5% CO{sub 2}) for up to 72 h. Cell viability, proliferation, H{sub 2}O{sub 2} production and angiogenesis were analyzed. Sex-specific differences in the expressionmore » of VEGFR2 and modulation of NF-kappa B pathway were measured. Male HUVECs have decreased survival, greater oxidative stress and impairment in angiogenesis compared to similarly exposed female cells. There is differential expression of VEGFR2 between male and female HUVECs and greater activation of the NF-kappa B pathway in female HUVECs under hyperoxic conditions. The results indicate that sex differences exist between male and female HUVECs in vitro after hyperoxia exposure. Since endothelial dysfunction has a major role in the pathogenesis of BPD, these differences could explain in part the mechanisms behind sex-specific differences in the incidence of this disease. - Highlights: • Cellular sex effects viability and oxidative stress in HUVECs exposed to hyperoxia. • Male HUVECs show greater impairment in angiogenesis compared to female cells. • Sex-specific modulation of VEGFR2 and the NF-kappaB pathway was noted.« less

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

NASA Astrophysics Data System (ADS)

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

1998-04-01

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

The angiogenic factor CCN1 promotes adhesion and migration of circulating CD34+ progenitor cells: potential role in angiogenesis and endothelial regeneration.

PubMed

Grote, Karsten; Salguero, Gustavo; Ballmaier, Matthias; Dangers, Marc; Drexler, Helmut; Schieffer, Bernhard

2007-08-01

Tissue regeneration involves the formation of new blood vessels regulated by angiogenic factors. We reported recently that the expression of the angiogenic factor CCN1 is up-regulated under various pathophysiologic conditions within the cardiovascular system. Because CD34+ progenitor cells participate in cardiovascular tissue regeneration, we investigated whether CCN1-detected for the first time in human plasma-promotes the recruitment of CD34+ progenitor cells to endothelial cells, thereby enhancing endothelial proliferation and neovascularization. In this study, we demonstrated that CCN1 and supernatants from CCN1-stimulated human CD34+ progenitor cells promoted proliferation of endothelial cells and angiogenesis in vitro and in vivo. In addition, CCN1 induced migration and transendothelial migration of CD34+ cells and the release of multiple growth factors, chemokines, and matrix metalloproteinase-9 (MMP-9) from these cells. Moreover, the CCN1-specific integrins alpha(M)beta(2) and alpha(V)beta(3) are expressed on CD34+ cells and CCN1 stimulated integrin-dependent signaling. Furthermore, integrin antagonists (RGD-peptides) suppressed both binding of CCN1 to CD34+ cells and CCN1-induced adhesion of CD34+ cells to endothelial cells. These data suggest that CCN1 promotes integrin-dependent recruitment of CD34+ progenitor cells to endothelial cells, which may contribute to paracrine effects on angiogenesis and tissue regeneration.

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.

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

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.

Pericyte-targeting drug delivery and tissue engineering.

PubMed

Kang, Eunah; Shin, Jong Wook

2016-01-01

Pericytes are contractile mural cells that wrap around the endothelial cells of capillaries and venules. Depending on the triggers by cellular signals, pericytes have specific functionality in tumor microenvironments, properties of potent stem cells, and plasticity in cellular pathology. These features of pericytes can be activated for the promotion or reduction of angiogenesis. Frontier studies have exploited pericyte-targeting drug delivery, using pericyte-specific peptides, small molecules, and DNA in tumor therapy. Moreover, the communication between pericytes and endothelial cells has been applied to the induction of vessel neoformation in tissue engineering. Pericytes may prove to be a novel target for tumor therapy and tissue engineering. The present paper specifically reviews pericyte-specific drug delivery and tissue engineering, allowing insight into the emerging research targeting pericytes.

Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation

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

Okazaki, Hideki; Tokumaru, Sho; Hanakawa, Yasushi

2011-09-02

Highlights: {yields} VEGF-A enhanced lymphatic endothelial cell migration and increased tube formation. {yields} VEGF-A treated lymphatic endothelial cell showed activation of STAT3. {yields} Dominant-negative STAT3 inhibited VEGF-A-induced lymphatic endothelial cell migration and tube formation. -- Abstract: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor that regulates endothelial functions, and signal transducers and activators of transcription (STATs) are known to be important during VEGF receptor signaling. The aim of this study was to determine whether STAT3 regulates VEGF-induced lymphatic endothelial cell (LEC) migration and tube formation. VEGF-A (33 ng/ml) enhanced LEC migration by 2-fold and increased tube lengthmore » by 25% compared with the control, as analyzed using a Boyden chamber and Matrigel assay, respectively. Western blot analysis and immunostaining revealed that VEGF-A induced the nuclear translocation of phosphorylated STAT3 in LECs, and this translocation was blocked by the transfection of LECs with an adenovirus vector expressing a dominant-negative mutant of STAT3 (Ax-STAT3F). Transfection with Ax-STAT3F also almost completely inhibited VEGF-A-induced LEC migration and tube formation. These results indicate that STAT3 is essential for VEGF-A-induced LEC migration and tube formation and that STAT3 regulates LEC functions.« less

CD147 Promotes Entry of Pentamer-Expressing Human Cytomegalovirus into Epithelial and Endothelial Cells

PubMed Central

Pritchard, Sarah R.; Wisner, Todd W.; Liu, Jing; Jardetzky, Ted S.; Johnson, David C.

2018-01-01

ABSTRACT Human cytomegalovirus (HCMV) replicates in many diverse cell types in vivo, and entry into different cells involves distinct entry mechanisms and different envelope glycoproteins. HCMV glycoprotein gB is thought to act as the virus fusogen, apparently after being triggered by different gH/gL proteins that bind distinct cellular receptors or entry mediators. A trimer of gH/gL/gO is required for entry into all cell types, and entry into fibroblasts involves trimer binding to platelet-derived growth factor receptor alpha (PDGFRα). HCMV entry into biologically relevant epithelial and endothelial cells and monocyte-macrophages also requires a pentamer, gH/gL complexed with UL128, UL130, and UL131, and there is evidence that the pentamer binds unidentified receptors. We screened an epithelial cell cDNA library and identified the cell surface protein CD147, which increased entry of pentamer-expressing HCMV into HeLa cells but not entry of HCMV that lacked the pentamer. A panel of CD147-specific monoclonal antibodies inhibited HCMV entry into epithelial and endothelial cells, but not entry into fibroblasts. shRNA silencing of CD147 in endothelial cells inhibited HCMV entry but not entry into fibroblasts. CD147 colocalized with HCMV particles on cell surfaces and in endosomes. CD147 also promoted cell-cell fusion induced by expression of pentamer and gB in epithelial cells. However, soluble CD147 did not block HCMV entry and trimer and pentamer did not bind directly to CD147, supporting the hypothesis that CD147 acts indirectly through other proteins. CD147 represents the first HCMV entry mediator that specifically functions to promote entry of pentamer-expressing HCMV into epithelial and endothelial cells. PMID:29739904

SMAD1 and SMAD5 Expression Is Coordinately Regulated by FLI1 and GATA2 during Endothelial Development.

PubMed

Marks-Bluth, Jonathon; Khanna, Anchit; Chandrakanthan, Vashe; Thoms, Julie; Bee, Thomas; Eich, Christina; Kang, Young Chan; Knezevic, Kathy; Qiao, Qiao; Fitch, Simon; Oxburgh, Leif; Ottersbach, Katrin; Dzierzak, Elaine; de Bruijn, Marella F T R; Pimanda, John E

2015-06-01

The bone morphogenetic protein (BMP)/SMAD signaling pathway is a critical regulator of angiogenic sprouting and is involved in vascular development in the embryo. SMAD1 and SMAD5, the core mediators of BMP signaling, are vital for this activity, yet little is known about their transcriptional regulation in endothelial cells. Here, we have integrated multispecies sequence conservation, tissue-specific chromatin, in vitro reporter assay, and in vivo transgenic data to identify and validate Smad1+63 and the Smad5 promoter as tissue-specific cis-regulatory elements that are active in the developing endothelium. The activity of these elements in the endothelium was dependent on highly conserved ETS, GATA, and E-box motifs, and chromatin immunoprecipitation showed high levels of enrichment of FLI1, GATA2, and SCL at these sites in endothelial cell lines and E11 dorsal aortas in vivo. Knockdown of FLI1 and GATA2 but not SCL reduced the expression of SMAD1 and SMAD5 in endothelial cells in vitro. In contrast, CD31(+) cKit(-) endothelial cells harvested from embryonic day 9 (E9) aorta-gonad-mesonephros (AGM) regions of GATA2 null embryos showed reduced Smad1 but not Smad5 transcript levels. This is suggestive of a degree of in vivo selection where, in the case of reduced SMAD1 levels, endothelial cells with more robust SMAD5 expression have a selective advantage. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Specific Binding, Uptake, and Transport of ICAM-1-Targeted Nanocarriers Across Endothelial and Subendothelial Cell Components of the Blood-Brain Barrier

PubMed Central

Hsu, Janet; Rappaport, Jeff; Muro, Silvia

2014-01-01

Purpose The blood-brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain endothelial cells (ECs) helps transporting drugs and carriers into and across this barrier. While most receptors tested are associated with clathrin-mediated pathways, clathrin-independent routes are rather unexplored. We have examined the potential for one of these pathways, cell adhesion molecule (CAM)-mediated endocytosis induced by targeting intercellular adhesion molecule 1 (ICAM-1), to transport drug carriers into and across BBB models. Methods Model polymer nanocarriers (NCs) coated with control IgG or antibodies against ICAM-1 (IgG NCs vs. anti-ICAM NCs; ~250-nm) were incubated with human brain ECs, astrocytes (ACs), or pericytes (PCs) grown as monocultures or bilayered (endothelial+subendothelial) co-cultures. Results ICAM-1 was present and overexpressed in disease-like conditions on ECs and, at a lesser extent, on ACs and PCs which are BBB subendothelial components. Specific targeting and CAM-mediated uptake of anti-ICAM NCs occurred in these cells, although this was greater for ECs. Anti-ICAM NCs were transported across endothelial monolayers and endothelial+subendothelial co-cultures modeling the BBB. Conclusions CAM-mediated transport induced by ICAM-1 targeting operates in endothelial and subendothelial cellular components of the BBB, which may provide an avenue to overcome this barrier. PMID:24558007

Specific binding, uptake, and transport of ICAM-1-targeted nanocarriers across endothelial and subendothelial cell components of the blood-brain barrier.

PubMed

Hsu, Janet; Rappaport, Jeff; Muro, Silvia

2014-07-01

The blood-brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain endothelial cells (ECs) helps transport drugs and carriers into and across this barrier. While most receptors tested are associated with clathrin-mediated pathways, clathrin-independent routes are rather unexplored. We have examined the potential for one of these pathways, cell adhesion molecule (CAM)-mediated endocytosis induced by targeting intercellular adhesion molecule -1 (ICAM-1), to transport drug carriers into and across BBB models. Model polymer nanocarriers (NCs) coated with control IgG or antibodies against ICAM-1 (IgG NCs vs. anti-ICAM NCs; ~250-nm) were incubated with human brain ECs, astrocytes (ACs), or pericytes (PCs) grown as monocultures or bilayered (endothelial+subendothelial) co-cultures. ICAM-1 was present and overexpressed in disease-like conditions on ECs and, at a lesser extent, on ACs and PCs which are BBB subendothelial components. Specific targeting and CAM-mediated uptake of anti-ICAM NCs occurred in these cells, although this was greater for ECs. Anti-ICAM NCs were transported across endothelial monolayers and endothelial+subendothelial co-cultures modeling the BBB. CAM-mediated transport induced by ICAM-1 targeting operates in endothelial and subendothelial cellular components of the BBB, which may provide an avenue to overcome this barrier.

Evaluation of RGD-targeted albumin carriers for specific delivery of auristatin E to tumor blood vessels.

PubMed

Temming, Kai; Meyer, Damon L; Zabinski, Roger; Dijkers, Eli C F; Poelstra, Klaas; Molema, Grietje; Kok, Robbert J

2006-01-01

Induction of apoptosis in endothelial cells is considered an attractive strategy to therapeutically interfere with a solid tumor's blood supply. In the present paper, we constructed cytotoxic conjugates that specifically target angiogenic endothelial cells, thus preventing typical side effects of apoptosis-inducing drugs. For this purpose, we conjugated the potent antimitotic agent monomethyl-auristatin-E (MMAE) via a lysosomal cleavable linker to human serum albumin (HSA) and further equipped this drug-albumin conjugate with cyclic c(RGDfK) peptides for multivalent interaction with alphavbeta3-integrin. The RGD-peptides were conjugated via either an extended poly(ethylene glycol) linker or a short alkyl linker. The resulting drug-targeting conjugates RGDPEG-MMAE-HSA and RGD-MMAE-HSA demonstrated high binding affinity and specificity for alphavbeta3-integrin expressing human umbilical vein endothelial cells (HUVEC). Both types of conjugates were internalized by endothelial cells and killed the target cells at low nM concentrations. Furthermore, we observed RGD-dependent binding of the conjugates to C26 carcinoma. Upon i.v. administration to C26-tumor bearing mice, both drug-targeting conjugates displayed excellent tumor homing properties. Our results demonstrate that RGD-modified albumins are suitable carriers for cell selective intracellular delivery of cytotoxic compounds, and further studies will be conducted to assess the antivascular and tumor inhibitory potential of RGDPEG-MMAE-HSA and RGD-MMAE-HSA.

[Study on the specific immunity induced by dendritic cell vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell in mice].

PubMed

Zhao, Jun; Lu, Jing; Liu, Ya-qin; Yang, Hong-yan; Huang, You-tian; Zhao, Ji-min; Li, Shan; Zhai, Jing-ming; Zhao, Ming-yao; Zhang, Xi; Dong, Zi-ming

2011-01-01

To explore the specific cellular and humoral immunity induced by dendritic cells (DC) vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell of mice. Mouse brain microvascular endothelial cell bEnd.3 was cultured and identified for preparation endothelial cell bEnd.3 antigen. The level of mRNA expression of vascular endothelial growth factor receptor 2 (VEGF-R₂) and integrin αV was detected by reverse transcription (RT)-PCR. The BALB/c mice were immuned with DC loading bEnd.3 antigen 4 times in 4 weeks (bEnd.3-DC group), while the mice only were immuned with DC or injected with phosphate buffer saline (PBS group) as control group. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, cytotoxic T lymphocyte (CTL) response of spleen lymphocytes in vitro, the percentage of CD₃+CD₈+ surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and western blot. The expression of VEGF-R₂ and integrin αV gene in bEnd.3 cells were expressed highly. After the vaccine was injected, the tumors of mice in PBS group grew faster than those in other groups, while the tumors in bEnd.3-DC group grew slowly and disappeared after 2 weeks. The volume of tumors in DC group grew slower than those in PBS group [(0.11 ± 0.13) cm³ versus (3.38 ± 0.34) cm³]. The CTL response of spleen lymphocytes in vitro showed that bEnd.3-DC cells could kill bEnd.3 cells, the special lysis rate was more than 60%. The percentage of CD₃+CD₈+ spleen lymphocytes in bEnd.3-DC group [(38.6 ± 0.7)%] was higher than those in other groups (P < 0.05). The titer of serum antibody of bEnd.3-DC group was 1:3200, while it was 1:800 in DC group and there were not any in PBS group. Immunocytochemistry analysis indicated there were specific antigen-antibody reaction to bEnd.3 cell in bEnd.3-DC group. Western blot analysis revealed that there were specific bands at 220,000 (VEGF-R₂). bEnd.3-DC vaccine can inhibit the tumor growth of U14 cervical cancer cell of mice, which indicates that the special cellular and humoral immunity are induced by bEnd.3-DC antigen which maybe have some antigens in bEnd.3 cells that reacts with endothelial cell proliferation-related antigens.

SRF selectively controls tip cell invasive behavior in angiogenesis.

PubMed

Franco, Claudio A; Blanc, Jocelyne; Parlakian, Ara; Blanco, Raquel; Aspalter, Irene M; Kazakova, Natalia; Diguet, Nicolas; Mylonas, Elena; Gao-Li, Jacqueline; Vaahtokari, Anne; Penard-Lacronique, Virgine; Fruttiger, Markus; Rosewell, Ian; Mericskay, Mathias; Gerhardt, Holger; Li, Zhenlin

2013-06-01

Efficient angiogenic sprouting is essential for embryonic, postnatal and tumor development. Serum response factor (SRF) is known to be important for embryonic vascular development. Here, we studied the effect of inducible endothelial-specific deletion of Srf in postnatal and adult mice. We find that endothelial SRF activity is vital for postnatal growth and survival, and is equally required for developmental and pathological angiogenesis, including during tumor growth. Our results demonstrate that SRF is selectively required for endothelial filopodia formation and cell contractility during sprouting angiogenesis, but seems dispensable for vascular remodeling. At the molecular level, we observe that vascular endothelial growth factor A induces nuclear accumulation of myocardin-related transcription factors (MRTFs) and regulates MRTF/SRF-dependent target genes including Myl9, which is important for endothelial cell migration in vitro. We conclude that SRF has a unique function in regulating migratory tip cell behavior during sprouting angiogenesis. We hypothesize that targeting the SRF pathway could provide an opportunity to selectively target tip cell filopodia-driven angiogenesis to restrict tumor growth.

VEGF-C and TGF-β reciprocally regulate mesenchymal stem cell commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes.

PubMed

Igarashi, Yasuyuki; Chosa, Naoyuki; Sawada, Shunsuke; Kondo, Hisatomo; Yaegashi, Takashi; Ishisaki, Akira

2016-04-01

The direction of mesenchymal stem cell (MSC) differentiation is regulated by stimulation with various growth factors and cytokines. We recently established MSC lines, [transforming growth factor-β (TGF-β)-responsive SG‑2 cells, bone morphogenetic protein (BMP)-responsive SG‑3 cells, and TGF-β/BMP-non-responsive SG‑5 cells], derived from the bone marrow of green fluorescent protein-transgenic mice. In this study, to compare gene expression profiles in these MSC lines, we used DNA microarray analysis to characterize the specific gene expression profiles observed in the TGF-β-responsive SG‑2 cells. Among the genes that were highly expressed in the SG‑2 cells, we focused on vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), the gene product of FMS-like tyrosine kinase 4 (Flt4). We found that VEGF-C, a specific ligand of VEGFR3, significantly induced the cell proliferative activity, migratory ability (as shown by Transwell migration assay), as well as the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the SG‑2 cells. Additionally, VEGF-C significantly increased the expression of prospero homeobox 1 (Prox1) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), which are lymphatic endothelial cell markers, and decreased the expression of osteogenic differentiation marker genes in these cells. By contrast, TGF-β significantly increased the expression of early-phase osteogenic differentiation marker genes in the SG‑2 cells and markedly decreased the expression of lymphatic endothelial cell markers. The findings of our study strongly suggest the following: i) that VEGF-C promotes the proliferative activity and migratory ability of MSCs; and ii) VEGF-C and TGF-β reciprocally regulate MSC commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes, respectively. Our findings provide new insight into the molecular mechanisms underlying the regenerative ability of MSCs.

Iron ion irradiation increases promotes adhesion of monocytic cells to arterial vascular endothelium

NASA Astrophysics Data System (ADS)

Kucik, Dennis; Khaled, Saman; Gupta, Kiran; Wu, Xing; Yu, Tao; Chang, Polly; Kabarowski, Janusz

Radiation causes inflammation, and chronic, low-level vascular inflammation is a risk factor for atherosclerosis. Consistent with this, exposure to radiation from a variety of sources is associated with increased risk of heart disease and stroke. Part of the inflammatory response to radiation is a change in the adhesiveness of the endothelial cells that line the blood vessels, triggering inappropriate accumulation of leukocytes, leading to later, damaging effects of inflammation. Although some studies have been done on the effects of gamma irradiation on vascular endothelium, the response of endothelium to heavy ion radiation likely to be encountered in prolonged space flight has not been determined. We investigated how irradiation of aortic endothelial cells with iron ions affects adhesiveness of cultured aortic endothelial cells for monocytic cells and the consequences of this for development of atherosclerosis. Aortic endothelial cells were irradiated with 600 MeV iron ions at Brookhaven National Laboratory and adhesion-related changes were measured. Cells remained viable for at least 72 hours, and were even able to repair acute damage to cell junctions. We found that iron ion irradiation altered expression levels of specific endothelial cell adhesion molecules. Further, these changes had functional consequences. Using a flow chamber adhesion assay to measure adhesion of monocytic cells to endothelial cells under physiological shear stress, we found that adhesivity of vascular endothelium was enhanced in as little as 24 hours after irradiation. Further, the radiation dose dependence was not monotonic, suggesting that it was not simply the result of endothelial cell damage. We also irradiated aortic arches and carotid arteries of Apolipoprotein-E-deficient mice. Histologic analysis of these mice will be conducted to determine whether effects of radiation on endothelial adhesiveness result in consequences for development of atherosclerosis. (Supported by NSBRI: NCC-9-58-162)

Vascular Repair by Circumferential Cell Therapy Using Magnetic Nanoparticles and Tailored Magnets.

PubMed

Vosen, Sarah; Rieck, Sarah; Heidsieck, Alexandra; Mykhaylyk, Olga; Zimmermann, Katrin; Bloch, Wilhelm; Eberbeck, Dietmar; Plank, Christian; Gleich, Bernhard; Pfeifer, Alexander; Fleischmann, Bernd K; Wenzel, Daniela

2016-01-26

Cardiovascular disease is often caused by endothelial cell (EC) dysfunction and atherosclerotic plaque formation at predilection sites. Also surgical procedures of plaque removal cause irreversible damage to the EC layer, inducing impairment of vascular function and restenosis. In the current study we have examined a potentially curative approach by radially symmetric re-endothelialization of vessels after their mechanical denudation. For this purpose a combination of nanotechnology with gene and cell therapy was applied to site-specifically re-endothelialize and restore vascular function. We have used complexes of lentiviral vectors and magnetic nanoparticles (MNPs) to overexpress the vasoprotective gene endothelial nitric oxide synthase (eNOS) in ECs. The MNP-loaded and eNOS-overexpressing cells were magnetic, and by magnetic fields they could be positioned at the vascular wall in a radially symmetric fashion even under flow conditions. We demonstrate that the treated vessels displayed enhanced eNOS expression and activity. Moreover, isometric force measurements revealed that EC replacement with eNOS-overexpressing cells restored endothelial function after vascular injury in eNOS(-/-) mice ex and in vivo. Thus, the combination of MNP-based gene and cell therapy with custom-made magnetic fields enables circumferential re-endothelialization of vessels and improvement of vascular function.

Traction force dynamics predict gap formation in activated endothelium

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

Valent, Erik T.; Nieuw Amerongen, Geerten P. van; Hinsbergh, Victor W.M. van

In many pathological conditions the endothelium becomes activated and dysfunctional, resulting in hyperpermeability and plasma leakage. No specific therapies are available yet to control endothelial barrier function, which is regulated by inter-endothelial junctions and the generation of acto-myosin-based contractile forces in the context of cell-cell and cell-matrix interactions. However, the spatiotemporal distribution and stimulus-induced reorganization of these integral forces remain largely unknown. Traction force microscopy of human endothelial monolayers was used to visualize contractile forces in resting cells and during thrombin-induced hyperpermeability. Simultaneously, information about endothelial monolayer integrity, adherens junctions and cytoskeletal proteins (F-actin) were captured. This revealed a heterogeneousmore » distribution of traction forces, with nuclear areas showing lower and cell-cell junctions higher traction forces than the whole-monolayer average. Moreover, junctional forces were asymmetrically distributed among neighboring cells. Force vector orientation analysis showed a good correlation with the alignment of F-actin and revealed contractile forces in newly formed filopodia and lamellipodia-like protrusions within the monolayer. Finally, unstable areas, showing high force fluctuations within the monolayer were prone to form inter-endothelial gaps upon stimulation with thrombin. To conclude, contractile traction forces are heterogeneously distributed within endothelial monolayers and force instability, rather than force magnitude, predicts the stimulus-induced formation of intercellular gaps. - Highlights: • Endothelial monolayers exert dynamic- and heterogeneous traction forces. • High traction forces correlate with junctional areas and the F-actin cytoskeleton. • Newly formed inter-endothelial gaps are characterized by opposing traction forces. • Force stability is a key feature controlling endothelial permeability.« less

JunB is required for endothelial cell morphogenesis by regulating core-binding factor β

PubMed Central

Licht, Alexander H.; Pein, Oliver T.; Florin, Lore; Hartenstein, Bettina; Reuter, Hendrik; Arnold, Bernd; Lichter, Peter; Angel, Peter; Schorpp-Kistner, Marina

2006-01-01

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis. PMID:17158955

Genetics Home Reference: Fuchs endothelial dystrophy

MedlinePlus

... a protein that is part of type VIII collagen. Type VIII collagen is largely found within the cornea, surrounding the endothelial cells. Specifically, type VIII collagen is a major component of a tissue at ...

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.

EGF and hydrocortisone as critical factors for the co-culture of adipogenic differentiated ASCs and endothelial cells.

PubMed

Volz, Ann-Cathrin; Huber, Birgit; Schwandt, Alina Maria; Kluger, Petra Juliane

In vitro composed vascularized adipose tissue is and will continue to be in great demand e.g. for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Up to date, the lack of adequate culture conditions, mainly a culture medium, decelerates further achievements. In our study, we evaluated the influence of epidermal growth factor (EGF) and hydrocortisone (HC), often supplemented in endothelial cell (EC) specific media, on the co-culture of adipogenic differentiated adipose-derived stem cells (ASCs) and microvascular endothelial cells (mvECs). In ASCs, EGF and HC are thought to inhibit adipogenic differentiation and have lipolytic activities. Our results showed that in indirect co-culture for 14 days, adipogenic differentiated ASCs further incorporated lipids and partly gained an univacuolar morphology when kept in media with low levels of EGF and HC. In media with high EGF and HC levels, cells did not incorporate further lipids, on the contrary, cells without lipid droplets appeared. Glycerol release, to measure lipolysis, also increased with elevated amounts of EGF and HC in the culture medium. Adipogenic differentiated ASCs were able to release leptin in all setups. MvECs were functional and expressed the cell specific markers, CD31 and von Willebrand factor (vWF), independent of the EGF and HC content as long as further EC specific factors were present. Taken together, our study demonstrates that adipogenic differentiated ASCs can be successfully co-cultured with mvECs in a culture medium containing low or no amounts of EGF and HC, as long as further endothelial cell and adipocyte specific factors are available. Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerisation.

PubMed

Cai, Shijie; Alp, Nicholas J; McDonald, Denise; Smith, Ian; Kay, Jonathan; Canevari, Laura; Heales, Simon; Channon, Keith M

2002-09-01

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent, eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS-GFP fusion protein were significantly increased following GTPCH gene transfer. These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be a rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states.

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.

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

NASA Technical Reports Server (NTRS)

Chen, Chu-Huang; Pellis, Neal R.

1997-01-01

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

Differential Activation of Acid Sphingomyelinase and Ceramide Release Determines Invasiveness of Neisseria meningitidis into Brain Endothelial Cells

PubMed Central

Simonis, Alexander; Hebling, Sabrina; Gulbins, Erich; Schneider-Schaulies, Sibylle; Schubert-Unkmeir, Alexandra

2014-01-01

The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains. PMID:24945304

Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension

PubMed Central

Wagner, Charlotte; Jobs, Alexander; Schweda, Frank; Kurtz, Lisa; Kurt, Birguel; Sequeira Lopez, Maria L.; Gomez, R. Ariel; van Veen, Toon A.B.; de Wit, Cor; Kurtz, Armin

2011-01-01

Renin-producing juxtaglomerular cells are connected to each other and to endothelial cells of afferent arterioles by gap junctions containing Connexin 40 (Cx40), abundantly expressed by these two cell types. Here, we generated mice with cell-specific deletion of Cx40 in endothelial and in renin-producing cells, as its global deletion caused local dissociation of renin-producing cells from endothelial cells, renin hypersecretion, and hypertension. In mice lacking endothelial Cx40, the blood pressure, renin-producing cell distribution, and the control of renin secretion were similar to wild-type mice. In contrast, mice deficient for Cx40 in renin-producing cells were hypertensive and these cells were ectopically localized. Although plasma renin activity and kidney renin mRNA levels of these mice were not different from controls, the negative regulation of renin secretion by pressure was inverted to a positive feedback in kidneys lacking Cx40 in renin-producing cells. Thus, our findings show that endothelial Cx40 is not essential for the control of renin expression and/or release. Cx40 in renin-producing cells is required for their correct positioning in the juxtaglomerular area and the control of renin secretion by pressure. PMID:20686449

Myocardial pressure overload induces systemic inflammation through endothelial cell IL-33

PubMed Central

Chen, Wei-Yu; Hong, Jaewoo; Gannon, Joseph; Kakkar, Rahul; Lee, Richard T.

2015-01-01

Hypertension increases the pressure load on the heart and is associated with a poorly understood chronic systemic inflammatory state. Interleukin 33 (IL-33) binds to membrane-bound ST2 (ST2L) and has antihypertrophic and antifibrotic effects in the myocardium. In contrast, soluble ST2 appears to act as a decoy receptor for IL-33, blocking myocardial and vascular benefits, and is a prognostic biomarker in patients with cardiovascular diseases. Here we report that a highly local intramyocardial IL-33/ST2 conversation regulates the heart’s response to pressure overload. Either endothelial-specific deletion of IL33 or cardiomyocyte-specific deletion of ST2 exacerbated cardiac hypertrophy with pressure overload. Furthermore, pressure overload induced systemic circulating IL-33 as well as systemic circulating IL-13 and TGF-beta1; this was abolished by endothelial-specific deletion of IL33 but not by cardiomyocyte-specific deletion of IL33. Our study reveals that endothelial cell secretion of IL-33 is crucial for translating myocardial pressure overload into a selective systemic inflammatory response. PMID:25941360

Transcriptional Responses of Candida albicans to Epithelial and Endothelial Cells▿ †

PubMed Central

Park, Hyunsook; Liu, Yaoping; Solis, Norma; Spotkov, Joshua; Hamaker, Jessica; Blankenship, Jill R.; Yeaman, Michael R.; Mitchell, Aaron P.; Liu, Haoping; Filler, Scott G.

2009-01-01

Candida albicans interacts with oral epithelial cells during oropharyngeal candidiasis and with vascular endothelial cells when it disseminates hematogenously. We set out to identify C. albicans genes that govern interactions with these host cells in vitro. The transcriptional response of C. albicans to the FaDu oral epithelial cell line and primary endothelial cells was determined by microarray analysis. Contact with epithelial cells caused a decrease in transcript levels of genes related to protein synthesis and adhesion, whereas contact with endothelial cells did not significantly influence any specific functional category of genes. Many genes whose transcripts were increased in response to either host cell had not been previously characterized. We constructed mutants with homozygous insertions in 22 of these uncharacterized genes to investigate their function during host-pathogen interaction. By this approach, we found that YCK2, VPS51, and UEC1 are required for C. albicans to cause normal damage to epithelial cells and resist antimicrobial peptides. YCK2 is also necessary for maintenance of cell polarity. VPS51 is necessary for normal vacuole formation, resistance to multiple stressors, and induction of maximal endothelial cell damage. UEC1 encodes a unique protein that is required for resistance to cell membrane stress. Therefore, some C. albicans genes whose transcripts are increased upon contact with epithelial or endothelial cells are required for the organism to damage these cells and withstand the stresses that it likely encounters during growth in the oropharynx and bloodstream. PMID:19700637

Immobilization of DNA aptamers via plasma polymerized allylamine film to construct an endothelial progenitor cell-capture surface.

PubMed

Qi, Pengkai; Yan, Wei; Yang, Ying; Li, Yalong; Fan, Yi; Chen, Junying; Yang, Zhilu; Tu, Qiufen; Huang, Nan

2015-02-01

The endothelial progenitor cells (EPCs) capture stent has drawn increasing attentions and become one of the most promising concepts for the next generation vascular stent. In this regard, it is of great significance to immobilize a molecule with the ability to bind EPC for rapid in vivo endothelialization with high specificity. In this work, a facile two-step method aimed at constructing a coating with specific EPC capturing aptamers is reported. The processes involves as the first-step deposition of plasma polymerized allylamine (PPAam) on a substrate to introduce amine groups, followed by the electrostatic adsorption of a 34 bases single strand DNA sequence to the PPAam surface as a second step (PPAam-DNA). Grazing incidence attenuated total reflection Fourier transform infrared spectroscopy (GATR-FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed the successful immobilization of the aptamers. Quartz crystal microbalance with dissipation (QCM-D) real time monitoring result shows that about 175 ng/cm(2) aptamers were conjugated onto the PPAam surface. The interactions between the modified surfaces and human umbilical vein endothelial cells (ECs), smooth muscle cells (SMCs), and murine induced EPCs derived from mesenchymal stem cells (MSCs) were also investigated. It was demonstrated that PPAam-DNA samples could capture more EPCs, and present a cellular friendly surface for the proliferation of both EPCs and ECs but no effect on the hyperplasia of SMCs. Also, the co-culture results of 3 types of cells confirmed that the aptamer could specifically bond EPCs rather than ECs and SMCs, suggesting the competitive adhesion advantage of EPCs to ECs and SMCs. These data demonstrate that the EPC aptamer has large potential for designing an EPC captured stent and other vascular grafts with targeted in situ endothelialization. Copyright © 2014 Elsevier B.V. All rights reserved.

Regulator of Calcineurin 1 in Periodontal Disease

PubMed Central

Peters, Ulrike; Solominidou, Eleni; Korkmaz, Yüksel; Rüttermann, Stefan; Klocke, Astrid; Flemmig, Thomas Frank; Beikler, Thomas

2016-01-01

Nuclear factor of activated T-cells (NFAT) and NF-kB pathway associated processes are involved in the pathogenesis of various inflammatory disorders, for example, periodontal disease. The activation of these pathways is controlled by the regulator of calcineurin 1 (RCAN1). The aim of this study was to elucidate the role of RCAN1 in periodontal disease. Healthy and inflamed periodontal tissues were analyzed by immunohistochemistry and immunofluorescence using specific rabbit polyclonal anti-RCAN1 antibodies. For expression analysis human umbilical vein endothelial cells (HUVEC) were used. HUVEC were incubated for 2 h with Vascular Endothelial Growth Factor (VEGF) or with wild type and laboratory strains of Porphyromonas gingivalis (P. gingivalis). Expression analysis of rcan1 and cox2 was done by real time PCR using specific primers for rcan1.4 and cox2. The expression of rcan1 was found to be significantly suppressed in endothelial cells of chronically inflamed periodontal tissues compared to healthy controls. Rcan1 and cox2 were significantly induced by VEGF and wild type and laboratory P. gingivalis strains. Interestingly, the magnitude of the rcan1 and cox2 induction was strain dependent. The results of this study indicate that RCAN1 is suppressed in endothelial cells of chronically inflamed periodontal tissues. During an acute infection, however, rcan1 seems to be upregulated in endothelial cells, indicating a modulating role in immune homeostasis of periodontal tissues. PMID:27403036

Gene Electrotransfer of Plasmid with Tissue Specific Promoter Encoding shRNA against Endoglin Exerts Antitumor Efficacy against Murine TS/A Tumors by Vascular Targeted Effects.

PubMed

Stimac, Monika; Dolinsek, Tanja; Lampreht, Ursa; Cemazar, Maja; Sersa, Gregor

2015-01-01

Vascular targeted therapies, targeting specific endothelial cell markers, are promising approaches for the treatment of cancer. One of the targets is endoglin, transforming growth factor-β (TGF-β) co-receptor, which mediates proliferation, differentiation and migration of endothelial cells forming neovasculature. However, its specific, safe and long-lasting targeting remains the challenge. Therefore, in our study we evaluated the transfection efficacy, vascular targeted effects and therapeutic potential of the plasmid silencing endoglin with the tissue specific promoter, specific for endothelial cells marker endothelin-1 (ET) (TS plasmid), in comparison to the plasmid with constitutive promoter (CON plasmid), in vitro and in vivo. Tissue specificity of TS plasmid was demonstrated in vitro on several cell lines, and its antiangiogenic efficacy was demonstrated by reducing tube formation of 2H11 endothelial cells. In vivo, on a murine mammary TS/A tumor model, we demonstrated good antitumor effect of gene electrotransfer (GET) of either of both plasmids in treatment of smaller tumors still in avascular phase of growth, as well as on bigger tumors, already well vascularized. In support to the observations on predominantly vascular targeted effects of endoglin, histological analysis has demonstrated an increase in necrosis and a decrease in the number of blood vessels in therapeutic groups. A significant antitumor effect was observed in tumors in avascular and vascular phase of growth, possibly due to both, the antiangiogenic and the vascular disrupting effect. Furthermore, the study indicates on the potential use of TS plasmid in cancer gene therapy since the same efficacy as of CON plasmid was determined.

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

Tong, Meng, E-mail: tong.59@osu.edu; Han, Byungdo B.; Holpuch, Andrew S.

The presence of the EMT (epithelial-mesenchymal transition), EndMT (endothelial-mesenchymal transition) and VM (vasculogenic mimicry) demonstrates the multidirectional extent of phenotypic plasticity in cancers. Previous findings demonstrating the crosstalk between head and neck squamous cell carcinoma (HNSCC) and vascular endothelial growth factor (VEGF) imply that HNSCC cells share some functional commonalities with endothelial cells. Our current results reveal that cultured HNSCC cells not only possess endothelial-specific markers, but also display endotheliod functional features including low density lipoprotein uptake, formation of tube-like structures on Matrigel and growth state responsiveness to VEGF and endostatin. HNSCC cell subpopulations are also highly responsive to transformingmore » growth factor-β1 and express its auxiliary receptor, endoglin. Furthermore, the endotheliod characteristics observed in vitro recapitulate phenotypic features observed in human HNSCC tumors. Conversely, cultured normal human oral keratinocytes and intact or ulcerated human oral epithelia do not express comparable endotheliod characteristics, which imply that assumption of endotheliod features is restricted to transformed keratinocytes. In addition, this phenotypic state reciprocity facilitates HNSCC progression by increasing production of factors that are concurrently pro-proliferative and pro-angiogenic, conserving cell energy stores by LDL internalization and enhancing cell mobility. Finally, recognition of this endotheliod phenotypic transition provides a solid rationale to evaluate the antitumorigenic potential of therapeutic agents formerly regarded as exclusively angiostatic in scope. - Highlights: ► HNSCC tumor cells express endothelial specific markers VE-cadherin, CD31 and vimentin. ► Similarly, cultured HNSCC cells retain expression of these markers. ► HNSCC cells demonstrate functional endotheliod characteristics i.e. AcLDL uptake. ► HNSCC cell subpopulations are highly responsive to TGF- β1, VEGF and endostatin. ► TGF-β1 facilitates cadherin switching and augments invasiveness of HNSCC subpopulations.« less

The chemokine receptor CX3CR1 coordinates monocyte recruitment and endothelial regeneration after arterial injury.

PubMed

Getzin, Tobias; Krishnasamy, Kashyap; Gamrekelashvili, Jaba; Kapanadze, Tamar; Limbourg, Anne; Häger, Christine; Napp, L Christian; Bauersachs, Johann; Haller, Hermann; Limbourg, Florian P

2018-02-01

Regeneration of arterial endothelium after injury is critical for the maintenance of normal blood flow, cell trafficking, and vascular function. Using mouse models of carotid injury, we show that the transition from a static to a dynamic phase of endothelial regeneration is marked by a strong increase in endothelial proliferation, which is accompanied by induction of the chemokine CX 3 CL1 in endothelial cells near the wound edge, leading to progressive recruitment of Ly6C lo monocytes expressing high levels of the cognate CX 3 CR1 chemokine receptor. In Cx3cr1 -deficient mice recruitment of Ly6C lo monocytes, endothelial proliferation and regeneration of the endothelial monolayer after carotid injury are impaired, which is rescued by acute transfer of normal Ly6C lo monocytes. Furthermore, human non-classical monocytes induce proliferation of endothelial cells in co-culture experiments in a VEGFA-dependent manner, and monocyte transfer following carotid injury promotes endothelial wound closure in a hybrid mouse model in vivo Thus, CX 3 CR1 coordinates recruitment of specific monocyte subsets to sites of endothelial regeneration, which promote endothelial proliferation and arterial regeneration. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Chemokines and skin diseases.

PubMed

Sugaya, Makoto

2015-04-01

Chemokines are small molecules that induce chemotaxis and activation of certain subsets of leukocytes. The expression patterns of chemokines and chemokine receptors are specific to certain organs and cells. Therefore, chemokines are important to elucidate the mechanism of organ-specific human diseases. CCL17 expressed by Langerhans cells, blood endothelial cells, and fibroblasts plays a key role in attracting Th2 cells and tumor cells of adult T-cell leukemia/lymphoma and mycosis fungoides/Sézary syndrome into the skin, developing various Th2-type inflammatory skin diseases as well as cutaneous lymphoma. CCL11 and CCL26 expressed by skin-resident cells, such as fibroblasts, blood endothelial cells, and keratinocytes, induce infiltration of CCR3-expressing cells such as Th2 cells and eosinophils. CCL11 may also serve as an autocrine as well as a paracrine in anaplastic large cell lymphoma. CX3CL1 expressed on blood endothelial cells leads to infiltration of CX3CR1(+) immune cells, such as mast cells, neutrophils, and macrophages, playing important roles in wound healing, tumor immunity, and vasculitis. Biologics targeting chemokines and their receptors are promising strategies for various skin diseases that are resistant to the current therapy.

Asbestos-induced endothelial cell activation and injury. Demonstration of fiber phagocytosis and oxidant-dependent toxicity.

PubMed

Garcia, J G; Gray, L D; Dodson, R F; Callahan, K S

1988-10-01

Vascular endothelial cell injury is important in the development of a variety of chronic interstitial lung disorders. However, the involvement of such injury in the inflammatory response associated with the inhalation of asbestos fibers is unclear and the mechanism of asbestos fiber cytotoxicity remains unknown. In the present study, human umbilical vein endothelial cells were challenged with amosite asbestos and several parameters of cellular function were examined. Electron microscopic examination revealed that endothelial cell exposure to asbestos resulted in active phagocytosis of these particulates. Biochemical evidence of dose-dependent asbestos-mediated endothelial cell activation was indicated by increased metabolism of arachidonic acid. For example, amosite asbestos (500 micrograms/ml) produced a ninefold increase in prostacyclin (PGI2) levels over those levels in non-exposed cells. Incubation of human endothelial cells with asbestos fibers induced specific 51Cr release in both a dose- and time-dependent fashion indicative of cellular injury. Injury induced by amosite asbestos was not significantly attenuated by treatment of the endothelial cell monolayer with either the iron chelator deferoxamine, which prevents hydroxyl radical (.OH) formation, or by the superoxide anion (O2-) scavenger, superoxide dismutase. However, significant dose-dependent protection was observed with the hydrogen peroxide (H2O2) scavenger, catalase. Chelation of elemental iron present within amosite asbestos fibers by deferoxamine produced a 33% reduction in asbestos cytotoxicity, suggesting a potential role for hydroxyl radical-mediated injury via the iron-catalyzed Haber-Weiss reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequence preferences to control cell-specific organization of endothelial cells, smooth muscle cells, and fibroblasts.

PubMed

Kanie, Kei; Kato, Ryuji; Zhao, Yingzi; Narita, Yuji; Okochi, Mina; Honda, Hiroyuki

2011-06-01

Effective surface modification with biocompatible molecules is known to be effective in reducing the life-threatening risks related to artificial cardiovascular implants. In recent strategies in regenerative medicine, the enhancement and support of natural repair systems at the site of injury by designed biocompatible molecules have succeeded in rapid and effective injury repair. Therefore, such a strategy could also be effective for rapid endothelialization of cardiovascular implants to lower the risk of thrombosis and stenosis. To achieve this enhancement of the natural repair system, a biomimetic molecule that mimics proper cellular organization at the implant location is required. In spite of the fact that many reported peptides have cell-attracting properties on material surfaces, there have been few peptides that could control cell-specific adhesion. For the advanced cardiovascular implants, peptides that can mimic the natural mechanism that controls cell-specific organization have been strongly anticipated. To obtain such peptides, we hypothesized the cellular bias toward certain varieties of amino acids and examined the cell preference (in terms of adhesion, proliferation, and protein attraction) of varieties and of repeat length on SPOT peptide arrays. To investigate the role of specific peptides in controlling the organization of various cardiovascular-related cells, we compared endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts (FBs). A clear, cell-specific preference was found for amino acids (longer than 5-mer) using three types of cells, and the combinational effect of the physicochemical properties of the residues was analyzed to interpret the mechanism. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis.

PubMed

Lee, Heon-Woo; Chong, Diana C; Ola, Roxana; Dunworth, William P; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L; Eichmann, Anne; Jin, Suk-Won

2017-04-01

Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2 , and Alk3 in mouse retinal vessels. Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2 . Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2 / acvr1 or Alk3 / Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels. © 2017 The Authors.

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein Induced Retinal Angiogenesis

PubMed Central

Lee, Heon-Woo; Chong, Diana C.; Ola, Roxana; Dunworth, William P.; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M.; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L.; Eichmann, Anne; Jin, Suk-Won

2017-01-01

Objective Increasing evidence suggests that Bone Morphogenetic Protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early post-natal angiogenesis by analysis of inducible, endothelial specific deletion of the BMP receptor components Bmpr2, Alk1, Alk2 and Alk3 in mouse retinal vessels. Approach and Results Expression analysis of several BMP ligands showed that pro-angiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of BMP Type 2 receptor (Bmpr2). Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branchpoints behind the front, leading to attenuated radial expansion. To identify critical BMPR1s associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of three BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for pro-angiogenic BMP signaling in retinal vessels. Conclusions Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential pro-angiogenic cue for retinal vessels. PMID:28232325

Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

NASA Astrophysics Data System (ADS)

Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

2016-11-01

Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

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.

Placental labyrinth formation in mice requires endothelial FLRT2/UNC5B signaling.

PubMed

Tai-Nagara, Ikue; Yoshikawa, Yusuke; Numata, Naoko; Ando, Tomofumi; Okabe, Keisuke; Sugiura, Yuki; Ieda, Masaki; Takakura, Nobuyuki; Nakagawa, Osamu; Zhou, Bin; Okabayashi, Koji; Suematsu, Makoto; Kitagawa, Yuko; Bastmeyer, Martin; Sato, Kohji; Klein, Rüdiger; Navankasattusas, Sutip; Li, Dean Y; Yamagishi, Satoru; Kubota, Yoshiaki

2017-07-01

The placental labyrinth is the interface for gas and nutrient exchange between the embryo and the mother; hence its proper development is essential for embryogenesis. However, the molecular mechanism underlying development of the placental labyrinth, particularly in terms of its endothelial organization, is not well understood. Here, we determined that fibronectin leucine-rich transmembrane protein 2 (FLRT2), a repulsive ligand of the UNC5 receptor family for neurons, is unexpectedly expressed in endothelial cells specifically in the placental labyrinth. Mice lacking FLRT2 in endothelial cells exhibited embryonic lethality at mid-gestation, with systemic congestion and hypoxia. Although they lacked apparent deformities in the embryonic vasculature and heart, the placental labyrinths of these embryos exhibited aberrant alignment of endothelial cells, which disturbed the feto-maternal circulation. Interestingly, this vascular deformity was related to endothelial repulsion through binding to the UNC5B receptor. Our results suggest that the proper organization of the placental labyrinth depends on coordinated inter-endothelial repulsion, which prevents uncontrolled layering of the endothelium. © 2017. Published by The Company of Biologists Ltd.

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

PubMed Central

Kraehling, Jan R.; Chidlow, John H.; Rajagopal, Chitra; Sugiyama, Michael G.; Fowler, Joseph W.; Lee, Monica Y.; Zhang, Xinbo; Ramírez, Cristina M.; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W.; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L.; Fernández-Hernando, Carlos; Sessa, William C.

2016-01-01

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL. PMID:27869117

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.

PubMed

Kraehling, Jan R; Chidlow, John H; Rajagopal, Chitra; Sugiyama, Michael G; Fowler, Joseph W; Lee, Monica Y; Zhang, Xinbo; Ramírez, Cristina M; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L; Fernández-Hernando, Carlos; Sessa, William C

2016-11-21

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

RON kinase inhibition reduces renal endothelial injury in sickle cell disease mice

PubMed Central

Khaibullina, Alfia; Adjei, Elena A.; Afangbedji, Nowah; Ivanov, Andrey; Kumari, Namita; Almeida, Luis E.F.; Quezado, Zenaide M.N.; Nekhai, Sergei; Jerebtsova, Marina

2018-01-01

Sickle cell disease patients are at increased risk of developing a chronic kidney disease. Endothelial dysfunction and inflammation associated with hemolysis lead to vasculopathy and contribute to the development of renal disease. Here we used a Townes sickle cell disease mouse model to examine renal endothelial injury. Renal disease in Townes mice was associated with glomerular hypertrophy, capillary dilation and congestion, and significant endothelial injury. We also detected substantial renal macrophage infiltration, and accumulation of macrophage stimulating protein 1 in glomerular capillary. Treatment of human cultured macrophages with hemin or red blood cell lysates significantly increased expression of macrophage membrane-associated protease that might cleave and activate circulating macrophage stimulating protein 1 precursor. Macrophage stimulating protein 1 binds to and activates RON kinase, a cell surface receptor tyrosine kinase. In cultured human renal glomerular endothelial cells, macrophage stimulating protein 1 induced RON downstream signaling, resulting in increased phosphorylation of ERK and AKT kinases, expression of Von Willebrand factor, increased cell motility, and re-organization of F-actin. Specificity of macrophage stimulating protein 1 function was confirmed by treatment with RON kinase inhibitor BMS-777607 that significantly reduced downstream signaling. Moreover, treatment of sickle cell mice with BMS-777607 significantly reduced glomerular hypertrophy, capillary dilation and congestion, and endothelial injury. Taken together, our findings demonstrated that RON kinase is involved in the induction of renal endothelial injury in sickle cell mice. Inhibition of RON kinase activation may provide a novel approach for prevention of the development of renal disease in sickle cell disease. PMID:29519868

Role of VPO1, a newly identified heme-containing peroxidase, in ox-LDL induced endothelial cell apoptosis

PubMed Central

Bai, Yong-Ping; Hu, Chang-Ping; Yuan, Qiong; Peng, Jun; Shi, Rui-Zheng; Yang, Tian-Lun; Cao, Ze-Hong; Li, Yuan-Jian; Cheng, Guangjie; Zhang, Guo-Gang

2013-01-01

Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91phox subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91phox subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91phox subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis. PMID:21820048

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

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

Wang, Shu-Hong; Nan, Ke-Jun, E-mail: nankj@163.com; Wang, Yao-Chun

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

Pravastatin Protects Against Avascular Necrosis of Femoral Head via Autophagy.

PubMed

Liao, Yun; Zhang, Ping; Yuan, Bo; Li, Ling; Bao, Shisan

2018-01-01

Autophagy serves as a stress response and may contribute to the pathogenesis of avascular necrosis of the femoral head induced by steroids. Statins promote angiogenesis and ameliorate endothelial functions through apoptosis inhibition and necrosis of endothelial progenitor cells, however the process used by statins to modulate autophagy in avascular necrosis of the femoral head remains unclear. This manuscript determines whether pravastatin protects against dexamethasone-induced avascular necrosis of the femoral head by activating endothelial progenitor cell autophagy. Pravastatin was observed to enhance the autophagy activity in endothelial progenitor cells, specifically by upregulating LC3-II/Beclin-1 (autophagy related proteins), and autophagosome formation in vivo and in vitro . An autophagy inhibitor, 3-MA, reduced pravastatin protection in endothelial progenitor cells exposed to dexamethasone by attenuating pravastatin-induced autophagy. Adenosine monophosphate-activated protein kinase (AMPK) is a key autophagy regulator by sensing cellular energy changes, and indirectly suppressing activation of the mammalian target of rapamycin (mTOR). We found that phosphorylation of AMPK was upregulated however phosphorylation of mTOR was downregulated in pravastatin-treated endothelial progenitor cells, which was attenuated by AMPK inhibitor compound C. Furthermore, liver kinase B1 (a phosphorylase of AMPK) knockdown eliminated pravastatin regulated autophagy protein LC3-II in endothelial progenitor cells in vitro . We therefore demonstrated pravastatin rescued endothelial progenitor cells from dexamethasone-induced autophagy dysfunction through the AMPK-mTOR signaling pathway in a liver kinase B1-dependent manner. Our results provide useful information for the development of novel therapeutics for management of glucocorticoids-induced avascular necrosis of the femoral head.

Alteration of human umbilical vein endothelial cell gene expression in different biomechanical environments.

PubMed

Shoajei, Shahrokh; Tafazzoli-Shahdpour, Mohammad; Shokrgozar, Mohammad Ali; Haghighipour, Nooshin

2014-05-01

Biomechanical environments affect the function of cells. In this study we analysed the effects of five mechanical stimuli on the gene expression of human umbilical vein endothelial cells (HUVECs) in mRNA level using real-time PCR. The following loading regimes were applied on HUVECs for 48 h: intermittent (0-5 dyn/cm(2) , 1 Hz) and uniform (5 dyn/cm(2) ) shear stresses concomitant by 10% intermittent equiaxial stretch (1 Hz), uniform shear stress alone (5 dyn/cm(2) ), and intermittent uniaxial and equiaxial stretches (10%, 1 Hz). A new bioreactor was made to apply uniform/cyclic shear and tensile loadings. Three endothelial suggestive specific genes (vascular endothelial growth factor receptor-2 (VEGFR-2, also known as FLK-1), von Willebrand Factor (vWF) and vascular endothelial-cadherin (VE-cadherin)), and two smooth muscle genes (α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SMMHC)) were chosen for assessment of alteration in gene expression of endothelial cells and transdifferentiation toward smooth cells following load applications. Shear stress alone enhanced the endothelial gene expression significantly, while stretching alone was identified as a transdifferentiating factor. Cyclic equiaxial stretch contributed less to elevation of smooth muscle genes compared to uniaxial stretch. Cyclic shear stress in comparison to uniform shear stress concurrent with cyclic stretch was more influential on promotion of endothelial genes expression. Influence of different mechanical stimuli on gene expression may open a wider horizon to regulate functions of cell for tissue engineering purposes. © 2013 International Federation for Cell Biology.

Pleiotropy of tissue-specific growth factors: from neurons to vessels via the bone marrow

PubMed Central

Duda, Dan G.; Jain, Rakesh K.

2005-01-01

Recent evidence has demonstrated that endothelial-specific growth factors affect the development of apparently unrelated organs and cells. Expanding this evidence further, new findings in this issue of the JCI show that neurotrophic factors can affect neovascularization. Neurotrophic factors achieve proangiogenic effects not only by directly affecting endothelial cells, but also by recruiting hematopoietic precursors. Further understanding of the biology of angiogenic factors, as well as of the function of hematopoietic cells in tissue neovascularization, will lead to improved therapeutic strategies for the treatment of diseases ranging from ischemia to cancer. PMID:15765145

Aldosterone Promotes Cardiac Endothelial Cell Proliferation In Vivo

PubMed Central

Gravez, Basile; Tarjus, Antoine; Pelloux, Véronique; Ouvrard‐Pascaud, Antoine; Delcayre, Claude; Samuel, Janelise; Clément, Karine; Farman, Nicolette; Jaisser, Fréderic; Messaoudi, Smail

2015-01-01

Background Experimentally, aldosterone in association with NaCl induces cardiac fibrosis, oxidative stress, and inflammation through mineralocorticoid receptor activation; however, the biological processes regulated by aldosterone alone in the heart remain to be identified. Methods and Results Mice were treated for 7 days with aldosterone, and then cardiac transcriptome was analyzed. Aldosterone regulated 60 transcripts (51 upregulated and 9 downregulated) in the heart (fold change ≥1.5, false discovery rate <0.01). To identify the biological processes modulated by aldosterone, a gene ontology analysis was performed. The majority of aldosterone‐regulated genes were involved in cell division. The cardiac Ki‐67 index (an index of proliferation) of aldosterone‐treated mice was higher than that of nontreated mice, confirming microarray predictions. Costaining of Ki‐67 with vinculin, CD68, α‐smooth muscle actin, CD31, or caveolin 1 revealed that the cycling cells were essentially endothelial cells. Aldosterone‐induced mineralocorticoid receptor–dependent proliferation was confirmed ex vivo in human endothelial cells. Moreover, pharmacological‐specific blockade of mineralocorticoid receptor by eplerenone inhibited endothelial cell proliferation in a preclinical model of heart failure (transverse aortic constriction). Conclusions Aldosterone modulates cardiac gene expression and induces the proliferation of cardiac endothelial cells in vivo. PMID:25564371

A Universal Aptamer Chimera for the Delivery of Functional microRNA-126.

PubMed

Rohde, Jan-H; Weigand, Julia E; Suess, Beatrix; Dimmeler, Stefanie

2015-06-01

microRNAs (miRs) regulate vascular diseases such as atherosclerosis and cancer. miR-126 is important for endothelial cell signaling and promotes angiogenesis, protects against atherosclerosis, and reduces breast cancer cell growth and metastasis. The overexpression of miR-126, therefore, may be an attractive therapeutic strategy for the treatment of cardiovascular disease or cancer. Here we report a novel strategy to deliver miR-126 to endothelial and breast cancer cells. We tested three different strategies to deliver miR-126 by linking the miR to an aptamer for the ubiquitously expressed transferrin receptor (transferrin receptor aptamer, TRA). Linking the precursor of miR-126 (pre-miR-126) to the TRA by annealing of a complementary stick led to efficient uptake and processing of miR-126, resulting in the delivery of 1.6×10(6)±0.3×10(6) copies miR-126-3p per ng RNA in human endothelial cells and 7.4×10(5)±2×10(5) copies miR-126-3p per ng in MCF7 breast cancer cells. The functionality of the active TRA-miR-126 chimera was further demonstrated by showing that the chimera represses the known miR-126 target VCAM-1 and improved endothelial cell sprouting in a spheroid assay. Moreover, the TRA-miR-126 chimera reduced proliferation and paracrine endothelial cell recruitment of breast cancer cells to a similar extent as miR-126-3p mimics introduced by conventional liposome-based transfection. Together, this data demonstrates that pre-miR-126 can be delivered by a non-specific aptamer to exert biological functions in two different cell models. The use of the TRA-miR-126 chimera or the combination of the delivery strategy with other endothelial or tumor specific aptamers may provide an interesting therapeutic option to treat vascular disease or cancers.

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.

Conversion of adult endothelium to immunocompetent haematopoietic stem cells.

PubMed

Lis, Raphael; Karrasch, Charles C; Poulos, Michael G; Kunar, Balvir; Redmond, David; Duran, Jose G Barcia; Badwe, Chaitanya R; Schachterle, William; Ginsberg, Michael; Xiang, Jenny; Tabrizi, Arash Rafii; Shido, Koji; Rosenwaks, Zev; Elemento, Olivier; Speck, Nancy A; Butler, Jason M; Scandura, Joseph M; Rafii, Shahin

2017-05-25

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1 + FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.

Conversion of adult endothelium to immunocompetent haematopoietic stem cells

PubMed Central

Lis, Raphael; Karrasch, Charles C.; Poulos, Michael G.; Kunar, Balvir; Redmond, David; Barcia Duran, Jose G.; Badwe, Chaitanya R.; Schachterle, Will; Ginsberg, Michael; Xiang, Jenny; Tabrizi, Arash Rafii; Shido, Koji; Rosenwaks, Zev; Elemento, Olivier; Speck, Nancy; Butler, Jason M.; Scandura, Joseph M.; Rafii, Shahin

2018-01-01

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully converting adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of genes encoding the transcription factors Fosb, Gfi1, Runx1, and Spi1 (also known as Fgrs) and vascular-niche-derived angiocrine factors. The induction phase (day 0–8) of conversion is initiated by expression of Fgrs in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (day 8–20), Runx1+ Fgrs-transduced endothelial cells commit to a haematopoietic fate yielding rEC-HSCs that no longer require Fgrs expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (at day 20–28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, are competent for clonal engraftment and serial primary and secondary multi-lineage reconstituting potential, including antigen-dependent adaptive immune function. Inhibition of TGF-β and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders. PMID:28514438

Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK

PubMed Central

Bhogal, Maninder; Lwin, Chan N.; Seah, Xin-Yi; Murugan, Elavazhagan; Adnan, Khadijah; Lin, Shu-Jun; Mehta, Jodhbir S.

2017-01-01

Purpose To establish a method for assessing graft viability, in-vivo, following corneal transplantation. Methods Optimization of calcein AM fluorescence and toxicity assessment was performed in cultured human corneal endothelial cells and ex-vivo corneal tissue. Descemet membrane endothelial keratoplasty grafts were incubated with calcein AM and imaged pre and post preparation, and in-situ after insertion and unfolding in a pig eye model. Global, macroscopic images of the entire graft and individual cell resolution could be attained by altering the magnification of a clinical confocal scanning laser microscope. Patterns of cell loss observed in situ were compared to those seen using standard ex-vivo techniques. Results Calcein AM showed a positive dose-fluorescence relationship. A dose of 2.67μmol was sufficient to allow clear discrimination between viable and non-viable areas (sensitivity of 96.6% with a specificity of 96.1%) and was not toxic to cultured endothelial cells or ex-vivo corneal tissue. Patterns of cell loss seen in-situ closely matched those seen on ex-vivo assessment with fluorescence viability imaging, trypan blue/alizarin red staining or scanning electron microscopy. Iatrogenic graft damage from preparation and insertion varied between 7–35% and incarceration of the graft tissue within surgical wounds was identified as a significant cause of endothelial damage. Conclusions In-situ graft viability assessment using clinical imaging devices provides comparable information to ex-vivo methods. This method shows high sensitivity and specificity, is non-toxic and can be used to evaluate immediate cell viability in new grafting techniques in-vivo. PMID:28977017

Horizontal alignment of 5' -> 3' intergene distance segment tropy with respect to the gene as the conserved basis for DNA transcription.

PubMed

Sarin, Hemant

2017-03-01

To study the conserved basis for gene expression in comparative cell types at opposite ends of the cell pressuromodulation spectrum, the lymphatic endothelial cell and the blood microvascular capillary endothelial cell. The mechanism for gene expression is studied in terms of the 5' -> 3' direction paired point tropy quotients ( prpT Q s) and the final 5' -> 3' direction episodic sub-episode block sums split-integrated weighted average-averaged gene overexpression tropy quotient ( esebssiwaagoT Q ). The final 5' -> 3' esebssiwaagoT Q classifies an lymphatic endothelial cell overexpressed gene as a supra-pressuromodulated gene ( esebssiwaagoT Q ≥ 0.25 < 0.75) every time and classifies a blood microvascular capillary endothelial cell overexpressed gene every time as an infra-pressuromodulated gene ( esebssiwaagoT Q < 0.25) (100% sensitivity; 100% specificity). Horizontal alignment of 5' -> 3' intergene distance segment tropy wrt the gene is the basis for DNA transcription in the pressuromodulated state.

The influence of surface integrin binding patterns on specific biomaterial-cell interactions

NASA Astrophysics Data System (ADS)

Beranek, Maggi Marie

As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second aim, the response of surface-bound integrins to flow-related shear stress was examined. Based on fluorescent analysis, total alphavbeta 3, alpha1beta1, and alpha5beta 1 appeared to increase on stainless steel after 90-minute low shear stress exposure, whereas only alpha5beta1 appeared to increase when exposed to high shear. 65/35 poly(D, L-lactide-co-glycolide) exhibited increased total binding of alpha5beta1 and alphaMbeta2, when exposed to either shear stress level. Exposure to either shear stress regimen appeared to increase binding of all integrins on the combinational surface. These responses to shear stress suggest differential integrin binding affinity compared to stainless steel. Using antibodies specific to the integrin subunits, the apparent increase in surface-bound integrins was found to be related to a surface disassociation of alpha and beta subunits. The third aim evaluated human aortic endothelial cells and acute monocytic leukemia cells (THP-1) cell binding to the tested biomaterial surfaces under both static and flow conditions. Both stainless steel and the combinational surface had increased endothelial cell binding compared to monocyte attachment. Pre-incubation of the surface with the specific integrins significantly inhibited human aortic endothelial cell binding. Aim four was designed to investigate the influence of surface bound integrins on human aortic endothelial cell migration under shear stress. If biomaterial surface integrin binding patterns are specific, then pre-bound surface integrins should competitively inhibit binding of cellular integrins to the surface. Cell migration distance on to alphavbeta3, alpha 1beta1, and alpha5beta1 pre-incubated stainless steel was decreased ten-fold, and decreased by three-fold on both 65/35 poly(D, L-lactide-coglycolide) and combinational surfaces compared to the respective bare surfaces. In contrast, migration distance on to alphaMbeta2 pre-coated stainless steel and combinational surface was decreased by only sixty percent and only fifty percent on alphaMbeta2 precoated 65/35 poly(D, L -lactide-co-glycolide). These results suggested that surface binding sites are selective and critical in governing endothelial cell migration. In conclusion, these results support the hypothesis that a surface that encourages specific integrin binding would promote differential cell binding. The novel integrin binding model used in this investigation may be a methodology that can be employed to evaluate potential vascular biomaterials.

Flow shear stress differentially regulates endothelial uptake of nanocarriers targeted to distinct epitopes of PECAM-1.

PubMed

Han, Jingyan; Shuvaev, Vladimir V; Davies, Peter F; Eckmann, David M; Muro, Silvia; Muzykantov, Vladimir R

2015-07-28

Targeting nanocarriers (NC) to endothelial cell adhesion molecules including Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1 or CD31) improves drug delivery and pharmacotherapy of inflammation, oxidative stress, thrombosis and ischemia in animal models. Recent studies unveiled that hydrodynamic conditions modulate endothelial endocytosis of NC targeted to PECAM-1, but the specificity and mechanism of effects of flow remain unknown. Here we studied the effect of flow on endocytosis by human endothelial cells of NC targeted by monoclonal antibodies Ab62 and Ab37 to distinct epitopes on the distal extracellular domain of PECAM. Flow in the range of 1-8dyn/cm(2), typical for venous vasculature, stimulated the uptake of spherical Ab/NC (~180nm diameter) carrying ~50 vs 200 Ab62 and Ab37 per NC, respectively. Effect of flow was inhibited by disruption of cholesterol-rich plasmalemma domains and deletion of PECAM-1 cytosolic tail. Flow stimulated endocytosis of Ab62/NC and Ab37/NC via eliciting distinct signaling pathways mediated by RhoA/ROCK and Src Family Kinases, respectively. Therefore, flow stimulates endothelial endocytosis of Ab/NC in a PECAM-1 epitope specific manner. Using ligands of binding to distinct epitopes on the same target molecule may enable fine-tuning of intracellular delivery based on the hemodynamic conditions in the vascular area of interest. Copyright © 2015 Elsevier B.V. All rights reserved.

TMEM16A Contributes to Endothelial Dysfunction by Facilitating Nox2 NADPH Oxidase-Derived Reactive Oxygen Species Generation in Hypertension.

PubMed

Ma, Ming-Ming; Gao, Min; Guo, Kai-Min; Wang, Mi; Li, Xiang-Yu; Zeng, Xue-Lin; Sun, Lu; Lv, Xiao-Fei; Du, Yan-Hua; Wang, Guan-Lei; Zhou, Jia-Guo; Guan, Yong-Yuan

2017-05-01

Ca 2+ -activated Cl - channels play a crucial role in various physiological processes. However, the role of TMEM16A in vascular endothelial dysfunction during hypertension is unclear. In this study, we investigated the specific involvement of TMEM16A in regulating endothelial function and blood pressure and the underlying mechanism. Reverse transcription-polymerase chain reaction, Western blotting, coimmunoprecipitation, confocal imaging, patch-clamp recordings, and TMEM16A endothelial-specific transgenic and knockout mice were used. We found that TMEM16A was expressed abundantly and functioned as a Ca 2+ -activated Cl - channel in endothelial cells. Angiotensin II induced endothelial dysfunction with an increase in TMEM16A expression. The knockout of endothelial-specific TMEM16A significantly lowered the blood pressure and ameliorated endothelial dysfunction in angiotensin II-induced hypertension, whereas the overexpression of endothelial-specific TMEM16A resulted in the opposite effects. These results were related to the increased reactive oxygen species production, Nox2-containing NADPH oxidase activation, and Nox2 and p22phox protein expression that were facilitated by TMEM16A on angiotensin II-induced hypertensive challenge. Moreover, TMEM16A directly bound with Nox2 and reduced the degradation of Nox2 through the proteasome-dependent degradation pathway. Therefore, TMEM16A is a positive regulator of endothelial reactive oxygen species generation via Nox2-containing NADPH oxidase, which induces endothelial dysfunction and hypertension. Modification of TMEM16A may be a novel therapeutic strategy for endothelial dysfunction-associated diseases. © 2017 American Heart Association, Inc.

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.

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

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy.

PubMed

Sackey-Aboagye, Bridget; Olsen, Abby L; Mukherjee, Sarmistha M; Ventriglia, Alexander; Yokosaki, Yasuyuki; Greenbaum, Linda E; Lee, Gi Yun; Naga, Hani; Wells, Rebecca G

2016-01-01

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9β1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN.

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy

PubMed Central

Sackey-Aboagye, Bridget; Olsen, Abby L.; Mukherjee, Sarmistha M.; Ventriglia, Alexander; Yokosaki, Yasuyuki; Greenbaum, Linda E.; Lee, Gi Yun; Naga, Hani

2016-01-01

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9β1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN. PMID:27741254

Outside-in HLA class I signaling regulates ICAM-1 clustering and endothelial cell-monocyte interactions via mTOR in transplant antibody-mediated rejection.

PubMed

Salehi, Sahar; Sosa, Rebecca A; Jin, Yi-Ping; Kageyama, Shoichi; Fishbein, Michael C; Rozengurt, Enrique; Kupiec-Weglinski, Jerzy W; Reed, Elaine F

2018-05-01

Antibody-mediated rejection (AMR) resulting in transplant allograft vasculopathy (TAV) is the major obstacle for long-term survival of solid organ transplants. AMR is caused by donor-specific antibodies to HLA, which contribute to TAV by initiating outside-in signaling transduction pathways that elicit monocyte recruitment to activated endothelium. Mechanistic target of rapamycin (mTOR) inhibitors can attenuate TAV; therefore, we sought to understand the mechanistic underpinnings of mTOR signaling in HLA class I Ab-mediated endothelial cell activation and monocyte recruitment. We used an in vitro model to assess monocyte binding to HLA I Ab-activated endothelial cells and found mTOR inhibition reduced ezrin/radixin/moesin (ERM) phosphorylation, intercellular adhesion molecule 1 (ICAM-1) clustering, and monocyte firm adhesion to HLA I Ab-activated endothelium. Further, in a mouse model of AMR, in which C57BL/6. RAG1 -/- recipients of BALB/c cardiac allografts were passively transferred with donor-specific MHC I antibodies, mTOR inhibition significantly reduced vascular injury, ERM phosphorylation, and macrophage infiltration of the allograft. Taken together, these studies indicate mTOR inhibition suppresses ERM phosphorylation in endothelial cells, which impedes ICAM-1 clustering in response to HLA class I Ab and prevents macrophage infiltration into cardiac allografts. These findings indicate a novel therapeutic application for mTOR inhibitors to disrupt endothelial cell-monocyte interactions during AMR. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

PMab-48 Recognizes Dog Podoplanin of Lymphatic Endothelial Cells.

PubMed

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

2018-02-01

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

Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

PubMed

Kułdo, J M; Ásgeirsdóttir, S A; Zwiers, P J; Bellu, A R; Rots, M G; Schalk, J A C; Ogawara, K I; Trautwein, C; Banas, B; Haisma, H J; Molema, G; Kamps, J A A M

2013-02-28

In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression. Copyright © 2012 Elsevier B.V. All rights reserved.

Endothelial β-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and Central Nervous System Homeostasis.

PubMed

Tran, Khiem A; Zhang, Xianming; Predescu, Dan; Huang, Xiaojia; Machado, Roberto F; Göthert, Joachim R; Malik, Asrar B; Valyi-Nagy, Tibor; Zhao, You-Yang

2016-01-12

The blood-brain barrier (BBB) formed by brain endothelial cells interconnected by tight junctions is essential for the homeostasis of the central nervous system. Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. Using a mouse model with tamoxifen-inducible endothelial cell-restricted disruption of ctnnb1 (iCKO), we show here that endothelial β-catenin signaling is essential for maintaining BBB integrity and central nervous system homeostasis in adult mice. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and central nervous system inflammation, and all had postictal death. Disruption of endothelial β-catenin induced BBB breakdown and downregulation of the specific tight junction proteins claudin-1 and -3 in adult brain endothelial cells. The clinical relevance of the data is indicated by the observation of decreased expression of claudin-1 and nuclear β-catenin in brain endothelial cells of hemorrhagic lesions of hemorrhagic stroke patients. These results demonstrate the prerequisite role of endothelial β-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective β-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity, and central nervous system inflammation. © 2015 American Heart Association, Inc.

Angiotensin receptors and β-catenin regulate brain endothelial integrity in malaria

PubMed Central

Basu-Roy, Upal; Ty, Maureen; Alique, Matilde; Fernandez-Arias, Cristina; Movila, Alexandru; Gomes, Pollyanna; Edagha, Innocent; Wassmer, Samuel C.; Walther, Thomas

2016-01-01

Cerebral malaria is characterized by cytoadhesion of Plasmodium falciparum–infected red blood cells (Pf-iRBCs) to endothelial cells in the brain, disruption of the blood-brain barrier, and cerebral microhemorrhages. No available antimalarial drugs specifically target the endothelial disruptions underlying this complication, which is responsible for the majority of malaria-associated deaths. Here, we have demonstrated that ruptured Pf-iRBCs induce activation of β-catenin, leading to disruption of inter–endothelial cell junctions in human brain microvascular endothelial cells (HBMECs). Inhibition of β-catenin–induced TCF/LEF transcription in the nucleus of HBMECs prevented the disruption of endothelial junctions, confirming that β-catenin is a key mediator of P. falciparum adverse effects on endothelial integrity. Blockade of the angiotensin II type 1 receptor (AT1) or stimulation of the type 2 receptor (AT2) abrogated Pf-iRBC–induced activation of β-catenin and prevented the disruption of HBMEC monolayers. In a mouse model of cerebral malaria, modulation of angiotensin II receptors produced similar effects, leading to protection against cerebral malaria, reduced cerebral hemorrhages, and increased survival. In contrast, AT2-deficient mice were more susceptible to cerebral malaria. The interrelation of the β-catenin and the angiotensin II signaling pathways opens immediate host-targeted therapeutic possibilities for cerebral malaria and other diseases in which brain endothelial integrity is compromised. PMID:27643439

Progress and challenges in the development of a cell-based therapy for hemophilia A

PubMed Central

Fomin, Marina E.; Togarrati, Padma Priya; Muench, Marcus O.

2015-01-01

Hemophilia A results from an insufficiency of factor VIII (FVIII). Although replacement therapy with plasma-derived or recombinant FVIII is a life-saving therapy for hemophilia A patients, such therapy is a life-long treatment rather than a cure for the disease. In this review we discuss the possibilities, progress and challenges that remain in the development of a cell-based cure for hemophilia A. The success of cell therapy depends on the type and availability of donor cells, the age of the host and method of transplantation, and the levels of engraftment and production of FVIII by the graft. Early therapy, possibly even prenatal transplantation, may yield the highest levels of engraftment by avoiding immunological rejection of the graft. Potential cell sources of FVIII include a specialized subset of endothelial cells known as liver sinusoidal endothelial cells (LSECs) present in the adult and fetal liver, or patient-specific endothelial cells derived from induced pluripotent stem cells (iPSCs) that have undergone gene editing to produce FVIII. Achieving sufficient engraftment of transplanted LSECs is one of the obstacles to successful cell therapy for hemophilia A. We discuss recent results from transplants performed in animals that show production of functional and clinically relevant levels of FVIII obtained from donor LSECs. Hence, the possibility of treating hemophilia A can be envisioned through persistent production of FVIII from transplanted donor cells derived from a number of potential cell sources or through creation of donor endothelial cells from patient-specific iPSCs. PMID:25297648

Modifications in astrocyte morphology and calcium signaling induced by a brain capillary endothelial cell line.

PubMed

Yoder, Elizabeth J

2002-04-15

Astrocytes extend specialized endfoot processes to perisynaptic and perivascular regions, and thus are positioned to mediate the bidirectional flow of metabolic, ionic, and other transmissive substances between neurons and the blood stream. While mutual structural and functional interactions between neurons and astrocytes have been documented, less is known about the interactions between astrocytes and cerebrovascular cells. For example, although the ability of astrocytes to induce structural and functional changes in endothelial cells is established, the reciprocity of brain endothelial cells to induce changes in astrocytes is undetermined. This issue is addressed in the present study. Changes in primary cultures of neonatal mouse cortical astrocytes were investigated following their coculture with mouse brain capillary endothelial (bEnd3) cells. The presence of bEnd3 cells altered the morphology of astrocytes by transforming them from confluent monolayers into networks of elongated multicellular columns. These columns did not occur when either bEnd3 cells or astrocytes were cocultured with other cell types, suggesting that astrocytes undergo specific morphological consequences when placed in close proximity to brain endothelial cells. In addition to these structural changes, the pharmacological profile of astrocytes was modified by coculture with bEnd3 cells. Astrocytes in the cocultures showed an increased Ca2+ responsiveness to bradykinin and glutamate, but no change in responsiveness to ATP, as compared to controls. Coculturing the astrocytes with a neuronal cell line resulted in increased responsiveness of the glial responses to glutamate but not to bradykinin. These studies indicate that brain endothelial cells induce changes in astrocyte morphology and pharmacology. Copyright 2002 Wiley-Liss, Inc.

Effect of high-glucose conditions on human periodontal ligament endothelial cells: in vitro analysis.

PubMed

Maruyama, Kosuke; Sato, Soh

2017-01-01

Endothelial cells participate in key aspects of vascular biology, such as maintenance of capillary permeability and regulation of inflammation. According to previous reports, endothelial cells have revealed highly specific characteristics depending on the organs and tissues. In particular, periodontal endothelial cells have a higher permeability than vascular endothelial cells of other types of tissue. Periodontal disease is not only a chronic disease in oral, but also affect the entire body. Diabetes and periodontal disease are closely related, with periodontal disease even been referred to as the sixth complication of disease. However, no reports have investigated the pathophysiology of microvascular in periodontal tissue once diabetes has developed. Therefore, the aim of the present study is to investigate changes in the properties of human periodontal endothelial cells (HPDLECs) that were cultured under high-glucose conditions. We isolated HPDLECs from human periodontal ligament cells. HPDLECs were cultured under high-glucose (5.5, 11.0, 22.0 mM) and investigated proliferation, apoptosis, tube formation and the expression of cell adhesion molecules. A 5.5 mM (100 mg/dl) control was used in this study. HPDLECs stimulated with high glucose concentration exhibited suppression of cell proliferation and an increased percentage of apoptosis-positive cells. This results suggested that apoptosis was caused by TNF-α expression. The expression levels cell adhesion molecules increased. These results suggest that when HPDLECs are stimulated with a high glucose concentrations, PKC in the intracellular cell substrate is activated, increasing the expression of intercellular and vascular adhesion molecules. Thus, the results of this study demonstrate that diabetes exacerbates periodontal disease.

Systemic Hypoxia Changes the Organ-Specific Distribution of Vascular Endothelial Growth Factor and Its Receptors

NASA Astrophysics Data System (ADS)

Marti, Hugo H.; Risau, Werner

1998-12-01

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF in vitro. Here we demonstrate that VEGF is induced in vivo by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver. In situ hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia in vivo is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.

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

Dynamical mechanisms of conducted vasoreactivity: minimalistic modeling study

NASA Astrophysics Data System (ADS)

Kuryshova, Ekaterina A.; Rogatina, Kristina V.; Postnov, Dmitry E.

2018-04-01

Endothelial cells are cells lining the inner surface of the blood and lymphatic vessels, they separate the bloodstream from the deeper layers of the vascular wall. Earlier endothelium was considered only as a passive barrier between blood and tissues. However, it has now become apparent that endothelial cells, specifically reacting to different molecular signals generated locally and remotely, perform a variety of functions. Simulation of large vascular networks requires the development of specialized models of autoregulation of vascular tone. On the one hand, such models should have a strong support for cellular dynamics, on the other - be as computationally efficient as possible. A model of a two-dimensional cylindrical array of endothelial cells is proposed on the basis of the integral description by means of the whole-cell CVC. The process of propagation of hyperpolarizing and depolarizing pulses is investigated depending on the statistics of cell distribution between the two main types. Endothelial cells are considered as a dynamic system possessing bistability. Based on the articles, the results of the distribution of the resting-potential values were repeated, the propagation of the hyperpolarizing pulse was observed, the endothelial cell chain supported the propagation of the wave switching to a hyperpolarized state, and then the return wave returned to its original state.

Generation and characterization of a porcine endometrial endothelial cell line susceptible to porcine reproductive and respiratory syndrome virus.

PubMed

Feng, Lili; Zhang, Xinyu; Xia, Xiaoli; Li, Yangyang; He, Shan; Sun, Huaichang

2013-01-01

Previous studies on the underlying mechanism for porcine reproductive and respiratory syndrome virus (PRRSV)-induced reproductive failure have been focused on the viral replication in the endothelial macrophages, and the susceptibility of porcine endometrial endothelial (PEE) cells to PRRSV has not yet been investigated. Therefore, in the present study we generated a PEE cell line by transfection of the primary cells with a SV40 large T antigen expression vector. The PEE cell line maintained the endothelial morphology with a significantly faster growth rate, shorter population doubling time and higher plating efficiency than the primary cells. The endothelial origination of the cell line was confirmed by detection of the endothelial cell-specific markers. The PEE cell line had been passed successively for 60 generations with an unlimited growth potential. To further characterize the PEE cell line, cells of different passages were infected with different PRRSV strains and analyzed for the viral antigen and replication. Overt cytopathic effect was observed from 36h postinfection (HPI) and the viral antigen detected as early as 12 HPI. The infectious virus was recovered from the infected PEE cells with a titer higher than that in MARC-145 cells. Since the data presented indicate a high susceptibility of PEE cells to PRRSV, we conclude that the PEE cell line generated will be useful for growth of PRRSV and further studies on the underlying mechanism for PRRSV infection of PEE cells. The finding of the susceptibility of PEE cells to PRRSV may provide an alternative explanation for PRRSV-induced reproductive failure. Copyright © 2012 Elsevier B.V. All rights reserved.

Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

PubMed

Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

2017-01-01

Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

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

PubMed Central

2013-01-01

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

Loss of autocrine endothelial-derived VEGF significantly reduces hemangiosarcoma development in conditional p53-deficient mice

PubMed Central

Farhang Ghahremani, Morvarid; Radaelli, Enrico; Haigh, Katharina; Bartunkova, Sonia; Haenebalcke, Lieven; Marine, Jean-Christophe; Goossens, Steven; Haigh, Jody J

2014-01-01

Malignant transformation of the endothelium is rare, and hemangiosarcomas comprise only 1% of all sarcomas. For this reason and due to the lack of appropriate mouse models, the genetic mechanisms of malignant endothelial transformation are poorly understood. Here, we describe a hemangiosarcoma mouse model generated by deleting p53 specifically in the endothelial and hematopoietic lineages. This strategy led to a high incidence of hemangiosarcoma, with an average latency of 25 weeks. To study the in vivo roles of autocrine or endothelial cell autonomous VEGF signaling in the initiation and/or progression of hemangiosarcomas, we genetically deleted autocrine endothelial sources of VEGF in this mouse model. We found that loss of even a single conditional VEGF allele results in substantial rescue from endothelial cell transformation. These findings highlight the important role of threshold levels of autocrine VEGF signaling in endothelial malignancies and suggest a new approach for hemangiosarcoma treatment using targeted autocrine VEGF inhibition. PMID:24626176

Loss of autocrine endothelial-derived VEGF significantly reduces hemangiosarcoma development in conditional p53-deficient mice.

PubMed

Farhang Ghahremani, Morvarid; Radaelli, Enrico; Haigh, Katharina; Bartunkova, Sonia; Haenebalcke, Lieven; Marine, Jean-Christophe; Goossens, Steven; Haigh, Jody J

2014-01-01

Malignant transformation of the endothelium is rare, and hemangiosarcomas comprise only 1% of all sarcomas. For this reason and due to the lack of appropriate mouse models, the genetic mechanisms of malignant endothelial transformation are poorly understood. Here, we describe a hemangiosarcoma mouse model generated by deleting p53 specifically in the endothelial and hematopoietic lineages. This strategy led to a high incidence of hemangiosarcoma, with an average latency of 25 weeks. To study the in vivo roles of autocrine or endothelial cell autonomous VEGF signaling in the initiation and/or progression of hemangiosarcomas, we genetically deleted autocrine endothelial sources of VEGF in this mouse model. We found that loss of even a single conditional VEGF allele results in substantial rescue from endothelial cell transformation. These findings highlight the important role of threshold levels of autocrine VEGF signaling in endothelial malignancies and suggest a new approach for hemangiosarcoma treatment using targeted autocrine VEGF inhibition.

Vasculogenesis of decidua side population cells of first-trimester pregnancy.

PubMed

Wang, Qiushi; Shen, Licong; Huang, Wei; Song, Yong; Xiao, Li; Xu, Wenming; Liu, Ying

2013-05-07

Sufficient uterine blood supply is essential for the fetus to develop normally in the uterus. Several mechanisms are involved in the process of vessel development in deciduas and villus. We focus on whether first-trimester decidua side population (SP) cells contain cells capable of differentiating into endothelial cells. Eight decidua samples were collected from healthy women, 22- to 30-years old, undergoing elective terminations of early pregnancy (six to eight gestational weeks). The cell suspensions from human deciduas were stained by Hoechst 33342 and sorted by flow cytometry, further cultured under differentiation conditions and analyzed for specific markers. These cells were implanted into ischemic limbs of nude mice to test the capacity of angiogenesis in vivo by DiI tracers and immunohistochemistry. Decidua CD31(-)CD146(-) SP cells of first-trimester human pregnancy can differentiate into endothelial cells, express the corresponding specific markers of endothelial cells, such as CD31 and CD146, and form tube-like structures on Matrigel and part of newly formed vessels in the ischemic limbs of nude mice. Vascular endothelial growth factor was more effective in promoting proliferation of CD31(-)CD146(-)SP cells compared with other growth factors, and estrogen and progesterone at a final concentration of 10 μmol/L and 30 μmol/L, respectively, promoted the migration of CD31()-CD146(-)SP cells in a dose-dependent manner. CD31(-)CD146(-) SP cells may be involved in the formation of new vessels in the maternal aspect of the placenta in the first trimester.

Endothelial cell markers in vascular neoplasms: an immunohistochemical study comparing factor VIII-related antigen, blood group specific antigens, 6-keto-PGF1 alpha, and Ulex europaeus 1 lectin.

PubMed

Little, D; Said, J W; Siegel, R J; Fealy, M; Fishbein, M C

1986-06-01

Markers for endothelial cells including Ulex europaeus 1 lectin, blood group A, B, and H, and the prostaglandin metabolite 6-keto-PGF1 alpha were evaluated in paraffin secretions from formalin-fixed benign and malignant vascular neoplasms using a variety of immunohistochemical techniques, and results compared with staining for factor VIII-related antigen. Staining for Ulex appeared more sensitive than factor VIII-related antigen in identifying poorly differentiated neoplasms including haemangiosarcomas and spindle cell proliferations in Kaposi's sarcoma. Staining for blood group related antigens correlated with blood group in all cases. Ulex europaeus 1 lectin was the only marker for endothelial cells in lymphangiomas.

O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.

PubMed

Makino, Ayako; Dai, Anzhi; Han, Ying; Youssef, Katia D; Wang, Weihua; Donthamsetty, Reshma; Scott, Brian T; Wang, Hong; Dillmann, Wolfgang H

2015-11-01

Cardiovascular disease is the primary cause of morbidity and mortality in diabetes, and endothelial dysfunction is commonly seen in these patients. Increased O-linked N-acetylglucosamine (O-GlcNAc) protein modification is one of the central pathogenic features of diabetes. Modification of proteins by O-GlcNAc (O-GlcNAcylation) is regulated by two key enzymes: β-N-acetylglucosaminidase [O-GlcNAcase (OGA)], which catalyzes the reduction of protein O-GlcNAcylation, and O-GlcNAc transferase (OGT), which induces O-GlcNAcylation. However, it is not known whether reducing O-GlcNAcylation can improve endothelial dysfunction in diabetes. To examine the effect of endothelium-specific OGA overexpression on protein O-GlcNAcylation and coronary endothelial function in diabetic mice, we generated tetracycline-inducible, endothelium-specific OGA transgenic mice, and induced OGA by doxycycline administration in streptozotocin-induced type 1 diabetic mice. OGA protein expression was significantly decreased in mouse coronary endothelial cells (MCECs) isolated from diabetic mice compared with control MCECs, whereas OGT protein level was markedly increased. The level of protein O-GlcNAcylation was increased in diabetic compared with control mice, and OGA overexpression significantly decreased the level of protein O-GlcNAcylation in MCECs from diabetic mice. Capillary density in the left ventricle and endothelium-dependent relaxation in coronary arteries were significantly decreased in diabetes, while OGA overexpression increased capillary density to the control level and restored endothelium-dependent relaxation without changing endothelium-independent relaxation. We found that connexin 40 could be the potential target of O-GlcNAcylation that regulates the endothelial functions in diabetes. These data suggest that OGA overexpression in endothelial cells improves endothelial function and may have a beneficial effect on coronary vascular complications in diabetes. Copyright © 2015 the American Physiological Society.

Isolation and characterization of vascular endothelial cells derived from fetal tooth buds of miniature swine.

PubMed

Nasu, Masanori; Nakahara, Taka; Tominaga, Noriko; Tamaki, Yuichi; Ide, Yoshiaki; Tachibana, Toshiaki; Ishikawa, Hiroshi

2013-03-01

The aim of the present study was to isolate endothelial cells from tooth buds (unerupted deciduous teeth) of miniature swine. Mandibular molar tooth buds harvested from swine fetuses at fetal days 90-110 were cultured in growth medium supplemented with 15% fetal bovine serum in 100-mm culture dishes until the primary cells outgrown from the tooth buds reached confluence. A morphologically defined set of pavement-shaped primary cells were picked up manually with filter paper containing trypsin/ethylenediamine tetraacetic acid solution and transferred to a separate dish. A characterization of the cellular characteristics and a functional analysis of the cultured cells at passages 3 to 5 were performed using immunofluorescence, a reverse transcriptase polymerase chain reaction assay, a tube formation assay, and transmission electron microscopy. The isolated cells grew in a pavement arrangement and showed the characteristics of contact inhibition upon reaching confluence. The population doubling time was ~48 h at passage 3. As shown by immunocytostaining and western blotting with specific antibodies, the cells produced the endothelial marker proteins such as vascular endothelial cadherin, von Willebrand factor, and vascular endothelial growth factor receptor-2. Observation with time-lapse images showed that small groups of cells aggregated and adhered to each other to form tube-like structures. Moreover, as revealed through transmission electron microscopy, these adherent cells had formed junctional complexes. These endothelial cells from the tooth buds of miniature swine are available as cell lines for studies on tube formation and use in regenerative medical science.

Unique gene expression profiles of donor-matched human retinal and choroidal vascular endothelial cells.

PubMed

Smith, Justine R; Choi, Dongseok; Chipps, Timothy J; Pan, Yuzhen; Zamora, David O; Davies, Michael H; Babra, Bobby; Powers, Michael R; Planck, Stephen R; Rosenbaum, James T

2007-06-01

Consistent with clinical observations that posterior uveitis frequently involves the retinal vasculature and recent recognition of vascular heterogeneity, the hypothesis for this study was that retinal vascular endothelium was a cell population of unique molecular phenotype. Donor-matched cultures of primary retinal and choroidal endothelial cells from six human cadavers were incubated with either Toxoplasma gondii tachyzoites (10:1, parasites per cell) or Escherichia coli lipopolysaccharide (100 ng/mL); control cultures were simultaneously incubated with medium. Gene expression profiling of endothelial cells was performed using oligonucleotide arrays containing probes designed to detect 8746 human transcripts. After normalization, differential gene expression was assessed by the significance analysis of microarrays, with the false-discovery rate set at 5%. For selected genes, differences in the level of expression between retinal and choroidal cells were evaluated by real-time RT-PCR. Graphic descriptive analysis demonstrated a strong correlation between gene expression of unstimulated retinal and choroidal endothelial cells, but also highlighted distinctly different patterns of expression that were greater than differences noted between donors or between unstimulated and stimulated cells. Overall, 779 (8.9%) of 8746 transcripts were differentially represented. Of note, the 330 transcripts that were present at higher levels in retinal cells included a larger percentage of transcripts encoding molecules involved in the immune response. Differential gene expression was confirmed for 12 transcripts by RT-PCR. Retinal and choroidal vascular endothelial cells display distinctive gene expression profiles. The findings suggest the possibility of treating posterior uveitis by targeting specific interactions between the retinal endothelial cell and an infiltrating leukocyte.

Decellularized ovine arteries as biomatrix scaffold support endothelial of mesenchymal stem cells.

PubMed

Zhang, Wenbo; Huo, Yanhong; Wang, Xinling; Jia, Yingmin; Su, Li; Wang, Caixia; Li, Ying; Yang, Yonghong; Liu, Yuanyuan

2016-11-01

The differentiation rate of adipose-derived mesenchymal stem cells (Ad-MSCs) into endothelial cells is always lower under normal condition, which limits further clinical application of Ad-MSCs for angiogenesis regenerative medicine and needs to be enhanced. In the present study, the tissue-specific-derived decellularized ovine arteries matrix (DCS) was used as scaffold to investigate the pro-endothelial differentiation ability of decellularized ovine arteries matrix as well as the underlying mechanisms. The prepared decellularized ovine arteries matrix by the combination of enzymatic and chemical decellularization approaches preserved macroscopic 3D architecture, native composition and ultrastructure of natural ovine arteries. The RT-PCR, histopathological and immunofluorescence assay results suggested that DCS could increase the proliferation ability of MSC. What's more, the DCS could also induce the endothelial differentiation of MSC, which was further enhanced by adding VEGF. Our results showed that natural 3D matrix from decellularized ovine arteries could induce the endothelial differentiation of AD-MSCs alone or with the combination of VEGF. Our results indicated that the decellularized ovine arteries matrix would serve as an efficient culture system for promoting endothelial differentiation of Ad-MSCs.

Increased endothelial cell adhesion and elongation on micron-patterned nano-rough poly(dimethylsiloxane) films.

PubMed

Ranjan, Ashwini; Webster, Thomas J

2009-07-29

The success of synthetic vascular grafts is largely determined by their ability to promote vital endothelial cell functions such as adhesion, alignment, proliferation, and extracellular matrix (ECM) deposition. Developing such biomaterials requires the design and fabrication of materials that mimic select properties of native extracellular matrices. Furthermore, cells of the native endothelium have elongated and aligned morphology in the direction of blood flow, yet few materials promote this type of morphology initially, but rather rely on blood flow to orient endothelial cells. Therefore, the objective of this in vitro study was to design a biomaterial that mimics the conditions of the micro- and nano-environment of vascular intima tissue suitable for endothelial cell adhesion and elongation to improve the efficacy of small synthetic vascular grafts. Towards this end, patterned poly(dimethylsiloxane) (PDMS) films consisting of periodic arrays of nano-grooves (500 nm), with spacings ranging from 22 to 80 microm, and alternating nano- and micron roughness were fabricated using a novel electron beam physical vapor deposition method followed by polymer casting. By varying pattern spacing, the area of micron- and nano-rough surface was controlled. In vitro rat aortic endothelial cell adhesion and elongation studies indicated that endothelial cell function was enhanced on patterned PDMS surfaces with the widest spacing and greatest surface area of nano-roughness, as compared to more narrow pattern spacings and non-patterned PDMS surfaces. Specifically, endothelial cells adherent on PDMS patterned films of the widest spacing (greatest nano-rough area) displayed almost twice as much elongation as cells on non-patterned surfaces. For these reasons, the present study highlighted design criteria (the use of micron patterns of nano-features on PDMS) that may contribute to the intelligent design of new-generation vascular grafts.

Internalization of targeted quantum dots by brain capillary endothelial cells in vivo.

PubMed

Paris-Robidas, Sarah; Brouard, Danny; Emond, Vincent; Parent, Martin; Calon, Frédéric

2016-04-01

Receptors located on brain capillary endothelial cells forming the blood-brain barrier are the target of most brain drug delivery approaches. Yet, direct subcellular evidence of vectorized transport of nanoformulations into the brain is lacking. To resolve this question, quantum dots were conjugated to monoclonal antibodies (Ri7) targeting the murine transferrin receptor. Specific transferrin receptor-mediated endocytosis of Ri7-quantum dots was first confirmed in N2A and bEnd5 cells. After intravenous injection in mice, Ri7-quantum dots exhibited a fourfold higher volume of distribution in brain tissues, compared to controls. Immunofluorescence analysis showed that Ri7-quantum dots were sequestered throughout the cerebral vasculature 30 min, 1 h, and 4 h post injection, with a decline of signal intensity after 24 h. Transmission electron microscopic studies confirmed that Ri7-quantum dots were massively internalized by brain capillary endothelial cells, averaging 37 ± 4 Ri7-quantum dots/cell 1 h after injection. Most quantum dots within brain capillary endothelial cells were observed in small vesicles (58%), with a smaller proportion detected in tubular structures or in multivesicular bodies. Parenchymal penetration of Ri7-quantum dots was extremely low and comparable to control IgG. Our results show that systemically administered Ri7-quantum dots complexes undergo extensive endocytosis by brain capillary endothelial cells and open the door for novel therapeutic approaches based on brain endothelial cell drug delivery. © The Author(s) 2015.

Cytochrome P450-2C11 mRNA is not expressed in endothelial cells dissected from rat renal arterioles.

PubMed

Heil, Sandra G; De Vriese, An S; Kluijtmans, Leo A J; Dijkman, Henry; van Strien, Denise; Akkers, Robert; Blom, Henk J

2005-01-01

Cytochrome P450 (CYP) isoenzymes (CYP2C and CYP2J) are involved in the production of epoxyeicosatrienoic acids, which are postulated as endothelium-derived hyperpolarizing factors (EDHFs). We hypothesized that if CYP2C11 is involved in the EDHF-mediated responses, its mRNA should be expressed in endothelial cells. We, therefore, examined the mRNA expression of CYP2C11 in endothelial cells of renal arterioles. Laser microdissection was applied to isolate endothelial cells from the renal arterioles of 4 male and 4 female Wistar rats. As a positive control of CYP2C11 expression, hepatocytes were also dissected from these rats. RNA was isolated and real-time quantitative polymerase chain reaction (Q-PCR) analysis was applied. Q-PCR analysis showed that CYP2C11 mRNA was not expressed in laser microdissected endothelial cells of renal arterioles of male and female rats. CYP2C11 mRNA expression was highly abundant in hepatocytes dissected from male livers, but in female livers hardly any CYP2C11 mRNA was detected. We have shown that endothelial cells can be dissected from small renal arterioles by laser microdissection to study the mRNA expression of specific genes by Q-PCR. Using this novel tool, we demonstrated that the CYP2C11 mRNA was not expressed in the endothelial cells of renal arterioles. Therefore, we speculate that CYP2C11 does not contribute to the EDHF-mediated responses in renal arterioles. Copyright (c) 2005 S. Karger AG, Basel.

Cell selection and characterization of a novel human endothelial cell specific nanobody.

PubMed

Ahmadvand, Davoud; Rasaee, Mohammad J; Rahbarizadeh, Fatemeh; Kontermann, Roland E; Sheikholislami, Farzaneh

2009-05-01

Antibody-based targeting of angiogenesis and vascular targeting therapy of cancer are extremely attractive conceptually and open new important diagnostic and therapeutic opportunities. Compelling evidence suggests that CD105 represents an ideal target for anti-angiogenic therapy and its presence in solid tumor vasculature has prognostic value. Camelids produce functional antibodies devoid of light chains and constant heavy chain domain (CH1). Nanobodies, the antigen-binding fragments of such heavy chain antibodies, are therefore comprised in one single domain. The aim of this study was to explore the possibilities of using anti-endoglin nanobody as an angiogenesis inhibitor. The anti-CD105 nanobody (AR-86a) was isolated from immune library by selections on purified antigens and target cells. Immunocytochemistry and FACS analysis showed that the purified nanobody reacted specifically with human umbilical vein endothelial cells (HUVECs) but not with other cell lines such as MDA-MB-453, Mel III, T-47D, MCF-7, AGO and HT 29. Further, selected nanobody potently inhibited proliferation of human endothelial cells and formation of capillary-like structures. This selected high affinity anti-endoglin nanobody may offer high specificity towards tumors with reduced side effects, and may be less likely to elicit drug resistance compared to conventional therapy.

Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

PubMed Central

Gonzalez-Rodriguez, David; Barakat, Abdul I.

2015-01-01

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

Surface engineering of cardiovascular stent with endothelial cell selectivity for in vivo re-endothelialisation.

PubMed

Wei, Yu; Ji, Ying; Xiao, Lin-Lin; Lin, Quan-kui; Xu, Jian-ping; Ren, Ke-feng; Ji, Jian

2013-04-01

The in vivo endothelialisation of materials provides a promising strategy for the rapid re-endothelialisation of a cardiovascular implantation. Although many studies have focused on improving the rapid endothelialisation through the immobilisation of bioactive molecules, it should be noted that the endothelial cells (ECs) will compete with other cell types in vivo. Thus, the efforts to partially enhance the EC growth without considering the cell competition might be misleading and meaningless in vivo. In this study, we demonstrated that the competitive growth of human umbilical vein endothelial cells (HUVECs) over human aortic smooth muscle cells (HASMCs) could be increased through the synergic action of the nonspecific resistance to phosphorylcholine and the specific recognition of the REDV peptide. Further in vivo data indicate that the competitive ability of ECs over SMCs, instead of the number of ECs, is a significantly more important criterion for the development of a pure endothelial layer in vivo and thus the attainment of a better anti-restenosis effect. Consequently, the surface tailoring of a stent to obtain high endothelial cell selectivity is likely an effective design criterion for in situ endothelialisation and a possible future solution for the problem of in-stent restenosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

Effect of amplitude and duration of impulsive pressure on endothelial permeability in in vitro fluid percussion trauma.

PubMed

Nakadate, Hiromichi; Inuzuka, Koji; Akanuma, Suguru; Kakuta, Akira; Aomura, Shigeru

2014-04-16

Intracranial pressure changes during head impact cause brain injuries such as vasogenic edema and cerebral contusion. However, the influence of impulsive pressure on endothelial function has not yet been fully studied in vitro. In this study, we developed a pressure loading device that produced positive and negative pressures by modifying an in vitro fluid percussion model and examined the effects of the amplitude and duration of the pressures on endothelial permeability. Human umbilical vein endothelial cells were subjected to three types of positive pressure (average amplitude/average duration of 352 kPa/23 ms, 73 kPa/27 ms, and 70 kPa/44 ms) and three types of negative pressure (-72 kPa/41 ms, -67 kPa/104 ms, and -91 kPa/108 ms), and the transendothelial electrical resistance (TEER) was measured between 15 min and 24 h after pressure loading for quantifying the formation of an integral monolayer of endothelial cells. After loading, vascular endothelial- (VE-) cadherin, an endothelium-specific cell-cell adhesion molecule involved in endothelial barrier function, was stained and observed using fluorescence microscopy. The pressure loading device could produce positive pressure pulses with amplitudes of 53-1348 kPa and durations of 9-29.1 ms and negative pressure pulses with amplitudes of -52 - -93 kPa and durations of 42.9-179.5 ms. The impulsive pressure reduced the TEER associated with the change in VE-cadherin localization. Additionally, TEER decreased considerably at 15 min and 6 h post-loading, with these changes being significant in positive pressure with larger amplitude and shorter duration and in all types of negative pressures compared to pre-loading. The changes in intracranial pressure during head impact impair endothelial barrier function by the disruption of the integrity of endothelial cell-cell junctions, and the degree of increase in endothelial permeability depends on the amplitude, duration, and direction (compressive and tensile) of the impulsive pressure.

Oxidative stress activates endothelial innate immunity via sterol regulatory element binding protein 2 (SREBP2) transactivation of microRNA-92a.

PubMed

Chen, Zhen; Wen, Liang; Martin, Marcy; Hsu, Chien-Yi; Fang, Longhou; Lin, Feng-Mao; Lin, Ting-Yang; Geary, McKenna J; Geary, Greg G; Zhao, Yongli; Johnson, David A; Chen, Jaw-Wen; Lin, Shing-Jong; Chien, Shu; Huang, Hsien-Da; Miller, Yury I; Huang, Po-Hsun; Shyy, John Y-J

2015-03-03

Oxidative stress activates endothelial innate immunity and disrupts endothelial functions, including endothelial nitric oxide synthase-derived nitric oxide bioavailability. Here, we postulated that oxidative stress induces sterol regulatory element-binding protein 2 (SREBP2) and microRNA-92a (miR-92a), which in turn activate endothelial innate immune response, leading to dysfunctional endothelium. Using cultured endothelial cells challenged by diverse oxidative stresses, hypercholesterolemic zebrafish, and angiotensin II-infused or aged mice, we demonstrated that SREBP2 transactivation of microRNA-92a (miR-92a) is oxidative stress inducible. The SREBP2-induced miR-92a targets key molecules in endothelial homeostasis, including sirtuin 1, Krüppel-like factor 2, and Krüppel-like factor 4, leading to NOD-like receptor family pyrin domain-containing 3 inflammasome activation and endothelial nitric oxide synthase inhibition. In endothelial cell-specific SREBP2 transgenic mice, locked nucleic acid-modified antisense miR-92a attenuates inflammasome, improves vasodilation, and ameliorates angiotensin II-induced and aging-related atherogenesis. In patients with coronary artery disease, the level of circulating miR-92a is inversely correlated with endothelial cell-dependent, flow-mediated vasodilation and is positively correlated with serum level of interleukin-1β. Our findings suggest that SREBP2-miR-92a-inflammasome exacerbates endothelial dysfunction during oxidative stress. Identification of this mechanism may help in the diagnosis or treatment of disorders associated with oxidative stress, innate immune activation, and endothelial dysfunction. © 2014 American Heart Association, Inc.

Fluid Mechanical Forces and Endothelial Mitochondria: A Bioengineering Perspective.

PubMed

Scheitlin, Christopher G; Nair, Devi M; Crestanello, Juan A; Zweier, Jay L; Alevriadou, B Rita

2014-12-01

Endothelial cell dysfunction is the hallmark of every cardiovascular disease/condition, including atherosclerosis and ischemia/reperfusion injury. Fluid shear stress acting on the vascular endothelium is known to regulate cell homeostasis. Altered hemodynamics is thought to play a causative role in endothelial dysfunction. The dysfunction is associated with/preceded by mitochondrial oxidative stress. Studies by our group and others have shown that the form and/or function of the mitochondrial network are affected when endothelial cells are exposed to shear stress in the absence or presence of additional physicochemical stimuli. The present review will summarize the current knowledge on the interconnections among intracellular Ca 2+ - nitric oxide - mitochondrial reactive oxygen species, mitochondrial fusion/fission, autophagy/mitophagy, and cell apoptosis vs. survival. More specifically, it will list the evidence on potential regulation of the above intracellular species and processes by the fluid shear stress acting on the endothelium under either physiological flow conditions or during reperfusion (following a period of ischemia). Understanding how the local hemodynamics affects mitochondrial physiology and the cell redox state may lead to development of novel therapeutic strategies for prevention or treatment of the endothelial dysfunction and, hence, of cardiovascular disease.

Identification of exosite residues of factor Xa involved in recognition of PAR-2 on endothelial cells.

PubMed

Manithody, Chandrashekhara; Yang, Likui; Rezaie, Alireza R

2012-03-27

Recent results have indicated that factor Xa (FXa) cleaves protease-activated receptor 2 (PAR-2) to elicit protective intracellular signaling responses in endothelial cells. In this study, we investigated the molecular determinants of the specificity of FXa interaction with PAR-2 by monitoring the cleavage of PAR-2 by FXa in endothelial cells transiently transfected with a PAR-2 cleavage reporter construct in which the extracellular domain of the receptor was fused to cDNA encoding for alkaline phosphatase. Comparison of the cleavage efficiency of PAR-2 by a series of FXa mutants containing mutations in different surface loops indicated that the acidic residues of 39-loop (Glu-36, Glu-37, and Glu-39) and the basic residues of 60-loop (Lys-62 and Arg-63), 148-loop (Arg-143, Arg-150, and Arg-154), and 162-helix (Arg-165 and Lys-169) contribute to the specificity of receptor recognition by FXa on endothelial cells. This was evidenced by significantly reduced activity of mutants toward PAR-2 expressed on transfected cells. The extent of loss in the PAR-2 cleavage activity of FXa mutants correlated with the extent of loss in their PAR-2-dependent intracellular signaling activity. Further characterization of FXa mutants indicated that, with the exception of basic residues of 162-helix, which play a role in the recognition specificity of the prothrombinase complex, none of the surface loop residues under study makes a significant contribution to the activity of FXa in the prothrombinase complex. These results provide new insight into mechanisms through which FXa specifically interacts with its macromolecular substrates in the clotting and signaling pathways.

A SAGE based approach to human glomerular endothelium: defining the transcriptome, finding a novel molecule and highlighting endothelial diversity.

PubMed

Sengoelge, Guerkan; Winnicki, Wolfgang; Kupczok, Anne; von Haeseler, Arndt; Schuster, Michael; Pfaller, Walter; Jennings, Paul; Weltermann, Ansgar; Blake, Sophia; Sunder-Plassmann, Gere

2014-08-27

Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis of gene expression (SAGE) was used for its unbiased approach to quantitative acquisition of transcripts. We generated a HGMEC SAGE library consisting of 68,987 transcript tags. Then taking advantage of large public databases and advanced bioinformatics we compared the HGMEC SAGE library with a SAGE library of non-cultured ex vivo human glomeruli (44,334 tags) which contained endothelial cells. The 823 tags common to both which would have the potential to be expressed in vivo were subsequently checked against 822,008 tags from 16 non-glomerular endothelial SAGE libraries. This resulted in 268 transcript tags differentially overexpressed in HGMEC compared to non-glomerular endothelia. These tags were filtered using a set of criteria: never before shown in kidney or any type of endothelial cell, absent in all nephron regions except the glomerulus, more highly expressed than statistically expected in HGMEC. Neurogranin, a direct target of thyroid hormone action which had been thought to be brain specific and never shown in endothelial cells before, fulfilled these criteria. Its expression in glomerular endothelium in vitro and in vivo was then verified by real-time-PCR, sequencing and immunohistochemistry. Our results represent an extensive molecular characterization of HGMEC beyond a mere database, underline the endothelial heterogeneity, and propose neurogranin as a potential link in the kidney-thyroid axis.

Combining 2D angiogenesis and 3D osteosarcoma microtissues to improve vascularization.

PubMed

Chaddad, Hassan; Kuchler-Bopp, Sabine; Fuhrmann, Guy; Gegout, Hervé; Ubeaud-Sequier, Geneviève; Schwinté, Pascale; Bornert, Fabien; Benkirane-Jessel, Nadia; Idoux-Gillet, Ysia

2017-11-15

Angiogenesis is now well known for being involved in tumor progression, aggressiveness, emergence of metastases, and also resistance to cancer therapies. In this study, to better mimic tumor angiogenesis encountered in vivo, we used 3D culture of osteosarcoma cells (MG-63) that we deposited on 2D endothelial cells (HUVEC) grown in monolayer. We report that endothelial cells combined with tumor cells were able to form a well-organized network, and that tubule-like structures corresponding to new vessels infiltrate tumor spheroids. These vessels presented a lumen and expressed specific markers as CD31 and collagen IV. The combination of 2D endothelial cells and 3D microtissues of tumor cells also increased expression of angiogenic factors as VEGF, CXCR4 and ICAM1. The cell environment is the key point to develop tumor vascularization in vitro and to be closer to tumor encountered in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

PubMed Central

Gerritsen, M. E.; Carley, W. W.; Ranges, G. E.; Shen, C. P.; Phan, S. A.; Ligon, G. F.; Perry, C. A.

1995-01-01

Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking. Images Figure 7 Figure 8 Figure 11 PMID:7543732

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.

HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcγRs1

PubMed Central

Valenzuela, Nicole M; Mulder, Arend; Reed, Elaine F

2013-01-01

Antibody-mediated rejection of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor specific antibody binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the antibody. We investigated the mechanisms underlying monocyte recruitment by HLA class I antibody-activated endothelium. We used a panel of murine monoclonal antibodies of different subclasses to crosslink HLA I on human aortic, venous and microvascular endothelial cells, and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. Mouse IgG2a but not mIgG1 could bind human FcγRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcγRI, and, to a lesser extent, FcγRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during antibody mediated rejection. We confirmed these observations using human HLA allele specific monoclonal antibodies and IgG purified from transplant patient sera. HLA I antibodies universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during antibody-mediated rejection. Importantly, the subclass of donor specific antibody may influence its pathogenesis. These results imply that hIgG1 and hIgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcγR interactions. PMID:23690477

HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcγRs.

PubMed

Valenzuela, Nicole M; Mulder, Arend; Reed, Elaine F

2013-06-15

Ab-mediated rejection (AMR) of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor-specific Ab binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the Ab. We investigated the mechanisms underlying monocyte recruitment by HLA class I (HLA I) Ab-activated endothelium. We used a panel of murine mAbs of different subclasses to crosslink HLA I on human aortic, venous, and microvascular endothelial cells and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine (m)IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. mIgG2a but not mIgG1 could bind human FcγRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcγRI, and, to a lesser extent, FcγRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during AMR. We confirmed these observations using human HLA allele-specific mAbs and IgG purified from transplant patient sera. HLA I Abs universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during AMR. Importantly, the subclass of donor-specific Ab may influence its pathogenesis. These results imply that human IgG1 and human IgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcγR interactions.

Serine 1179 phosphorylation of endothelial nitric oxide synthase caused by 2,4,6-trinitrotoluene through PI3K/Akt signaling in endothelial cells

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

Sun Yang; Sumi, Daigo; Kumagai, Yoshito

2006-07-01

Although 2,4,6-trinitrotoluene (TNT) has been found to uncouple nitric oxide synthase (NOS), thereby leading to reactive oxygen species (ROS), cellular response against TNT still remains unclear. Exposure of bovine aortic endothelial cells (BAECs) to TNT (100 {mu}M) resulted in serine 1179 phosphorylation of endothelial NOS (eNOS). With specific inhibitors (wortmannin and LY294002), we found that PI3K/Akt signaling participated in the eNOS phosphorylation caused by TNT, whereas the ERK pathway did not. ROS were generated following exposure of BAECs to TNT. However, TNT-mediated phosphorylation of either eNOS or Akt was drastically blocked by NAC and PEG-CAT. Interestingly, pretreatment with apocynin, amore » specific inhibitor for NADPH oxidase, diminished the phosphorylation of eNOS and Akt. These results suggest that TNT affects NADPH oxidase, thereby generating hydrogen peroxide, which is capable of activating PI3K/Akt signaling associated with eNOS Ser 1179 phosphorylation.« less

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B.

PubMed

Koch, Alexander W; Mathivet, Thomas; Larrivée, Bruno; Tong, Raymond K; Kowalski, Joe; Pibouin-Fragner, Laurence; Bouvrée, Karine; Stawicki, Scott; Nicholes, Katrina; Rathore, Nisha; Scales, Suzie J; Luis, Elizabeth; del Toro, Raquel; Freitas, Catarina; Bréant, Christiane; Michaud, Annie; Corvol, Pierre; Thomas, Jean-Léon; Wu, Yan; Peale, Franklin; Watts, Ryan J; Tessier-Lavigne, Marc; Bagri, Anil; Eichmann, Anne

2011-01-18

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature. Copyright © 2011 Elsevier Inc. All rights reserved.

Efficient Direct Reprogramming of Mature Amniotic Cells into Endothelial Cells by ETS Factors and TGFβ Suppression

PubMed Central

Ginsberg, Michael; James, Daylon; Ding, Bi-Sen; Nolan, Daniel; Geng, Fuqiang; Butler, Jason M; Schachterle, William; Pulijaal, Venkat R; Mathew, Susan; Chasen, Stephen T; Xiang, Jenny; Rosenwaks, Zev; Shido, Koji; Elemento, Olivier; Rabbany, Sina Y; Rafii, Shahin

2012-01-01

ETS transcription factors ETV2, FLI1 and ERG1 specify pluripotent stem cells into endothelial cells (ECs). However, these ECs are unstable and drift towards non-vascular cell fates. We show that human mid-gestation c-Kit− lineage-committed amniotic cells (ACs) can be readily reprogrammed into induced vascular endothelial cells (iVECs). Transient ETV2 expression in ACs generated proliferative but immature iVECs, while co-expression with FLI1/ERG1 endowed iVECs with a vascular repertoire and morphology matching mature stable ECs. Brief TGFβ-inhibition functionalized VEGFR2 signaling, augmenting specification of ACs to iVECs. Genome-wide transcriptional analyses showed that iVECs are similar to adult ECs in which vascular-specific genes are turned on and non-vascular genes are silenced. Functionally, iVECs form long-lasting patent vasculature in Matrigel plugs and regenerating livers. Thus, short-term ETV2 expression and TGFβ-inhibition along with constitutive ERG1/FLI1 co-expression reprogram mature ACs into durable and functional iVECs with clinical-scale expansion potential. Public banking of HLA-typed iVECs would establish a vascular inventory for treatment of genetically diverse disorders. PMID:23084400

Lung endothelial HO-1 targeting in vivo using lentiviral miRNA regulates apoptosis and autophagy during oxidant injury

PubMed Central

Zhang, Yi; Jiang, Ge; Sauler, Maor; Lee, Patty J.

2013-01-01

The lung endothelium is a major target for inflammatory and oxidative stress. Heme oxygenase-1 (HO-1) induction is a crucial defense mechanism during oxidant challenges, such as hyperoxia. The role of lung endothelial HO-1during hyperoxia in vivo is not well defined. We engineered lentiviral vectors with microRNA (miRNA) sequences controlled by vascular endothelium cadherin (VE-cad) to study the specific role of lung endothelial HO-1. Wild-type (WT) murine lung endothelial cells (MLECs) or WT mice were treated with lentivirus and exposed to hyperoxia (95% oxygen). We detected HO-1 knockdown (∼55%) specifically in the lung endothelium. MLECs and lungs showed approximately a 2-fold increase in apoptosis and ROS generation after HO-1 silencing. We also demonstrate for the first time that silencing endothelial HO-1 has the same effect on lung injury and survival as silencing HO-1 in multiple lung cell types and that HO-1 regulates caspase 3 activation and autophagy in endothelium during hyperoxia. These studies demonstrate the utility of endothelial-targeted gene silencing in vivo using lentiviral miRNA constructs to assess gene function and that endothelial HO-1 is an important determinant of survival during hyperoxia.—Zhang, Y., Jiang, G., Sauler, M., Lee, P. J. Lung endothelial HO-1 targeting in vivo using lentiviral miRNA regulates apoptosis and autophagy during oxidant injury. PMID:23771928

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier

PubMed Central

2013-01-01

Background Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Methods Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Results Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. Conclusions Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery. PMID:23773766

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier.

PubMed

Watson, P Marc D; Paterson, Judy C; Thom, George; Ginman, Ulrika; Lundquist, Stefan; Webster, Carl I

2013-06-18

Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery.

Glycosaminoglycan-Mediated Downstream Signaling of CXCL8 Binding to Endothelial Cells

PubMed Central

Derler, Rupert; Weber, Corinna; Strutzmann, Elisabeth; Miller, Ingrid; Kungl, Andreas

2017-01-01

The recruitment of leukocytes, mediated by endothelium bound chemokine gradients, is a vital process in inflammation. The highly negatively charged, unbranched polysaccharide family of glycosaminoglycans (GAGs), such as heparan sulfate and chondroitin sulfate mediate chemokine immobilization. Specifically the binding of CXCL8 (interleukin 8) to GAGs on endothelial cell surfaces is known to regulate neutrophil recruitment. Currently, it is not clear if binding of CXCL8 to GAGs leads to endothelial downstream signaling in addition to the typical CXCR1/CXCR2 (C-X-C motif chemokine receptor 1 and 2)-mediated signaling which activates neutrophils. Here we have investigated the changes in protein expression of human microvascular endothelial cells induced by CXCL8. Tumor necrosis factor alpha (TNFα) stimulation was used to mimic an inflammatory state which allowed us to identify syndecan-4 (SDC4) as the potential proteoglycan co-receptor of CXCL8 by gene array, real-time PCR and flow cytometry experiments. Enzymatic GAG depolymerization via heparinase III and chondroitinase ABC was used to emulate the effect of glycocalyx remodeling on CXCL8-induced endothelial downstream signaling. Proteomic analyses showed changes in the expression pattern of a number of endothelial proteins such as Zyxin and Caldesmon involved in cytoskeletal organization, cell adhesion and cell mobility. These results demonstrate for the first time a potential role of GAG-mediated endothelial downstream signaling in addition to the well-known CXCL8-CXCR1/CXCR2 signaling pathways in neutrophils. PMID:29207576

IFN-γ promotes transendothelial migration of CD4+ T cells across the blood-brain barrier.

PubMed

Sonar, Sandip Ashok; Shaikh, Shagufta; Joshi, Nupura; Atre, Ashwini N; Lal, Girdhari

2017-10-01

Transendothelial migration (TEM) of Th1 and Th17 cells across the blood-brain barrier (BBB) has a critical role in the development of experimental autoimmune encephalomyelitis (EAE). How cytokines produced by inflammatory Th1 and Th17 cells damage the endothelial BBB and promote transendothelial migration of immune cells into the central nervous system (CNS) during autoimmunity is not understood. We therefore investigated the effect of various cytokines on brain endothelial cells. Among the various cytokines tested, such as Th1 (IFN-γ, IL-1α, IL-1β, TNF-α, IL-12), Th2 (IL-3, IL-4, IL-6 and IL-13), Th17 (IL-17A, IL-17F, IL-21, IL-22, IL-23, GM-CSF) and Treg-specific cytokines (IL-10 and TGF-β), IFN-γ predominantly showed increased expression of ICAM-1, VCAM-1, MAdCAM-1, H2-K b and I-A b molecules on brain endothelial cells. Furthermore, IFN-γ induced transendothelial migration of CD4 + T cells from the apical (luminal side) to the basal side (abluminal side) of the endothelial monolayer to chemokine CCL21 in a STAT-1-dependent manner. IFN-γ also favored the transcellular route of TEM of CD4 + T cells. Multicolor immunofluorescence and confocal microscopic analysis showed that IFN-γ induced relocalization of ICAM-1, PECAM-1, ZO-1 and VE-cadherin in the endothelial cells, which affected the migration of CD4 + T cells. These findings reveal that the IFN-γ produced during inflammation could contribute towards disrupting the BBB and promoting TEM of CD4 + T cells. Our findings also indicate that strategies that interfere with the activation of CNS endothelial cells may help in controlling neuroinflammation and autoimmunity.

Endothelial permeability is controlled by spatially defined cytoskeletal mechanics: atomic force microscopy force mapping of pulmonary endothelial monolayer.

PubMed

Birukova, Anna A; Arce, Fernando T; Moldobaeva, Nurgul; Dudek, Steven M; Garcia, Joe G N; Lal, Ratnesh; Birukov, Konstantin G

2009-03-01

Actomyosin contraction directly regulates endothelial cell (EC) permeability, but intracellular redistribution of cytoskeletal tension associated with EC permeability is poorly understood. We used atomic force microscopy (AFM), EC permeability assays, and fluorescence microscopy to link barrier regulation, cell remodeling, and cytoskeletal mechanical properties in EC treated with barrier-protective as well as barrier-disruptive agonists. Thrombin, vascular endothelial growth factor, and hydrogen peroxide increased EC permeability, disrupted cell junctions, and induced stress fiber formation. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, hepatocyte growth factor, and iloprost tightened EC barriers, enhanced peripheral actin cytoskeleton and adherens junctions, and abolished thrombin-induced permeability and EC remodeling. AFM force mapping and imaging showed differential distribution of cell stiffness: barrier-disruptive agonists increased stiffness in the central region, and barrier-protective agents decreased stiffness in the center and increased it at the periphery. Attenuation of thrombin-induced permeability correlates well with stiffness changes from the cell center to periphery. These results directly link for the first time the patterns of cell stiffness with specific EC permeability responses.

Development of a microprocessing-assisted cell-systematic evolution of ligands by exponential enrichment method for human umbilical vein endothelial cells

NASA Astrophysics Data System (ADS)

Terazono, Hideyuki; Kim, Hyonchol; Nomura, Fumimasa; Yasuda, Kenji

2016-06-01

We developed a microprocessing-assisted technique to select single-strand DNA aptamers that bind to unknown targets on the cell surface by modifying the conventional systematic evolution of ligands by exponential enrichment (cell-SELEX). Our technique involves 1) the specific selection of target-cell-surface-bound aptamers without leakage of intracellular components by trypsinization and 2) cloning of aptamers by microprocessing-assisted picking of single cells using magnetic beads. After cell-SELEX, the enriched aptamers were conjugated with magnetic beads. The aptamer-magnetic beads conjugates attached to target cells were collected individually by microassisted procedures using microneedles under a microscope. After that, the sequences of the collected magnetic-bead-bound aptamers were identified. As a result, a specific aptamer for the surface of target cells, e.g., human umbilical vein endothelial cells (HUVECs), was chosen and its specificity was examined using other cell types, e.g., HeLa cells. The results indicate that this microprocessing-assisted cell-SELEX method for identifying aptamers is applicable in biological research and clinical diagnostics.

T cells bearing a chimeric antigen receptor against prostate-specific membrane antigen mediate vascular disruption and result in tumor regression

PubMed Central

Santoro, Stephen P.; Kim, Soorin; Motz, Gregory T.; Alatzoglou, Dimitrios; Li, Chunsheng; Irving, Melita; Powell, Daniel J.; Coukos, George

2014-01-01

Aberrant blood vessels enable tumor growth, provide a barrier to immune infiltration, and serve as a source of pro-tumorigenic signals. Targeting tumor blood vessels for destruction, or tumor vascular disruption therapy, can therefore provide significant therapeutic benefit. Here we describe the ability of chimeric antigen receptor (CAR)-bearing T cells to recognize human prostate-specific membrane antigen (hPSMA) on endothelial targets in vitro as well as in vivo. CAR T cells were generated using the anti-PSMA scFv, J591, and the intracellular signaling domains: CD3ζ, CD28, and/or CD137/4-1BB. We found that all anti-hPSMA CAR T cells recognized and eliminated PSMA+ endothelial targets in vitro, regardless of the signaling domain. T cells bearing the 3rd generation anti-hPSMA CAR, P28BBζ, were able to recognize and kill primary human endothelial cells isolated from gynecologic cancers. In addition, the P28BBζ CAR T cells mediated regression of hPSMA-expressing vascular neoplasms in mice. Finally, in murine models of ovarian cancers populated by murine vessels expressing hPSMA, the P28BBζ CAR T cells were able to ablate PSMA+ vessels, cause secondary depletion of tumor cells, and reduce tumor burden. Taken together, these results provide strong rationale for the use of CAR T cells as agents of tumor vascular disruption, specifically those targeting PSMA. PMID:25358763

Molecular crosstalk between tumour and brain parenchyma instructs histopathological features in glioblastoma.

PubMed

Bougnaud, Sébastien; Golebiewska, Anna; Oudin, Anaïs; Keunen, Olivier; Harter, Patrick N; Mäder, Lisa; Azuaje, Francisco; Fritah, Sabrina; Stieber, Daniel; Kaoma, Tony; Vallar, Laurent; Brons, Nicolaas H C; Daubon, Thomas; Miletic, Hrvoje; Sundstrøm, Terje; Herold-Mende, Christel; Mittelbronn, Michel; Bjerkvig, Rolf; Niclou, Simone P

2016-05-31

The histopathological and molecular heterogeneity of glioblastomas represents a major obstacle for effective therapies. Glioblastomas do not develop autonomously, but evolve in a unique environment that adapts to the growing tumour mass and contributes to the malignancy of these neoplasms. Here, we show that patient-derived glioblastoma xenografts generated in the mouse brain from organotypic spheroids reproducibly give rise to three different histological phenotypes: (i) a highly invasive phenotype with an apparent normal brain vasculature, (ii) a highly angiogenic phenotype displaying microvascular proliferation and necrosis and (iii) an intermediate phenotype combining features of invasion and vessel abnormalities. These phenotypic differences were visible during early phases of tumour development suggesting an early instructive role of tumour cells on the brain parenchyma. Conversely, we found that tumour-instructed stromal cells differentially influenced tumour cell proliferation and migration in vitro, indicating a reciprocal crosstalk between neoplastic and non-neoplastic cells. We did not detect any transdifferentiation of tumour cells into endothelial cells. Cell type-specific transcriptomic analysis of tumour and endothelial cells revealed a strong phenotype-specific molecular conversion between the two cell types, suggesting co-evolution of tumour and endothelial cells. Integrative bioinformatic analysis confirmed the reciprocal crosstalk between tumour and microenvironment and suggested a key role for TGFβ1 and extracellular matrix proteins as major interaction modules that shape glioblastoma progression. These data provide novel insight into tumour-host interactions and identify novel stroma-specific targets that may play a role in combinatorial treatment strategies against glioblastoma.

Molecular crosstalk between tumour and brain parenchyma instructs histopathological features in glioblastoma

PubMed Central

Bougnaud, Sébastien; Golebiewska, Anna; Oudin, Anaïs; Keunen, Olivier; Harter, Patrick N.; Mäder, Lisa; Azuaje, Francisco; Fritah, Sabrina; Stieber, Daniel; Kaoma, Tony; Vallar, Laurent; Brons, Nicolaas H.C.; Daubon, Thomas; Miletic, Hrvoje; Sundstrøm, Terje; Herold-Mende, Christel; Mittelbronn, Michel; Bjerkvig, Rolf; Niclou, Simone P.

2016-01-01

The histopathological and molecular heterogeneity of glioblastomas represents a major obstacle for effective therapies. Glioblastomas do not develop autonomously, but evolve in a unique environment that adapts to the growing tumour mass and contributes to the malignancy of these neoplasms. Here, we show that patient-derived glioblastoma xenografts generated in the mouse brain from organotypic spheroids reproducibly give rise to three different histological phenotypes: (i) a highly invasive phenotype with an apparent normal brain vasculature, (ii) a highly angiogenic phenotype displaying microvascular proliferation and necrosis and (iii) an intermediate phenotype combining features of invasion and vessel abnormalities. These phenotypic differences were visible during early phases of tumour development suggesting an early instructive role of tumour cells on the brain parenchyma. Conversely, we found that tumour-instructed stromal cells differentially influenced tumour cell proliferation and migration in vitro, indicating a reciprocal crosstalk between neoplastic and non-neoplastic cells. We did not detect any transdifferentiation of tumour cells into endothelial cells. Cell type-specific transcriptomic analysis of tumour and endothelial cells revealed a strong phenotype-specific molecular conversion between the two cell types, suggesting co-evolution of tumour and endothelial cells. Integrative bioinformatic analysis confirmed the reciprocal crosstalk between tumour and microenvironment and suggested a key role for TGFβ1 and extracellular matrix proteins as major interaction modules that shape glioblastoma progression. These data provide novel insight into tumour-host interactions and identify novel stroma-specific targets that may play a role in combinatorial treatment strategies against glioblastoma. PMID:27049916

Androgen actions on endothelium functions and cardiovascular diseases

PubMed Central

Cai, Jing-Jing; Wen, Juan; Jiang, Wei-Hong; Lin, Jian; Hong, Yuan; Zhu, Yuan-Shan

2016-01-01

The roles of androgens on cardiovascular physiology and pathophysiology are controversial as both beneficial and detrimental effects have been reported. Although the reasons for this discrepancy are unclear, multiple factors such as genetic and epigenetic variation, sex-specificity, hormone interactions, drug preparation and route of administration may contribute. Recently, growing evidence suggests that androgens exhibit beneficial effects on cardiovascular function though the mechanism remains to be elucidated. Endothelial cells (ECs) which line the interior surface of blood vessels are distributed throughout the circulatory system, and play a crucial role in cardiovascular function. Endothelial progenitor cells (EPCs) are considered an indispensable element for the reconstitution and maintenance of an intact endothelial layer. Endothelial dysfunction is regarded as an initiating step in development of atherosclerosis and cardiovascular diseases. The modulation of endothelial functions by androgens through either genomic or nongenomic signal pathways is one possible mechanism by which androgens act on the cardiovascular system. Obtaining insight into the mechanisms by which androgens affect EC and EPC functions will allow us to determine whether androgens possess beneficial effects on the cardiovascular system. This in turn may be critical in the prevention and therapy of cardiovascular diseases. This article seeks to review recent progress in androgen regulation of endothelial function, the sex-specificity of androgen actions, and its clinical applications in the cardiovascular system. PMID:27168746

Peptide-modified PELCL electrospun membranes for regulation of vascular endothelial cells.

PubMed

Zhou, Fang; Jia, Xiaoling; Yang, Yang; Yang, Qingmao; Gao, Chao; Zhao, Yunhui; Fan, Yubo; Yuan, Xiaoyan

2016-11-01

The efficiency of biomaterials used in small vascular repair depends greatly on their ability to interact with vascular endothelial cells (VECs). Rapid endothelialization of the vascular grafts is a promising way to prevent thrombosis and intimal hyperplasia. In this work, modification of electrospun membranes of poly(ethylene glycol)-b-poly(l-lactide-co-ε-caprolactone) (PELCL) by three different peptides for regulation of VECs were studied in order to obtain ideal bioactive biomaterials as small diameter vascular grafts. QK (a mimetic peptide to vascular endothelial growth factor), Arg-Glu-Asp-Val (REDV, a specific adhesive peptide to VECs) and Val-Ala-Pro-Gly (VAPG, a specific adhesive peptide to vascular smooth muscle cells) were investigated. Surface properties of the modified membranes and the response of VECs were verified. It was found that protein adsorption and platelet adhesion were effectively suppressed with the introduction of QK, REDV or VAPG peptides on the PELCL electrospun membranes. Both QK- and REDV-modified electrospun membranes could accelerate the proliferation of VECs in the first 9days, and the QK-modified electrospun membrane promoted cell proliferation more significantly than the REDV-modified one. The REDV-modified PELCL membrane was the most favorable for VECs adhesion than QK- and VAPG-modified membranes. It was suggested that QK- or REDV-modified PELCL electrospun membranes may have great potential applications in cardiovascular biomaterials for rapid endothelialization in situ. Copyright © 2016 Elsevier B.V. All rights reserved.

Nitric oxide releasing hydrogel promotes endothelial differentiation of mouse embryonic stem cells.

PubMed

Nie, Yan; Zhang, Kaiyue; Zhang, Shuaiqiang; Wang, Dan; Han, Zhibo; Che, Yongzhe; Kong, Deling; Zhao, Qiang; Han, Zhongchao; He, Zuo-Xiang; Liu, Na; Ma, Fengxia; Li, Zongjin

2017-11-01

Transplantation of endothelial cells (ECs) holds great promise for treating various kinds of ischemic diseases. However, the major challenge in ECs-based therapy in clinical applications is to provide high quality and enough amounts of cells. In this study, we developed a simple and efficient system to direct endothelial differentiation of mouse embryonic stem cells (ESCs) using a controllable chitosan nitric oxide (NO)-releasing hydrogel (CS-NO). ESCs were plated onto the hydrogel culture system, and the expressions of differentiation markers were measured. We found that the expression of Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) increased obviously under the controlled NO releasing environment. Moreover, the Flk-1 upregulation was accompanied by the activation of the phospho-inositide-3 kinase (PI3K)/Akt signaling. We also found that in the presence of the PI3K inhibitor (LY294002), the endothelial commitment of ESCs was abolished, indicating the importance of Akt phosphorylation in the endothelial differentiation of ESCs. Interestingly, in the absence of NO, the activation of Akt phosphorylation alone by using AKT activator (SC-79) did not profoundly promote the endothelial differentiation of ESCs, suggesting an interdependent relationship between NO and the Akt phosphorylation in driving endothelial fate specification of ESCs. Taken together, we demonstrated that NO releasing in a continuous and controlled manner is a simple and efficient method for directing the endothelial differentiation of ESCs without adding growth factors. Fascinating data continues to show that artificial stem cell niche not only serve as a physical supporting scaffold for stem cells proliferation, but also as a novel platform for directing stem cell differentiation. Because of the lack of proper microenvironment for generating therapeutic endothelial cells (ECs) in vitro, the source of ECs for transplantation is the major limitation in ECs-based therapy to clinical applications. The current study established a feeder cell-free, 2-dimensional culture system for promoting the differentiation processes of embryonic stem cells (ESCs) committed to the endothelial lineage via using a nitric oxide (NO) controlled releasing hydrogel (CS-NO). Notably, the NO releasing from the hydrogel could selectively up-regulate Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) in the absence of growth factors, which was of crucial importance in the endothelial differentiation of ESCs. In summary, the current study proposes a simple and efficient method for directing the endothelial differentiation of ESCs without extra growth factors. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Compound 49b Reduces Inflammatory Markers and Apoptosis after Ocular Blast Injury

DTIC Science & Technology

2014-09-01

drug, Compound 49b, have anti-apoptotic and anti-inflammatory properties in retinal endothelial cells and in a diabetic retinopathy model [7, 10...plays an important role in the development of early diabetic retinopathy and long-term histopathological alterations. Mol Vis, 2009; 15: 1418-28. 12...in other retinal damage models, specifically the streptozotocin- induced type 1 diabetic retinopathy model and retinal endothelial cells cultured in

Endothelial Depletion of Acvrl1 in Mice Leads to Arteriovenous Malformations Associated with Reduced Endoglin Expression

PubMed Central

Allinson, Kathleen R.; Redgrave, Rachael E.; Zhai, Zhenhua; Oh, S. Paul; Fruttiger, Marcus; Arthur, Helen M.

2014-01-01

Rare inherited cardiovascular diseases are frequently caused by mutations in genes that are essential for the formation and/or function of the cardiovasculature. Hereditary Haemorrhagic Telangiectasia is a familial disease of this type. The majority of patients carry mutations in either Endoglin (ENG) or ACVRL1 (also known as ALK1) genes, and the disease is characterized by arteriovenous malformations and persistent haemorrhage. ENG and ACVRL1 encode receptors for the TGFβ superfamily of ligands, that are essential for angiogenesis in early development but their roles are not fully understood. Our goal was to examine the role of Acvrl1 in vascular endothelial cells during vascular development and to determine whether loss of endothelial Acvrl1 leads to arteriovenous malformations. Acvrl1 was depleted in endothelial cells either in early postnatal life or in adult mice. Using the neonatal retinal plexus to examine angiogenesis, we observed that loss of endothelial Acvrl1 led to venous enlargement, vascular hyperbranching and arteriovenous malformations. These phenotypes were associated with loss of arterial Jag1 expression, decreased pSmad1/5/8 activity and increased endothelial cell proliferation. We found that Endoglin was markedly down-regulated in Acvrl1-depleted ECs showing endoglin expression to be downstream of Acvrl1 signalling in vivo. Endothelial-specific depletion of Acvrl1 in pups also led to pulmonary haemorrhage, but in adult mice resulted in caecal haemorrhage and fatal anaemia. We conclude that during development, endothelial Acvrl1 plays an essential role to regulate endothelial cell proliferation and arterial identity during angiogenesis, whilst in adult life endothelial Acvrl1 is required to maintain vascular integrity. PMID:24896812

FITC labeled silica nanoparticles as efficient cell tags: uptake and photostability study in endothelial cells.

PubMed

Veeranarayanan, Srivani; Poulose, Aby Cheruvathoor; Mohamed, Sheikh; Aravind, Athulya; Nagaoka, Yutaka; Yoshida, Yasuhiko; Maekawa, Toru; Kumar, D Sakthi

2012-03-01

The use of fluorescent nanomaterials has gained great importance in the field of medical imaging. Many traditional imaging technologies have been reported utilizing dyes in the past. These methods face drawbacks due to non-specific accumulation and photobleaching of dyes. We studied the uptake and internalization of two different sized (30 nm and 100 nm) FITC labeled silica nanoparticles in Human umbilical vein endothelial cell line. These nanomaterials show high biocompatability and are highly photostable inside live cells for increased period of time in comparison to the dye alone. To our knowledge, we report for the first time the use of 30 nm fluorescent silica nanoparticles as efficient endothelial tags along with the well studied 100 nm particles. We also have emphasized the good photostability of these materials in live cells.

Panoramic view of human corneal endothelial cell layer observed by a prototype slit-scanning wide-field contact specular microscope.

PubMed

Tanaka, Hiroshi; Okumura, Naoki; Koizumi, Noriko; Sotozono, Chie; Sumii, Yasuhiro; Kinoshita, Shigeru

2017-05-01

To observe the most peripheral region of the corneal endothelial cell (CEC) layer as long as optically recordable by use of a prototype slit-scanning wide-field contact specular microscope and produce a panoramic image to evaluate the variation of CEC density with ageing. Observational case series study. This study involved 15 eyes of 15 normal healthy subjects divided into three groups according to age: A (20-40 years), B (41-60 years) and C (>60 years). The corneal endothelial layer of each eye was recorded in a horizontal direction, from nasal to temporal, with a slit-scanning wide-field contact specular microscope (Konan) and endothelial cell density (ECD) in three specific regions (central, mid-peripheral, and peripheral) was automatically calculated via built-in analysis software. Corneal endothelial images from near the surgical limbus to limbus in all eyes were clearly recorded and panoramic images were made by combining still images. ECD in groups A, B and C were 2809±186, 2717±91 and 2580±129 cells/mm 2 at the centre, 2902±242, 2772±97 and 2604±187 cells/mm 2 at the mid-periphery and 2893±308, 2691±99 and 2533±112 cells/mm 2 at the periphery. Significance differences in ECD was found between groups A and C in all regions and groups between B and C at mid-peripheral region. A prototype slit-scanning wide-field contact specular microscope enabled us to record the endothelial layer from the surgical limbus to limbus of the cornea and compare specific areas among subjects, and showed that ECD in each region of the cornea decreases with ageing. UMIN000021264, Results. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

A distinct subset of proinflammatory neutrophils isolated from patients with systemic lupus erythematosus induces vascular damage and synthesizes type I Interferons*

PubMed Central

Denny, Michael F.; Yalavarthi, Srilakshmi; Zhao, Wenpu; Thacker, Seth G.; Anderson, Marc; Sandy, Ashley R.; McCune, W. Joseph; Kaplan, Mariana J.

2010-01-01

Neutrophil-specific genes are abundant in PBMC microarrays from lupus patients due to presence of low density granulocytes (LDGs) in mononuclear cell fractions. The functionality and pathogenicity of these LDGs have not been characterized. We developed a technique to purify LDGs from lupus PBMCs and assessed their phenotype, function and potential role in disease pathogenesis. LDGs, their autologous lupus neutrophils and healthy control neutrophils were compared in their microbicidal and phagocytic capacities, generation of reactive oxygen species, activation status, inflammatory cytokine profile and type I IFN expression and signatures. The capacity of LDGs to kill endothelial cells and their antiangiogenic potential were also assessed. LDGs display an activated phenotype, secrete increased levels of type I IFNs, TNF-α and IFN-γ, but show impaired phagocytic potential. LDGs induce significant endothelial cell cytotoxicity and synthesize sufficient levels of type I IFNs to disrupt the capacity of endothelial progenitor cells to differentiate into mature endothelial cells. Further, LDG depletion restores the functional capacity of endothelial progenitor cells. We conclude that lupus LDGs are proinflammatory and display pathogenic features, including the capacity to synthesize type I IFNs. They may play an important dual role in premature cardiovascular disease development in SLE by simultaneously mediating enhanced vascular damage while inhibiting vascular repair. PMID:20164424

Endothelial markers in malignant vascular tumours of the liver: superiority of QB-END/10 over von Willebrand factor and Ulex europaeus agglutinin 1.

PubMed

Anthony, P P; Ramani, P

1991-01-01

A new monoclonal antibody, QB-END/10, raised against the CD34 antigen in human endothelial cell membranes and haemopoietic progenitor cells, was studied for its usefulness as a marker of neoplastic vascular cells in 21 angiosarcomas and seven malignant haemangioendotheliomas of the liver. QB-END/10 was both more sensitive and more specific than Von Willebrand factor (VWF) and Ulex europaeus 1 agglutinin (UEA-1) in labelling endothelial cells and it did not cross react with epithelia as UEA-1 often does. Staining was uniformly strong and clear in all histological variants of these two tumours. QB-END/10 should prove particularly useful in the differential diagnosis of malignant vascular tumours of the liver.

Endothelial markers in malignant vascular tumours of the liver: superiority of QB-END/10 over von Willebrand factor and Ulex europaeus agglutinin 1.

PubMed Central

Anthony, P P; Ramani, P

1991-01-01

A new monoclonal antibody, QB-END/10, raised against the CD34 antigen in human endothelial cell membranes and haemopoietic progenitor cells, was studied for its usefulness as a marker of neoplastic vascular cells in 21 angiosarcomas and seven malignant haemangioendotheliomas of the liver. QB-END/10 was both more sensitive and more specific than Von Willebrand factor (VWF) and Ulex europaeus 1 agglutinin (UEA-1) in labelling endothelial cells and it did not cross react with epithelia as UEA-1 often does. Staining was uniformly strong and clear in all histological variants of these two tumours. QB-END/10 should prove particularly useful in the differential diagnosis of malignant vascular tumours of the liver. Images PMID:1705261

Endothelin Induces Rapid, Dynamin-mediated Budding of Endothelial Caveolae Rich in ET-B*

PubMed Central

Oh, Phil; Horner, Thierry; Witkiewicz, Halina; Schnitzer, Jan E.

2012-01-01

Clathrin-independent trafficking pathways for internalizing G protein-coupled receptors (GPCRs) remain undefined. Clathrin-mediated endocytosis of receptors including ligand-engaged GPCRs can be very rapid and comprehensive (<10 min). Caveolae-mediated endocytosis of ligands and antibodies has been reported to be much slower in cell culture (≫10 min). Little is known about the role of physiological ligands and specific GPCRs in regulating caveolae trafficking. Here, we find that one receptor for endothelin, ET-B but not ET-A, resides on endothelial cell surfaces in both tissue and cell culture primarily concentrated within caveolae. Reconstituted cell-free budding assays show that endothelins (ETs) induce the fission of caveolae from endothelial plasma membranes purified from rat lungs. Electron microcopy of lung tissue sections and tissue subcellular fractionation both show that endothelin administered intravascularly in rats also induces a significant loss of caveolae at the luminal surface of lung vascular endothelium. Endothelial cells in culture show that ET stimulates very rapid internalization of caveolae and cargo including caveolin, caveolae-targeting antibody, and itself. The ET-B inhibitor BQ788, but not the ET-A inhibitor BQ123, blocks the ET-induced budding of caveolae. Both the pharmacological inhibitor Dynasore and the genetic dominant negative K44A mutant of dynamin prevent this induced budding and internalization of caveolae. Also shRNA lentivirus knockdown of caveolin-1 expression prevents rapid internalization of ET and ET-B. It appears that endothelin can engage ET-B already highly concentrated in caveolae of endothelial cells to induce very rapid caveolae fission and endocytosis. This transport requires active dynamin function. Caveolae trafficking may occur more rapidly than previously documented when it is stimulated by a specific ligand to signaling receptors already located in caveolae before ligand engagement. PMID:22457360

The architecture and biological function of dual antibody-coated dendrimers: enhanced control of circulating tumor cells and their hetero-adhesion to endothelial cells for metastasis prevention.

PubMed

Xie, Jingjing; Zhao, Rongli; Gu, Songen; Dong, Haiyan; Wang, Jichuang; Lu, Yusheng; Sinko, Patrick J; Yu, Ting; Xie, Fangwei; Wang, Lie; Shao, Jingwei; Jia, Lee

2014-01-01

Dissemination of circulating tumor cells (CTCs) in blood and their hetero-adhesion to vascular endothelial bed of distant metastatic secondary organs are the critical steps to initiate cancer metastasis. The rarity of CTCs made their in vivo capture technically challenging. Current techniques by virtue of nanostructured scaffolds monovalently conjugated with a single antibody and/or drug seem less efficient and specific in capturing CTCs. Here, we report a novel platform developed to re-engineer nanoscale dendrimers for capturing CTCs in blood and interfering their adhesion to vascular endothelial bed to form micrometastatic foci. The nanoscale dendrimers were spatiotemporally accommodated with dual antibodies to target two surface biomarkers of colorectal CTCs. Physiochemical characterization, including spectra, fluorescence, electron microscope, dynamic light scattering, electrophoresis, and chromatography analyses, was conducted to demonstrate the successful conjugation of dual antibodies to dendrimer surface. The dual antibody conjugates were able to specifically recognize and bind CTCs, moderately down-regulate the activity of the captured CTCs by arresting them in S phase. The related adhesion assay displayed that the dual antibody conjugates interfered the hetero-adhesion of CTCs to fibronectin (Fn)-coated substrates and human umbilical vein endothelial cells (HUVECs). The dual antibody conjugates also showed the enhanced specificity and efficiency in vitro and in vivo in restraining CTCs in comparison with their single antibody counterparts. The present study showed a novel means to effectively prevent cancer metastatic initiation by binding, restraining CTCs and inhibiting their hetero-adhesion to blood vessels, not by traditional cytotoxic-killing of cancer cells.

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.

Inhibition of Autophagy Rescues Palmitic Acid-induced Necroptosis of Endothelial Cells*

PubMed Central

Khan, Muhammad Jadoon; Rizwan Alam, Muhammad; Waldeck-Weiermair, Markus; Karsten, Felix; Groschner, Lukas; Riederer, Monika; Hallström, Seth; Rockenfeller, Patrick; Konya, Viktoria; Heinemann, Akos; Madeo, Frank; Graier, Wolfgang F.; Malli, Roland

2012-01-01

Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca2+ triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA. PMID:22556413

Both cardiomyocyte and endothelial cell Nox4 mediate protection against hemodynamic overload-induced remodelling.

PubMed

Zhang, Min; Mongue-Din, Heloise; Martin, Daniel; Catibog, Norman; Smyrnias, Ioannis; Zhang, Xiaohong; Yu, Bin; Wang, Minshu; Brandes, Ralf P; Schröder, Katrin; Shah, Ajay M

2018-03-01

NADPH oxidase-4 (Nox4) is an important reactive oxygen species (ROS) source that is upregulated in the haemodynamically overloaded heart. Our previous studies using global Nox4 knockout (Nox4KO) mice demonstrated a protective role of Nox4 during chronic abdominal aortic banding, involving a paracrine enhancement of myocardial capillary density. However, other authors who studied cardiac-specific Nox4KO mice reported detrimental effects of Nox4 in response to transverse aortic constriction (TAC). It has been speculated that these divergent results are due to cell-specific actions of Nox4 (i.e. cardiomyocyte Nox4 detrimental but endothelial Nox4 beneficial) and/or differences in the model of pressure overload (i.e. abdominal banding vs. TAC). This study aimed to (i) investigate whether the effects of Nox4 on pressure overload-induced cardiac remodelling vary according to the pressure overload model and (ii) compare the roles of cardiomyocyte vs. endothelial cell Nox4. Global Nox4KO mice subjected to TAC developed worse cardiac remodelling and contractile dysfunction than wild-type littermates, consistent with our previous results with abdominal aortic banding. Next, we generated inducible cardiomyocyte-specific Nox4 KO mice (Cardio-Nox4KO) and endothelial-specific Nox4 KO mice (Endo-Nox4KO) and studied their responses to pressure overload. Both Cardio-Nox4KO and Endo-Nox4KO developed worse pressure overload-induced cardiac remodelling and dysfunction than wild-type littermates, associated with significant decrease in protein levels of HIF1α and VEGF and impairment of myocardial capillarization. Cardiomyocyte as well as endothelial cell Nox4 contributes to protection against chronic hemodynamic overload-induced cardiac remodelling, at least in part through common effects on myocardial capillary density. © The Author 2017 Published by Oxford University Press on behalf of the European Society of Cardiology.

Induction of Syndecan-4 by Organic-Inorganic Hybrid Molecules with a 1,10-Phenanthroline Structure in Cultured Vascular Endothelial Cells.

PubMed

Hara, Takato; Kojima, Takayuki; Matsuzaki, Hiroka; Nakamura, Takehiro; Yoshida, Eiko; Fujiwara, Yasuyuki; Yamamoto, Chika; Saito, Shinichi; Kaji, Toshiyuki

2017-02-08

Organic-inorganic hybrid molecules constitute analytical tools used in biological systems. Vascular endothelial cells synthesize and secrete proteoglycans, which are macromolecules consisting of a core protein and glycosaminoglycan side chains. Although the expression of endothelial proteoglycans is regulated by several cytokines/growth factors, there may be alternative pathways for proteoglycan synthesis aside from downstream pathways activated by these cytokines/growth factors. Here, we investigated organic-inorganic hybrid molecules to determine a variant capable of analyzing the expression of syndecan-4, a transmembrane heparan-sulfate proteoglycan, and identified 1,10-phenanthroline ( o -Phen) with or without zinc (Zn-Phen) or rhodium (Rh-Phen). Bovine aortic endothelial cells in culture were treated with these compounds, and the expression of syndecan-4 mRNA and core proteins was determined by real-time reverse transcription polymerase chain reaction and Western blot analysis, respectively. Our findings indicated that o -Phen and Zn-Phen specifically and strongly induced syndecan-4 expression in cultured vascular endothelial cells through activation of the hypoxia-inducible factor-1α/β pathway via inhibition of prolyl hydroxylase-domain-containing protein 2. These results demonstrated an alternative pathway involved in mediating induction of endothelial syndecan-4 expression and revealed organic-inorganic hybrid molecules as effective tools for analyzing biological systems.

Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity: Evidence From Mouse and Human Studies.

PubMed

Döring, Yvonne; Noels, Heidi; van der Vorst, Emiel P C; Neideck, Carlos; Egea, Virginia; Drechsler, Maik; Mandl, Manuela; Pawig, Lukas; Jansen, Yvonne; Schröder, Katrin; Bidzhekov, Kiril; Megens, Remco T A; Theelen, Wendy; Klinkhammer, Barbara M; Boor, Peter; Schurgers, Leon; van Gorp, Rick; Ries, Christian; Kusters, Pascal J H; van der Wal, Allard; Hackeng, Tilman M; Gäbel, Gabor; Brandes, Ralf P; Soehnlein, Oliver; Lutgens, Esther; Vestweber, Dietmar; Teupser, Daniel; Holdt, Lesca M; Rader, Daniel J; Saleheen, Danish; Weber, Christian

2017-07-25

The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreER T2 -driven)-specific or smooth muscle cell (SMC, SmmhcCreER T2 - or TaglnCre-driven)-specific deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/β-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. The cell-specific deletion of CXCR4 in arterial endothelial cells (n=12-15) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/β-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis. © 2017 American Heart Association, Inc.

The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells.

PubMed

Legeay, Samuel; Clere, Nicolas; Hilairet, Grégory; Do, Quoc-Tuan; Bernard, Philippe; Quignard, Jean-François; Apaire-Marchais, Véronique; Lapied, Bruno; Faure, Sébastien

2016-06-27

The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation.

Norrin expression in endothelial cells in the developing mouse retina.

PubMed

Lee, Hanjae; Jo, Dong Hyun; Kim, Jin Hyoung; Kim, Jeong Hun

2013-06-01

Norrin, a protein that acts on Frizzled-4 receptor, participates in angiogenesis in a variety of contexts through the Wnt-signaling pathway. Specifically, Norrin is found to play a crucial role in retinal vascularization. Norrin's pivotal role in angiogenesis led us to investigate its expression and the primary source in the developing retina. In this study we demonstrate, for the first time, that Norrin protein is expressed along the retinal blood vessels. The expression of Norrin coincided with the pattern of vascular growth in the developing mouse retina, and its expression was identified from the endothelial cells of the retinal capillaries. Furthermore, Norrin was also expressed on endothelial cells of the developing human retina. Given that Norrin is crucial in the normal development and maintenance of ocular capillaries, our finding provides a hint of the involvement of Norrin in the self generative and protective mechanism of the endothelial cells in the developing retina. Copyright © 2012 Elsevier GmbH. All rights reserved.

Nupr1/Chop signal axis is involved in mitochondrion-related endothelial cell apoptosis induced by methamphetamine

PubMed Central

Cai, D; Huang, E; Luo, B; Yang, Y; Zhang, F; Liu, C; Lin, Z; Xie, W-B; Wang, H

2016-01-01

Methamphetamine (METH) abuse has been a serious global public health problem for decades. Previous studies have shown that METH causes detrimental effects on the nervous and cardiovascular systems. METH-induced cardiovascular toxicity has been, in part, attributed to its destructive effect on vascular endothelial cells. However, the underlying mechanism of METH-caused endothelium disruption has not been investigated systematically. In this study, we identified a novel pathway involved in endothelial cell apoptosis induced by METH. We demonstrated that exposure to METH caused mitochondrial apoptosis in human umbilical vein endothelial cells and rat cardiac microvascular endothelial cells in vitro as well as in rat cardiac endothelial cells in vivo. We found that METH mediated endothelial cell apoptosis through Nupr1–Chop/P53–PUMA/Beclin1 signaling pathway. Specifically, METH exposure increased the expression of Nupr1, Chop, P53 and PUMA. Elevated p53 expression raised up PUMA expression, which initiated mitochondrial apoptosis by downregulating antiapoptotic Bcl-2, followed by upregulation of proapoptotic Bax, resulting in translocation of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria to cytoplasm and activation of caspase-dependent pathways. Interestingly, increased Beclin1, upregulated by Chop, formed a ternary complex with Bcl-2, thereby decreasing the dissociative Bcl-2. As a result, the ratio of dissociative Bcl-2 to Bax was also significantly decreased, which led to translocation of cyto c and initiated more drastic apoptosis. These findings were supported by data showing METH-induced apoptosis was significantly inhibited by silencing Nupr1, Chop or P53, or by PUMA or Beclin1 knockdown. Based on the present data, a novel mechanistic model of METH-induced endothelial cell toxicity is proposed. Collectively, these results highlight that the Nupr1–Chop/P53–PUMA/Beclin1 pathway is essential for mitochondrion-related METH-induced endothelial cell apoptosis and may be a potential therapeutic target for METH-caused cardiovascular toxicity. Future studies using knockout animal models are warranted to substantiate the present findings. PMID:27031958

Targeting NCK-Mediated Endothelial Cell Front-Rear Polarity Inhibits Neo-Vascularization

PubMed Central

Dubrac, Alexandre; Genet, Gael; Ola, Roxana; Zhang, Feng; Pibouin-Fragner, Laurence; Han, Jinah; Zhang, Jiasheng; Thomas, Jean-Léon; Chedotal, Alain; Schwartz, Martin A.; Eichmann, Anne

2015-01-01

Background Sprouting angiogenesis is a key process driving blood vessel growth in ischemic tissues and an important drug target in a number of diseases, including wet macular degeneration and wound healing. Endothelial cells forming the sprout must develop front-rear polarity to allow sprout extension. The adaptor proteins Nck1 and 2 are known regulators of cytoskeletal dynamics and polarity, but their function in angiogenesis is poorly understood. Here we show that the Nck adaptors are required for endothelial cell front-rear polarity and migration downstream of the angiogenic growth factors VEGF-A and Slit2. Methods and Results Mice carrying inducible, endothelial-specific Nck1/2 deletions fail to develop front-rear polarized vessel sprouts and exhibit severe angiogenesis defects in the postnatal retina and during embryonic development. Inactivation of NCK1 and 2 inhibits polarity by preventing Cdc42 and Pak2 activation by VEGF-A and Slit2. Mechanistically, NCK binding to ROBO1 is required for both Slit2 and VEGF induced front-rear polarity. Selective inhibition of polarized endothelial cell migration by targeting Nck1/2 prevents hypersprouting induced by Notch or Bmp signaling inhibition, as well as pathological ocular neovascularization and wound healing. Conclusions These data reveal a novel signal integration mechanism involving NCK1/2, ROBO1/2 and VEGFR2 that controls endothelial cell front-rear polarity during sprouting angiogenesis. PMID:26659946

Targeting NCK-Mediated Endothelial Cell Front-Rear Polarity Inhibits Neovascularization.

PubMed

Dubrac, Alexandre; Genet, Gael; Ola, Roxana; Zhang, Feng; Pibouin-Fragner, Laurence; Han, Jinah; Zhang, Jiasheng; Thomas, Jean-Léon; Chedotal, Alain; Schwartz, Martin A; Eichmann, Anne

2016-01-26

Sprouting angiogenesis is a key process driving blood vessel growth in ischemic tissues and an important drug target in a number of diseases, including wet macular degeneration and wound healing. Endothelial cells forming the sprout must develop front-rear polarity to allow sprout extension. The adaptor proteins Nck1 and 2 are known regulators of cytoskeletal dynamics and polarity, but their function in angiogenesis is poorly understood. Here, we show that the Nck adaptors are required for endothelial cell front-rear polarity and migration downstream of the angiogenic growth factors VEGF-A and Slit2. Mice carrying inducible, endothelial-specific Nck1/2 deletions fail to develop front-rear polarized vessel sprouts and exhibit severe angiogenesis defects in the postnatal retina and during embryonic development. Inactivation of NCK1 and 2 inhibits polarity by preventing Cdc42 and Pak2 activation by VEGF-A and Slit2. Mechanistically, NCK binding to ROBO1 is required for both Slit2- and VEGF-induced front-rear polarity. Selective inhibition of polarized endothelial cell migration by targeting Nck1/2 prevents hypersprouting induced by Notch or Bmp signaling inhibition, and pathological ocular neovascularization and wound healing, as well. These data reveal a novel signal integration mechanism involving NCK1/2, ROBO1/2, and VEGFR2 that controls endothelial cell front-rear polarity during sprouting angiogenesis. © 2015 American Heart Association, Inc.

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.

Crosstalk between reticular adherens junctions and platelet endothelial cell adhesion molecule-1 regulates endothelial barrier function.

PubMed

Fernández-Martín, Laura; Marcos-Ramiro, Beatriz; Bigarella, Carolina L; Graupera, Mariona; Cain, Robert J; Reglero-Real, Natalia; Jiménez, Anaïs; Cernuda-Morollón, Eva; Correas, Isabel; Cox, Susan; Ridley, Anne J; Millán, Jaime

2012-08-01

Endothelial cells provide a barrier between the blood and tissues, which is reduced during inflammation to allow selective passage of molecules and cells. Adherens junctions (AJ) play a central role in regulating this barrier. We aim to investigate the role of a distinctive 3-dimensional reticular network of AJ found in the endothelium. In endothelial AJ, vascular endothelial-cadherin recruits the cytoplasmic proteins β-catenin and p120-catenin. β-catenin binds to α-catenin, which links AJ to actin filaments. AJ are usually described as linear structures along the actin-rich intercellular contacts. Here, we show that these AJ components can also be organized in reticular domains that contain low levels of actin. Reticular AJ are localized in areas where neighboring cells overlap and encompass the cell adhesion receptor platelet endothelial cell adhesion molecule-1 (PECAM-1). Superresolution microscopy revealed that PECAM-1 forms discrete structures distinct from and distributed along AJ, within the voids of reticular domains. Inflammatory tumor necrosis factor-α increases permeability by mechanisms that are independent of actomyosin-mediated tension and remain incompletely understood. Reticular AJ, but not actin-rich linear AJ, were disorganized by tumor necrosis factor-α. This correlated with PECAM-1 dispersal from cell borders. PECAM-1 inhibition with blocking antibodies or small interfering RNA specifically disrupted reticular AJ, leaving linear AJ intact. This disruption recapitulated typical tumor necrosis factor-α-induced alterations of barrier function, including increased β-catenin phosphorylation, without altering the actomyosin cytoskeleton. We propose that reticular AJ act coordinately with PECAM-1 to maintain endothelial barrier function in regions of low actomyosin-mediated tension. Selective disruption of reticular AJ contributes to permeability increase in response to tumor necrosis factor-α.

Cell cycle progression in irradiated endothelial cells cultured from bovine aorta

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

Rubin, D.B.; Drab, E.A.; Ward, W.F.

1988-11-01

Logarithmically growing endothelial cells from bovine aortas were exposed to single doses of 0-10 Gy of 60Co gamma rays, and cell cycle phase distribution and progression were examined by flow cytometry and autoradiography. In some experiments, cells were synchronized in the cell cycle with hydroxyurea (1 mM). Cell number in sham-irradiated control cultures doubled in approximately 24 h. Estimated cycle stage times for control cells were 14.4 h for G1 phase, 7.2 h for S phase, and 2.4 h for G2 + M phase. Irradiated cells demonstrated a reduced distribution at the G1/S phase border at 4 h, and anmore » increased distribution in G2 + M phase at 24 h postirradiation. Autoradiographs of irradiated cells after continuous (3H)thymidine labeling indicated a block in G1 phase or at the G1/S-phase border. The duration of the block was dose dependent (2-3 min/cGy). Progression of the endothelial cells through S phase after removal of the hydroxyurea block also was retarded by irradiation, as demonstrated by increased distribution in early S phase and decreased distribution in late S phase. These results indicate that progression of asynchronous cultured bovine aortic endothelial cells through the DNA synthetic cycle is susceptible to radiation inhibition at specific sites in the cycle, resulting in redistribution and partial synchronization of the population. Thus aortic endothelial cells, diploid cells from a normal tissue, resemble many immortal cell types that have been examined in this regard in vitro.« less

Plasmalemma vesicle-associated protein: A crucial component of vascular homeostasis

PubMed Central

Guo, Ling; Zhang, Hongyan; Hou, Yinglong; Wei, Tianshu; Liu, Ju

2016-01-01

Endothelial subcellular structures, including caveolae, fenestrae and transendothelial channels, are crucial for regulating microvascular function. Plasmalemma vesicle-associated protein (PLVAP) is an endothelial cell-specific protein that forms the stomatal and fenestral diaphragms of blood vessels and regulates basal permeability, leukocyte migration and angiogenesis. Loss of PLVAP in mice leads to premature mortality due to disrupted homeostasis. Evidence from previous studies suggested that PLVAP is involved in cancer, traumatic spinal cord injury, acute ischemic brain disease, transplant glomerulopathy, Norrie disease and diabetic retinopathy. Specifically, PLVAP expression has been demonstrated to be upregulated in these diseases, accompanied by pro-angiogenic or pro-inflammatory responses. Therefore, PLVAP is considered a novel therapeutic target, in addition to an endothelial cell marker. The present review summarizes the structure and functions of PLVAP, and its roles in pathophysiological processes. PMID:27602081

Hydrodynamic shear stress and mass transport modulation of endothelial cell metabolism.

PubMed

Nollert, M U; Diamond, S L; McIntire, L V

1991-09-01

Mammalian cells responds to physical forces by altering their growth rate, morphology, metabolism, and genetic expression. We have studied the mechanism by which these cells detect the presence of mechanical stress and convert this force into intracellular signals. As our model systems, we have studied cultured human endothelial cells, which line the blood vessels and forms the interface between the blood and the vessel wall. These cell responds within minutes to the initiation of flow by increasing their arachidonic acid metabolism and increasing the level of the intracellular second messengers inositol trisphosphate and calcium ion concentration. With continued exposure to arterial levels of wall shear stress for up to 24 h, endothelial cells increase the expression of tissue plasminogen activator (tPA) and tPA messenger RNA (mRNA) and decrease the expression of endothelin peptide and endothelin mRNA. Since the initiation of flow also causes enhanced convective mass transfer to the endothelial cell monolayer, we have investigated the role of enhanced convection of adenosine trisphosphate (ATP) to the cell surface in eliciting a cellular response by monitoring cytosolic calcium concentrations on the single cell level and by computing the concentration profile of ATP in a parallel-plate flow geometry. Our result demonstrate that endothelial cells respond in very specific ways to the initiation of flow and that mass transfer and fluid shear stress can both play a role in the modulation of intracellular signal transduction and metabolism.

The secretome of endothelial progenitor cells promotes brain endothelial cell activity through PI3-kinase and MAP-kinase.

PubMed

Di Santo, Stefano; Seiler, Stefanie; Fuchs, Anna-Lena; Staudigl, Jennifer; Widmer, Hans Rudolf

2014-01-01

Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects.

Serum glucocorticoid inducible kinase (SGK)-1 protects endothelial cells against oxidative stress and apoptosis induced by hyperglycaemia.

PubMed

Ferrelli, Francesca; Pastore, Donatella; Capuani, Barbara; Lombardo, Marco F; Blot-Chabaud, Marcel; Coppola, Andrea; Basello, Katia; Galli, Angelica; Donadel, Giulia; Romano, Maria; Caratelli, Sara; Pacifici, Francesca; Arriga, Roberto; Di Daniele, Nicola; Sbraccia, Paolo; Sconocchia, Giuseppe; Bellia, Alfonso; Tesauro, Manfredi; Federici, Massimo; Della-Morte, David; Lauro, Davide

2015-02-01

Diabetic hyperglycaemia causes endothelial dysfunction mainly by impairing endothelial nitric oxide (NO) production. Moreover, hyperglycaemia activates several noxious cellular pathways including apoptosis, increase in reactive oxygen species (ROS) levels and diminishing Na(+)-K(+) ATPase activity which exacerbate vascular damage. Serum glucocorticoid kinase (SGK)-1, a member of the serine/threonine kinases, plays a pivotal role in regulating NO production through inducible NO synthase activation and other cellular mechanisms. Therefore, in this study, we aimed to investigate the protective role of SGK-1 against hyperglycaemia in human umbilical endothelial cells (HUVECs). We used retrovirus to infect HUVECs with either SGK-1, SGK-1Δ60 (lacking of the N-60 amino acids-increase SGK-1 activity) or SGK-1Δ60KD (kinase-dead constructs). We tested our hypothesis in vitro after high glucose and glucosamine incubation. Increase in SGK-1 expression and activity (SGK-1Δ60) resulted in higher production of NO, inhibition of ROS synthesis and lower apoptosis in endothelial cell after either hyperglycaemia or glucosamine treatments. Moreover, in this study, we showed increased GLUT-1 membrane translocation and Na(+)-K(+) ATPase activity in cell infected with SGK-1Δ60 construct. These results suggest that as in endothelial cells, an increased SGK-1 activity and expression reduces oxidative stress, improves cell survival and restores insulin-mediated NO production after different noxae stimuli. Therefore, SGK-1 may represent a specific target to further develop novel therapeutic options against diabetic vascular disease.

Effect of tributyltin on mammalian endothelial cell integrity.

PubMed

Botelho, G; Bernardini, C; Zannoni, A; Ventrella, V; Bacci, M L; Forni, M

2015-01-01

Tributyltin (TBT), is a man-made pollutants, known to accumulate along the food chain, acting as an endocrine disruptor in marine organisms, with toxic and adverse effects in many tissues including vascular system. Based on the absence of specific studies of TBT effects on endothelial cells, we aimed to evaluate the toxicity of TBT on primary culture of porcine aortic endothelial cells (pAECs), pig being an excellent model to study human cardiovascular disease. pAECs were exposed for 24h to TBT (100, 250, 500, 750 and 1000nM) showing a dose dependent decrease in cell viability through both apoptosis and necrosis. Moreover the ability of TBT (100 and 500nM) to influence endothelial gene expression was investigated at 1, 7 and 15h of treatment. Gene expression of tight junction molecules, occludin (OCLN) and tight junction protein-1 (ZO-1) was reduced while monocyte adhesion and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) levels increased significantly at 1h. IL-6 and estrogen receptors 1 and 2 (ESR-1 and ESR-2) mRNAs, after a transient decrease, reached the maximum levels after 15h of exposure. Finally, we demonstrated that TBT altered endothelial functionality greatly increasing monocyte adhesion. These findings indicate that TBT deeply alters endothelial profile, disrupting their structure and interfering with their ability to interact with molecules and other cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Progress in corneal wound healing

PubMed Central

Ljubimov, Alexander V.; Saghizadeh, Mehrnoosh

2015-01-01

Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and nanocarriers for corneal drug delivery are discussed. Attention is also paid to problems in wound healing understanding and treatment, such as lack of specific epithelial stem cell markers, reliable identification of stem cells, efficient prevention of haze and stromal scar formation, lack of data on wound regulating microRNAs in keratocytes and endothelial cells, as well as virtual lack of targeted systems for drug and gene delivery to select corneal cells. PMID:26197361

Progress in corneal wound healing.

PubMed

Ljubimov, Alexander V; Saghizadeh, Mehrnoosh

2015-11-01

Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β (TGF-β) system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and nanocarriers for corneal drug delivery are discussed. Attention is also paid to problems in wound healing understanding and treatment, such as lack of specific epithelial stem cell markers, reliable identification of stem cells, efficient prevention of haze and stromal scar formation, lack of data on wound regulating microRNAs in keratocytes and endothelial cells, as well as virtual lack of targeted systems for drug and gene delivery to select corneal cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Endothelial jagged-2 sustains hematopoietic stem and progenitor reconstitution after myelosuppression.

PubMed

Guo, Peipei; Poulos, Michael G; Palikuqi, Brisa; Badwe, Chaitanya R; Lis, Raphael; Kunar, Balvir; Ding, Bi-Sen; Rabbany, Sina Y; Shido, Koji; Butler, Jason M; Rafii, Shahin

2017-12-01

Angiocrine factors, such as Notch ligands, supplied by the specialized endothelial cells (ECs) within the bone marrow and splenic vascular niche play an essential role in modulating the physiology of adult hematopoietic stem and progenitor cells (HSPCs). However, the relative contribution of various Notch ligands, specifically jagged-2, to the homeostasis of HSPCs is unknown. Here, we show that under steady state, jagged-2 is differentially expressed in tissue-specific vascular beds, but its expression is induced in hematopoietic vascular niches after myelosuppressive injury. We used mice with EC-specific deletion of the gene encoding jagged-2 (Jag2) to demonstrate that while EC-derived jagged-2 was dispensable for maintaining the capacity of HSPCs to repopulate under steady-state conditions, by activating Notch2 it did contribute to the recovery of HSPCs in response to myelosuppressive conditions. Engraftment and/or expansion of HSPCs was dependent on the expression of endothelial-derived jagged-2 following myeloablation. Additionally, jagged-2 expressed in bone marrow ECs regulated HSPC cell cycle and quiescence during regeneration. Endothelial-deployed jagged-2 triggered Notch2/Hey1, while tempering Notch2/Hes1 signaling in HSPCs. Collectively, these data demonstrate that EC-derived jagged-2 activates Notch2 signaling in HSPCs to promote hematopoietic recovery and has potential as a therapeutic target to accelerate balanced hematopoietic reconstitution after myelosuppression.

Endothelial jagged-2 sustains hematopoietic stem and progenitor reconstitution after myelosuppression

PubMed Central

Guo, Peipei; Poulos, Michael G.; Palikuqi, Brisa; Badwe, Chaitanya R.; Lis, Raphael; Kunar, Balvir; Ding, Bi-Sen; Rabbany, Sina Y.; Butler, Jason M.

2017-01-01

Angiocrine factors, such as Notch ligands, supplied by the specialized endothelial cells (ECs) within the bone marrow and splenic vascular niche play an essential role in modulating the physiology of adult hematopoietic stem and progenitor cells (HSPCs). However, the relative contribution of various Notch ligands, specifically jagged-2, to the homeostasis of HSPCs is unknown. Here, we show that under steady state, jagged-2 is differentially expressed in tissue-specific vascular beds, but its expression is induced in hematopoietic vascular niches after myelosuppressive injury. We used mice with EC-specific deletion of the gene encoding jagged-2 (Jag2) to demonstrate that while EC-derived jagged-2 was dispensable for maintaining the capacity of HSPCs to repopulate under steady-state conditions, by activating Notch2 it did contribute to the recovery of HSPCs in response to myelosuppressive conditions. Engraftment and/or expansion of HSPCs was dependent on the expression of endothelial-derived jagged-2 following myeloablation. Additionally, jagged-2 expressed in bone marrow ECs regulated HSPC cell cycle and quiescence during regeneration. Endothelial-deployed jagged-2 triggered Notch2/Hey1, while tempering Notch2/Hes1 signaling in HSPCs. Collectively, these data demonstrate that EC-derived jagged-2 activates Notch2 signaling in HSPCs to promote hematopoietic recovery and has potential as a therapeutic target to accelerate balanced hematopoietic reconstitution after myelosuppression. PMID:29058691

Astrocyte–endothelial interactions and blood–brain barrier permeability*

PubMed Central

Abbott, N Joan

2002-01-01

The blood–brain barrier (BBB) is formed by brain endothelial cells lining the cerebral microvasculature, and is an important mechanism for protecting the brain from fluctuations in plasma composition, and from circulating agents such as neurotransmitters and xenobiotics capable of disturbing neural function. The barrier also plays an important role in the homeostatic regulation of the brain microenvironment necessary for the stable and co-ordinated activity of neurones. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of more complex tight junctions than in other capillary endothelia, and a number of specific transport and enzyme systems which regulate molecular traffic across the endothelial cells. Transporters characteristic of the BBB phenotype include both uptake mechanisms (e.g. GLUT-1 glucose carrier, L1 amino acid transporter) and efflux transporters (e.g. P-glycoprotein). In addition to a role in long-term barrier induction and maintenance, astrocytes and other cells can release chemical factors that modulate endothelial permeability over a time-scale of seconds to minutes. Cell culture models, both primary and cell lines, have been used to investigate aspects of barrier induction and modulation. Conditioned medium taken from growing glial cells can reproduce some of the inductive effects, evidence for involvement of diffusible factors. However, for some features of endothelial differentiation and induction, the extracellular matrix plays an important role. Several candidate molecules have been identified, capable of mimicking aspects of glial-mediated barrier induction of brain endothelium; these include TGFβ, GDNF, bFGF, IL-6 and steroids. In addition, factors secreted by brain endothelial cells including leukaemia inhibitory factor (LIF) have been shown to induce astrocytic differentiation. Thus endothelium and astrocytes are involved in two-way induction. Short-term modulation of brain endothelial permeability has been shown for a number of small chemical mediators produced by astrocytes and other nearby cell types. It is clear that endothelial cells are involved in both long- and short-term chemical communication with neighbouring cells, with the perivascular end feet of astrocytes being of particular importance. The role of barrier induction and modulation in normal physiology and in pathology is discussed. PMID:12162730

Molecular Engineering of Vector-Based Oncolytic and Imaging Approaches for Advanced Prostate Cancer

DTIC Science & Technology

2007-02-01

has begun to unravel, through the discovery of specific markers for lymphatic endothelial cells and their selective growth factors and receptors.13...Paraffin-embedded sections (5 lm) were stained for a vascular endothelial marker (biotinylated rat anti- mouse CD31 1:100, BD Biosciences, San Jose...CA), lymphatic endothelial markers (rabbit anti-LYVE-1 1:300, RELIATech, Braunschweig, Germany; rabbit anti-Prox1, 1:100, Novus Biologi- cals

Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival

PubMed Central

Sanchez, Erica L.; Carroll, Patrick A.; Thalhofer, Angel B.; Lagunoff, Michael

2015-01-01

Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi’s Sarcoma (KS). KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. KSHV requires the induction of multiple metabolic pathways, including glycolysis and fatty acid synthesis, for the survival of latently infected endothelial cells. Here we demonstrate that latent KSHV infection leads to increased levels of intracellular glutamine and enhanced glutamine uptake. Depletion of glutamine from the culture media leads to a significant increase in apoptotic cell death in latently infected endothelial cells, but not in their mock-infected counterparts. In cancer cells, glutamine is often required for glutaminolysis to provide intermediates for the tri-carboxylic acid (TCA) cycle and support for the production of biosynthetic and bioenergetic precursors. In the absence of glutamine, the TCA cycle intermediates alpha-ketoglutarate (αKG) and pyruvate prevent the death of latently infected cells. Targeted drug inhibition of glutaminolysis also induces increased cell death in latently infected cells. KSHV infection of endothelial cells induces protein expression of the glutamine transporter, SLC1A5. Chemical inhibition of SLC1A5, or knockdown by siRNA, leads to similar cell death rates as glutamine deprivation and, similarly, can be rescued by αKG. KSHV also induces expression of the heterodimeric transcription factors c-Myc-Max and related heterodimer MondoA-Mlx. Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG. Therefore, during latent infection of endothelial cells, KSHV activates and requires the Myc/MondoA-network to upregulate the glutamine transporter, SLC1A5, leading to increased glutamine uptake for glutaminolysis. These findings expand our understanding of the required metabolic pathways that are activated during latent KSHV infection of endothelial cells, and demonstrate a novel role for the extended Myc-regulatory network, specifically MondoA, during latent KSHV infection. PMID:26197457

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.

Interaction of hydroxyapatite nanoparticles with endothelial cells: internalization and inhibition of angiogenesis in vitro through the PI3K/Akt pathway

PubMed Central

Shi, Xingxing; Zhou, Kai; Huang, Fei; Wang, Chen

2017-01-01

Nano-hydroxyapatite (nano-HAP) has been proposed as a better candidate for bone tissue engineering; however, the interactions of nano-HAP with endothelial cells are currently unclear. In this study, HAP nanoparticles (HANPs; 20 nm np20 and 80 nm np80) and micro-sized HAP particles (m-HAP; 12 μm) were employed to explore and characterize cellular internalization, subcellular distribution, effects of HANPs on endothelial cell function and underlying mechanisms using human umbilical vein endothelial cells (HUVECs) as an in vitro model. It was found that HANPs were able to accumulate in the cytoplasm, and both adhesion and uptake of the HANPs followed a function of time; compared to np80, more np20 had been uptaken at the end of the observation period. HANPs were mainly uptaken via clathrin- and caveolin-mediated endocytosis, while macropinocytosis was the main pathway for m-HAP uptake. Unexpectedly, exposure to HANPs suppressed the angiogenic ability of HUVECs in terms of cell viability, cell cycle, apoptosis response, migration and capillary-like tube formation. Strikingly, HANPs reduced the synthesis of nitric oxide (NO) in HUVECs, which was associated with the inhibition of phosphatidylinositol 3-kinase (PI3K) and phosphorylation of eNOS. These findings provide additional insights into specific biological responses as HANPs interface with endothelial cells. PMID:28848353

The metabolism of N-acetylcysteine by human endothelial cells.

PubMed

Cotgreave, I; Moldéus, P; Schuppe, I

1991-06-21

When human umbilical endothelial cells were depleted of their glutathione by incubation in a sulfur amino acid-free medium, subsequent incubation of the cells with this deficient medium supplemented with N-acetylcysteine resulted in a dose-dependent stimulation of the synthesis of cellular glutathione. Similarly, the inclusion of N-acetylcysteine in the medium during the period of depletion of glutathione caused a dose-dependent retardation of the depletion kinetics. In contrast, the incubation of control cells in normal medium supplemented with N-acetylcysteine did not increase cellular glutathione levels above controls. These observations indicate the presence of an N-deacetylase in/on the cells with specificity for N-acetylcysteine. Due to the large surface area of the endothelium in the vasculature it seems likely that endothelial cell N-deacetylation plays a role in the metabolic disposition of N-acetylcysteine, particularly when administered intravenously. N-Acetylcysteine is, however, a relatively poor precursor to glutathione biosynthesis in comparison to cystine. Thus, any cytoprotective, antioxidant effect exerted by N-acetylcysteine on the human endothelium is likely to be due to direct scavenging of reactive intermediates rather than by stimulated glutathione synthesis in the endothelial cells themselves.

The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development.

PubMed

Kazanskaya, Olga; Ohkawara, Bisei; Heroult, Melanie; Wu, Wei; Maltry, Nicole; Augustin, Hellmut G; Niehrs, Christof

2008-11-01

The vertebrate embryonic vasculature develops from angioblasts, which are specified from mesodermal precursors and develop in close association with blood cells. The signals that regulate embryonic vasculogenesis and angiogenesis are incompletely understood. Here, we show that R-spondin 3 (Rspo3), a member of a novel family of secreted proteins in vertebrates that activate Wnt/beta-catenin signaling, plays a key role in these processes. In Xenopus embryos, morpholino antisense knockdown of Rspo3 induces vascular defects because Rspo3 is essential for regulating the balance between angioblast and blood cell specification. In mice, targeted disruption of Rspo3 leads to embryonic lethality caused by vascular defects. Specifically in the placenta, remodeling of the vascular plexus is impaired. In human endothelial cells, R-spondin signaling promotes proliferation and sprouting angiogenesis in vitro, indicating that Rspo3 can regulate endothelial cells directly. We show that vascular endothelial growth factor is an immediate early response gene and a mediator of R-spondin signaling. The results identify Rspo3 as a novel, evolutionarily conserved angiogenic factor in embryogenesis.

Mirtron microRNA-1236 inhibits VEGFR-3 signaling during inflammatory lymphangiogenesis.

PubMed

Jones, Dennis; Li, Yonghao; He, Yun; Xu, Zhe; Chen, Hong; Min, Wang

2012-03-01

Vascular endothelial growth factor receptor(VEGFR)-3 is a critical regulator of developmental and adult vasculogenesis and lymphangiogenesis through its interactions with select members of the VEGF family. The goal of this study was to investigate how VEGFR-3 expression is regulated during inflammatory lymphangiogenesis. In this study, we present for the first time evidence that VEGFR-3 can be negatively regulated by a mirtron, hsa-miR-1236 (miR-1236), which is expressed in primary human lymphatic endothelial cells. In human lymphatic endothelial cells, miR-1236 is upregulated in response to IL-1β, a negative regulator of VEGFR-3. miR-1236 binds the 3' untranslated region of Vegfr3, resulting in translational inhibition. Overexpression of miR-1236 significantly decreased expression of VEGFR-3, but not VEGFR-2, in human lymphatic endothelial cells. Compared to a control miR, overexpression of miR-1236 also led to decreased VEGFR-3 signaling. However, VEGFR-2-specific signaling was not affected. miR-1236 can attenuate human lymphatic endothelial cell migration and tube formation, as well as in vivo lymphangiogenesis. Our data suggest that miR-1236 may function as a negative regulator of VEGFR-3 signaling during inflammatory lymphangiogenesis.

Endocan and the respiratory system: a review.

PubMed

Kechagia, Maria; Papassotiriou, Ioannis; Gourgoulianis, Konstantinos I

2016-01-01

Endocan, formerly called endothelial cell-specific molecule 1, is an endothelial cell-associated proteoglycan that is preferentially expressed by renal and pulmonary endothelium. It is upregulated by proangiogenic molecules as well as by pro-inflammatory cytokines, and since it reflects endothelial activation and dysfunction, it is regarded as a novel tissue and blood-based relevant biomarker. As such, it is increasingly being researched and evaluated in a wide spectrum of healthy and disease pathophysiological processes. Here, we review the present scientific knowledge on endocan, with emphasis on the evidence that underlines its possible clinical value as a prognostic marker in several malignant, inflammatory and obstructive disorders of the respiratory system.

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.

Endothelial microparticles: Pathogenic or passive players in endothelial dysfunction in autoimmune rheumatic diseases?

PubMed

McCarthy, E M; Wilkinson, F L; Parker, B; Alexander, M Y

2016-11-01

Autoimmune rheumatic diseases are characterised by systemic inflammation and complex immunopathology, with an increased risk of cardiovascular disease, initiated by endothelial dysfunction in a chronic inflammatory environment. Endothelial microparticles (EMPs) are released into the circulation from activated endothelial cells and may therefore, reflect disease severity, vascular and endothelial dysfunction, that could influence disease pathogenesis via autocrine/paracrine signalling. The exact function of EMPs in rheumatic disease remains unknown, and this has initiated research to elucidate EMP composition and function, which may be determined by the mode of endothelial activation and the micro environment. To date, EMPs are thought to play a role in angiogenesis, thrombosis and inflammation by transferring specific proteins and microRNAs (miRs) to target cells. Here, we review the mechanisms underlying the generation and composition of EMPs and the clinical and experimental studies describing the involvement of EMPs in rheumatic diseases, since we have previously shown endothelial dysfunction and an elevated risk of cardiovascular disease are characteristics in systemic lupus erythematosus. We will also discuss the potential of EMPs as future biomarkers of cardiovascular risk in these diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis.

PubMed

Poulos, Michael G; Ramalingam, Pradeep; Gutkin, Michael C; Kleppe, Maria; Ginsberg, Michael; Crowley, Michael J P; Elemento, Olivier; Levine, Ross L; Rafii, Shahin; Kitajewski, Jan; Greenblatt, Matthew B; Shim, Jae-Hyuck; Butler, Jason M

2016-12-21

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens.

Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis

PubMed Central

Poulos, Michael G.; Ramalingam, Pradeep; Gutkin, Michael C.; Kleppe, Maria; Ginsberg, Michael; Crowley, Michael J. P.; Elemento, Olivier; Levine, Ross L.; Rafii, Shahin; Kitajewski, Jan; Greenblatt, Matthew B.; Shim, Jae-Hyuck; Butler, Jason M.

2016-01-01

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens. PMID:28000664

The glutathione mimic ebselen inhibits oxidative stress but not endoplasmic reticulum stress in endothelial cells.

PubMed

Ahwach, Salma Makhoul; Thomas, Melanie; Onstead-Haas, Luisa; Mooradian, Arshag D; Haas, Michael J

2015-08-01

Reactive oxygen species are associated with cardiovascular disease, diabetes, and atherosclerosis, yet the use of antioxidants in clinical trials has been ineffective at improving outcomes. In endothelial cells, high-dextrose-induced oxidative stress and endoplasmic reticulum stress promote endothelial dysfunction leading to the recruitment and activation of peripheral blood lymphocytes and the breakdown of barrier function. Ebselen, a glutathione peroxidase 1 (GPX1) mimic, has been shown to improve β-cell function in diabetes and prevent atherosclerosis. To determine if ebselen inhibits both oxidative stress and endoplasmic reticulum (ER) stress in endothelial cells, we examined its effects in human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC) with and without high-dextrose. Oxidative stress and ER stress were measured by 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride chemiluminescence and ER stress alkaline phosphatase assays, respectively. GPX1 over-expression and knockdown were performed by transfecting cells with a GPX1 expression construct or a GPX1-specific siRNA, respectively. Ebselen inhibited dextrose-induced oxidative stress but not ER stress in both HUVEC and HCAEC. Ebselen also had no effect on tunicamycin-induced ER stress in HCAEC. Furthermore, augmentation of GPX1 activity directly by sodium selenite supplementation or transfection of a GPX1 expression plasmid decreased dextrose-induced oxidative stress but not ER stress, while GPX1 knockout enhanced oxidative stress but had no effect on ER stress. These results suggest that ebselen targets only oxidative stress but not ER stress. Copyright © 2015. Published by Elsevier Inc.

Bone marrow endothelial progenitors augment atherosclerotic plaque regression in a mouse model of plasma lipid lowering

PubMed Central

Yao, Longbiao; Heuser-Baker, Janet; Herlea-Pana, Oana; Iida, Ryuji; Wang, Qilong; Zou, Ming-Hui; Barlic-Dicen, Jana

2012-01-01

The major event initiating atherosclerosis is hypercholesterolemia-induced disruption of vascular endothelium integrity. In settings of endothelial damage, endothelial progenitor cells (EPCs) are mobilized from bone marrow into circulation and home to sites of vascular injury where they aid endothelial regeneration. Given the beneficial effects of EPCs in vascular repair, we hypothesized that these cells play a pivotal role in atherosclerosis regression. We tested our hypothesis in the atherosclerosis-prone mouse model in which hypercholesterolemia, one of the main factors affecting EPC homeostasis, is reversible (Reversa mice). In these mice normalization of plasma lipids decreased atherosclerotic burden; however, plaque regression was incomplete. To explore whether endothelial progenitors contribute to atherosclerosis regression, bone marrow EPCs from a transgenic strain expressing green fluorescent protein under the control of endothelial cell-specific Tie2 promoter (Tie2-GFP+) were isolated. These cells were then adoptively transferred into atheroregressing Reversa recipients where they augmented plaque regression induced by reversal of hypercholesterolemia. Advanced plaque regression correlated with engraftment of Tie2-GFP+ EPCs into endothelium and resulted in an increase in atheroprotective nitric oxide and improved vascular relaxation. Similarly augmented plaque regression was also detected in regressing Reversa mice treated with the stem cell mobilizer AMD3100 which also mobilizes EPCs to peripheral blood. We conclude that correction of hypercholesterolemia in Reversa mice leads to partial plaque regression that can be augmented by AMD3100 treatment or by adoptive transfer of EPCs. This suggests that direct cell therapy or indirect progenitor cell mobilization therapy may be used in combination with statins to treat atherosclerosis. PMID:23081735

Inhibition of endothelial receptor expression and of T-cell ligand activity by mycophenolate mofetil.

PubMed

Blaheta, R A; Leckel, K; Wittig, B; Zenker, D; Oppermann, E; Harder, S; Scholz, M; Weber, S; Schuldes, H; Encke, A; Markus, B H

1998-12-01

The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.

Differential intra-endothelial delivery of polymer nanocarriers targeted to distinct PECAM-1 epitopes

PubMed Central

Garnacho, Carmen; Albelda, Steven M.; Muzykantov, Vladimir R.; Muro, Silvia

2008-01-01

Coupling drug carriers to antibodies for targeting endothelial cells (ECs) may improve treatment of vascular and pulmonary diseases. Selecting antibodies that deliver carriers to the cell surface or intracellularly may further optimize specifcity of interventions. We studied antibody-directed targeting of nanocarriers to platelet–endothelial cell adhesion molecule (PECAM)-1, an endothelial glycoprotein containing 6 Ig-like extracellular domains. PECAM-1 antibodies bind to ECs without internalization, but ECs internalize by endocytosis nanocarriers carrying multiple copies of anti-PECAM (anti-PECAM/NCs). To determine whether binding and intracellular transport of anti-PECAM/NCs depend on the epitope engaged, we targeted five PECAM-1 epitopes: mAb35, mAb37 and mAb62 (membrane-distal Ig domain 1), mAbGi34 (Ig domains 2/3), and mAb4G6 (membrane-proximal Ig domain 6). The antibodies bound to ECs regardless of the epitope proximity to the plasmalemma, whereas 130 nm diameter nanocarriers only targeted effectively distal domains (mAb4G6/NCs did not bind to ECs). ECs internalized mAb35, mAb62, and mAbGi34 carriers regardless of their size (0.13 to 5 µm diameter), yet they did not internalize mAb37/NCs. After internalization, mAb62/NCs trafficked to lysosomes within 2–3 h, whereas mAb35/NCs had prolonged residence in pre-lysosomal vesicles. Therefore, endothelial binding, endocytosis, and intracellular transport of anti-PECAM/NCs are epitope-specific. This paradigm will guide the design of endothelial drug delivery systems providing specific cellular localizations. PMID:18606202

Transfection of the Human Heme Oxygenase Gene Into Rabbit Coronary Microvessel Endothelial Cells: Protective Effect Against Heme and Hemoglobin Toxicity

NASA Astrophysics Data System (ADS)

Abraham, N. G.; Lavrovsky, Y.; Schwartzman, M. L.; Stoltz, R. A.; Levere, R. D.; Gerritsen, M. E.

1995-07-01

Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an ≈3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with >85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Efficacy for lung metastasis induced by the allogeneic bEnd3 vaccine in mice.

PubMed

Zhao, Jun; Lu, Jing; Zhou, Lurong; Zhao, Jimin; Dong, Ziming

2018-05-04

The mouse brain microvascular endothelial cell line bEnd.3 was used to develop a vaccine and its anti-tumor effect on lung metastases was observed in immunized mice. Mouse bEnd.3 cells cultured in-vitro and then fixed with glutaraldehyde was used to immunize mice; mice were challenged with the metastatic cancer cell line U14, and changes in metastatic cancer tissues were observed through hematoxylin and eosin staining. Carboxyfluorescein succinimidyl amino ester (CSFE) and propidium iodide (PI) were used to detect cytotoxic activity of spleen T lymphocytes; the ratio of CD3 + and CD8 + T-cell sub-sets was determined by flow cytometry. Enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and immunoblot were used to examine the specific response of the antisera of immunized mice. The number of metastatic nodules in bEnd.3 and human umbilical vein endothelial cell (HUVEC) vaccine groups was less than NIH3T3 vaccine group and phosphate buffered saline (PBS) control group. The bEnd.3-induced and HUVEC-induced cytotoxic T-lymphocytes (CTLs) showed significant lytic activity against bEnd.3 and HUVEC target cells, while the antisera of mice in bEnd.3 and HUVEC vaccine groups showed specific immune responses to membrane proteins and inhibited target cell proliferation in-vitro. Immunoblot results showed specific bands at 180KD and 220KD in bEnd.3 and at 130 kD and 220 kD in HUVEC lysates. Allogeneic bEnd.3 vaccine induced an active and specific immune response to tumor vascular endothelial cells that resulted in production of antibodies against the proliferation antigens VEGF-R II, integrin, Endog etc. Immunization with this vaccine inhibited lung metastasis of cervical cancer U14 cells and prolonged the survival of these mice.

The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

PubMed

Rezaei, Maryam; Cao, Jiahui; Friedrich, Katrin; Kemper, Björn; Brendel, Oliver; Grosser, Marianne; Adrian, Manuela; Baretton, Gustavo; Breier, Georg; Schnittler, Hans-Joachim

2018-01-01

The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased β-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

Deficiency of endothelial CXCR4 reduces reendothelialization and enhances neointimal hyperplasia after vascular injury in atherosclerosis-prone mice.

PubMed

Noels, Heidi; Zhou, Baixue; Tilstam, Pathricia V; Theelen, Wendy; Li, Xiaofeng; Pawig, Lukas; Schmitz, Corinna; Akhtar, Shamima; Simsekyilmaz, Sakine; Shagdarsuren, Erdenechimeg; Schober, Andreas; Adams, Ralf H; Bernhagen, Jürgen; Liehn, Elisa A; Döring, Yvonne; Weber, Christian

2014-06-01

The Cxcl12/Cxcr4 chemokine ligand/receptor axis mediates the mobilization of smooth muscle cell progenitors, driving injury-induced neointimal hyperplasia. This study aimed to investigate the role of endothelial Cxcr4 in neointima formation. β-Galactosidase staining using bone marrow x kinase (Bmx)-CreER(T2) reporter mice and double immunofluorescence revealed an efficient and endothelial-specific deletion of Cxcr4 in Bmx-CreER(T2+) compared with Bmx-CreER(T2-) Cxcr4-floxed apolipoprotein E-deficient (Apoe(-/-)) mice (referred to as Cxcr4(EC-KO)ApoE(-/-) and Cxcr4(EC-WT) ApoE(-/-), respectively). Endothelial Cxcr4 deficiency significantly increased wire injury-induced neointima formation in carotid arteries from Cxcr4(EC-KO)ApoE(-/-) mice. The lesions displayed a higher number of macrophages, whereas the smooth muscle cell and collagen content were reduced. This was associated with a significant reduction in reendothelialization and endothelial cell proliferation in injured Cxcr4(EC-KO)ApoE(-/-) carotids compared with Cxcr4(EC-WT)ApoE(-/-) controls. Furthermore, stimulation of human aortic endothelial cells with chemokine (C-X-C motif) ligand 12 (CXCL12) significantly enhanced their wound-healing capacity in an in vitro scratch assay, an effect that could be reversed with the CXCR4 antagonist AMD3100. Also, flow cytometric analysis showed a reduced mobilization of Sca1(+)Flk1(+)Cd31(+) and of Lin(-)Sca1(+) progenitors in Cxcr4(EC-KO) ApoE(-/-) mice after vascular injury, although Cxcr4 surface expression was unaltered. No differences could be detected in plasma concentrations of Cxcl12, vascular endothelial growth factor, sphingosine 1-phosphate, or Flt3 (fms-related tyrosine kinase 3) ligand, all cytokines with an established role in progenitor cell mobilization. Nonetheless, double immunofluorescence revealed a significant reduction in local endothelial Cxcl12 staining in injured carotids from Cxcr4(EC-KO)ApoE(-/-) mice. Endothelial Cxcr4 is crucial for efficient reendothelialization after vascular injury through endothelial wound healing and proliferation, and through the mobilization of Sca1(+)Flk1(+)Cd31(+) cells, often referred to as circulating endothelial progenitor cells. © 2014 American Heart Association, Inc.

Transcellular transport of cobalamin in aortic endothelial cells.

PubMed

Hannibal, Luciana; Bolisetty, Keerthana; Axhemi, Armend; DiBello, Patricia M; Quadros, Edward V; Fedosov, Sergey; Jacobsen, Donald W

2018-05-09

Cobalamin [Cbl (or B 12 )] deficiency causes megaloblastic anemia and a variety of neuropathies. However, homeostatic mechanisms of cyanocobalamin (CNCbl) and other Cbls by vascular endothelial cells are poorly understood. Herein, we describe our investigation into whether cultured bovine aortic endothelial cells (BAECs) perform transcytosis of B 12 , namely, the complex formed between serum transcobalamin and B 12 , designated as holo-transcobalamin (holo-TC). We show that cultured BAECs endocytose [ 57 Co]-CNCbl-TC (source material) via the CD320 receptor. The bound Cbl is transported across the cell both via exocytosis in its free form, [ 57 Co]-CNCbl, and via transcytosis as [ 57 Co]-CNCbl-TC. Transcellular mobilization of Cbl occurred in a bidirectional manner. A portion of the endocytosed [ 57 Co]-CNCbl was enzymatically processed by methylmalonic aciduria combined with homocystinuria type C (cblC) with subsequent formation of hydroxocobalamin, methylcobalamin, and adenosylcobalamin, which were also transported across the cell in a bidirectional manner. This demonstrates that transport mechanisms for Cbl in vascular endothelial cells do not discriminate between various β-axial ligands of the vitamin. Competition studies with apoprotein- and holo-TC and holo-intrinsic factor showed that only holo-TC was effective at inhibiting transcellular transport of Cbl. Incubation of BAECs with a blocking antibody against the extracellular domain of the CD320 receptor inhibited uptake and transcytosis by ∼40%. This study reveals that endothelial cells recycle uncommitted intracellular Cbl for downstream usage by other cell types and suggests that the endothelium is self-sufficient for the specific acquisition and subsequent distribution of circulating B 12 via the CD320 receptor. We posit that the endothelial lining of the vasculature is an essential component for the maintenance of serum-tissue homeostasis of B 12 .-Hannibal, L., Bolisetty, K., Axhemi, A., DiBello, P. M., Quadros, E. V., Fedosov, S., Jacobsen, D. W. Transcellular transport of cobalamin in aortic endothelial cells.

Biology and flow cytometry of proangiogenic hematopoietic progenitors cells.

PubMed

Rose, Jonathan A; Erzurum, Serpil; Asosingh, Kewal

2015-01-01

During development, hematopoiesis and neovascularization are closely linked to each other via a common bipotent stem cell called the hemangioblast that gives rise to both hematopoietic cells and endothelial cells. In postnatal life, this functional connection between the vasculature and hematopoiesis is maintained by a subset of hematopoietic progenitor cells endowed with the capacity to differentiate into potent proangiogenic cells. These proangiogenic hematopoietic progenitors comprise a specific subset of bone marrow (BM)-derived cells that homes to sites of neovascularization and possess potent paracrine angiogenic activity. There is emerging evidence that this subpopulation of hematopoietic progenitors plays a critical role in vascular health and disease. Their angiogenic activity is distinct from putative "endothelial progenitor cells" that become structural cells of the endothelium by differentiation into endothelial cells. Proangiogenic hematopoietic progenitor cell research requires multidisciplinary expertise in flow cytometry, hematology, and vascular biology. This review provides a comprehensive overview of proangiogenic hematopoietic progenitor cell biology and flow cytometric methods to detect these cells in the peripheral blood circulation and BM. © 2014 International Society for Advancement of Cytometry.

Eicosapentaenoic acid improves endothelial function and nitric oxide bioavailability in a manner that is enhanced in combination with a statin.

PubMed

Mason, R Preston; Dawoud, Hazem; Jacob, Robert F; Sherratt, Samuel C R; Malinski, Tadeusz

2018-07-01

The endothelium exerts many vasoprotective effects that are largely mediated by release of nitric oxide (NO). Endothelial dysfunction represents an early but reversible step in atherosclerosis and is characterized by a reduction in the bioavailability of NO. Previous studies have shown that eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), and statins individually improve endothelial cell function, but their effects in combination have not been tested. Through a series of in vitro experiments, this study evaluated the effects of a combined treatment of EPA and the active metabolite of atorvastatin (ATM) on endothelial cell function under conditions of oxidative stress. Specifically, the comparative and time-dependent effects of these agents on endothelial dysfunction were examined by measuring the levels of NO and peroxynitrite (ONOO - ) released from human umbilical vein endothelial cells (HUVECs). The data suggest that combined treatment with EPA and ATM is beneficial to endothelial function and was unique to EPA and ATM since similar improvements could not be recapitulated by substituting another O3FA docosahexaenoic acid (DHA) or other TG-lowering agents such as fenofibrate, niacin, or gemfibrozil. Comparable beneficial effects were observed when HUVECs were pretreated with EPA and ATM before exposure to oxidative stress. Interestingly, the kinetics of EPA-based protection of endothelial function in response to oxidation were found to be significantly different than those of DHA. Lastly, the beneficial effects on endothelial function generated by combined treatment of EPA and ATM were reproduced when this study was expanded to an ex vivo model utilizing rat glomerular endothelial cells. Taken together, these findings suggest that a combined treatment of EPA and ATM can inhibit endothelial dysfunction that occurs in response to conditions such as hyperglycemia, oxidative stress, and dyslipidemia. Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

Effect of amplitude and duration of impulsive pressure on endothelial permeability in in vitro fluid percussion trauma

PubMed Central

2014-01-01

Background Intracranial pressure changes during head impact cause brain injuries such as vasogenic edema and cerebral contusion. However, the influence of impulsive pressure on endothelial function has not yet been fully studied in vitro. In this study, we developed a pressure loading device that produced positive and negative pressures by modifying an in vitro fluid percussion model and examined the effects of the amplitude and duration of the pressures on endothelial permeability. Methods Human umbilical vein endothelial cells were subjected to three types of positive pressure (average amplitude/average duration of 352 kPa/23 ms, 73 kPa/27 ms, and 70 kPa/44 ms) and three types of negative pressure (−72 kPa/41 ms, −67 kPa/104 ms, and −91 kPa/108 ms), and the transendothelial electrical resistance (TEER) was measured between 15 min and 24 h after pressure loading for quantifying the formation of an integral monolayer of endothelial cells. After loading, vascular endothelial- (VE-) cadherin, an endothelium-specific cell-cell adhesion molecule involved in endothelial barrier function, was stained and observed using fluorescence microscopy. Results The pressure loading device could produce positive pressure pulses with amplitudes of 53–1348 kPa and durations of 9–29.1 ms and negative pressure pulses with amplitudes of −52–−93 kPa and durations of 42.9–179.5 ms. The impulsive pressure reduced the TEER associated with the change in VE-cadherin localization. Additionally, TEER decreased considerably at 15 min and 6 h post-loading, with these changes being significant in positive pressure with larger amplitude and shorter duration and in all types of negative pressures compared to pre-loading. Conclusions The changes in intracranial pressure during head impact impair endothelial barrier function by the disruption of the integrity of endothelial cell-cell junctions, and the degree of increase in endothelial permeability depends on the amplitude, duration, and direction (compressive and tensile) of the impulsive pressure. PMID:24739360

Tissue-specific mechanical and geometrical control of cell viability and actin cytoskeleton alignment

NASA Astrophysics Data System (ADS)

Wang, Dong; Zheng, Wenfu; Xie, Yunyan; Gong, Peiyuan; Zhao, Fang; Yuan, Bo; Ma, Wanshun; Cui, Yan; Liu, Wenwen; Sun, Yi; Piel, Matthieu; Zhang, Wei; Jiang, Xingyu

2014-08-01

Different tissues have specific mechanical properties and cells of different geometries, such as elongated muscle cells and polygonal endothelial cells, which are precisely regulated during embryo development. However, the mechanisms that underlie these processes are not clear. Here, we built an in vitro model to mimic the cellular microenvironment of muscle by combining both mechanical stretch and geometrical control. We found that mechanical stretch was a key factor that determined the optimal geometry of myoblast C2C12 cells under stretch, whereas vascular endothelial cells and fibroblasts had no such dependency. We presented the first experimental evidence that can explain why myoblasts are destined to take the elongated geometry so as to survive and maintain parallel actin filaments along the stretching direction. The study is not only meaningful for the research on myogenesis but also has potential application in regenerative medicine.

Inhibition of cell adhesion by anti–P-selectin aptamer: a new potential therapeutic agent for sickle cell disease

PubMed Central

Gutsaeva, Diana R.; Parkerson, James B.; Yerigenahally, Shobha D.; Kurz, Jeffrey C.; Schaub, Robert G.; Ikuta, Tohru

2011-01-01

Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti–P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti–P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti–P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti–P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti–P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti–P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti–P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti–P-selectin aptamer may be useful as a novel therapeutic agent for SCD. PMID:20926770

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

Establishment of cell-cell junctions depends on the oligomeric states of VE-cadherin

PubMed Central

Bibert, Stéphanie; Ayari, Hélène; Riveline, Daniel; Concord, Evelyne; Hermant, Bastien; Vernet, Thierry; Gulino-Debrac, Danièle

2008-01-01

Specifically expressed at intercellular adherens junctions of endothelial cells, VE-cadherin is a receptor that exhibits particular self-association properties. Indeed, in vitro studies demonstrated that the extracellular part of VE-cadherin elaborates Ca++-dependent hexameric structures. We hypothesized that this assembly could be at the basis of a new cadherin-mediated cell-cell adhesion mechanism. To verify this assumption, we first demonstrated that VE-cadherin can elaborate hexamers at the cell surface of confluent endothelial cells. Second, mutations were introduced within the extracellular part of VE-cadherin to destabilize the hexamer. Following an in vitro screening, three mutants were selected, among which, one is able to elaborate only dimers. The selected mutations were expressed as C-terminal Green Fluorescent Protein fusions in CHO cells. Despite their capacity to elaborate nascent cell-cell contacts, the mutants seem to be rapidly degraded and or internalized. Altogether, our results suggest that the formation of VE-cadherin hexamers protects this receptor and might allow the elaboration of mature endothelial cell-cell junctions. PMID:18343874

Endothelial progenitor cells from human dental pulp-derived iPS cells as a therapeutic target for ischemic vascular diseases.

PubMed

Yoo, Chae Hwa; Na, Hee-Jun; Lee, Dong-Seol; Heo, Soon Chul; An, Yuri; Cha, Junghwa; Choi, Chulhee; Kim, Jae Ho; Park, Joo-Cheol; Cho, Yee Sook

2013-11-01

Human dental pulp cells (hDPCs) are a valuable source for the generation of patient-specific human induced pluripotent stem cells (hiPSCs). An advanced strategy for the safe and efficient reprogramming of hDPCs and subsequent lineage-specific differentiation is a critical step toward clinical application. In present research, we successfully generated hDPC-iPSCs using only two non-oncogenic factors: Oct4 and Sox2 (2F hDPC-hiPSCs) and evaluated the feasibility of hDPC-iPSCs as substrates for endothelial progenitor cells (EPCs), contributing to EPC-based therapies. Under conventional differentiation conditions, 2F hDPC-hiPSCs showed higher differentiation efficiency, compared to hiPSCs from other cell types, into multipotent CD34(+) EPCs (2F-hEPCs) capable to differentiate into functional endothelial and smooth muscle cells. The angiogenic and neovasculogenic activities of 2F-hEPCs were confirmed using a Matrigel plug assay in mice. In addition, the therapeutic effects of 2F-hEPC transplantation were confirmed in mouse models of hind-limb ischemia and myocardial infarction. Importantly, 2F-EPCs effectively integrated into newly formed vascular structures and enhanced neovascularization via likely both direct and indirect paracrine mechanisms. 2F hDPC-hiPSCs have a robust capability for the generation of angiogenic and vasculogenic EPCs, representing a strategy for patient-specific EPC therapies and disease modeling, particularly for ischemic vascular diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cytoprotective signaling by activated protein C requires protease-activated receptor-3 in podocytes

PubMed Central

Madhusudhan, Thati; Wang, Hongjie; Straub, Beate K.; Gröne, Elisabeth; Zhou, Qianxing; Shahzad, Khurrum; Müller-Krebs, Sandra; Schwenger, Vedat; Gerlitz, Bruce; Grinnell, Brian W.; Griffin, John H.; Reiser, Jochen; Gröne, Hermann-Josef; Esmon, Charles T.; Nawroth, Peter P.

2012-01-01

The cytoprotective effects of activated protein C (aPC) are well established. In contrast, the receptors and signaling mechanism through which aPC conveys cytoprotection in various cell types remain incompletely defined. Thus, within the renal glomeruli, aPC preserves endothelial cells via a protease-activated receptor-1 (PAR-1) and endothelial protein C receptor-dependent mechanism. Conversely, the signaling mechanism through which aPC protects podocytes remains unknown. While exploring the latter, we identified a novel aPC/PAR-dependent cytoprotective signaling mechanism. In podocytes, aPC inhibits apoptosis through proteolytic activation of PAR-3 independent of endothelial protein C receptor. PAR-3 is not signaling competent itself as it requires aPCinduced heterodimerization with PAR-2 (human podocytes) or PAR-1 (mouse podocytes). This cytoprotective signaling mechanism depends on caveolin-1 dephosphorylation. In vivo aPC protects against lipopolysaccharide-induced podocyte injury and proteinuria. Genetic deletion of PAR-3 impairs the nephroprotective effect of aPC, demonstrating the crucial role of PAR-3 for aPC-dependent podocyte protection. This novel, aPC-mediated interaction of PARs demonstrates the plasticity and cell-specificity of cytoprotective aPC signaling. The evidence of specific, dynamic signaling complexes underlying aPC-mediated cytoprotection may allow the design of cell type specific targeted therapies. PMID:22117049

Circulating endothelial and progenitor cells: Evidence from acute and long-term exercise effects

PubMed Central

Koutroumpi, Matina; Dimopoulos, Stavros; Psarra, Katherini; Kyprianou, Theodoros; Nanas, Serafim

2012-01-01

Circulating bone-marrow-derived cells, named endothelial progenitor cells (EPCs), are capable of maintaining, generating, and replacing terminally differentiated cells within their own specific tissue as a consequence of physiological cell turnover or tissue damage due to injury. Endothelium maintenance and restoration of normal endothelial cell function is guaranteed by a complex physiological procedure in which EPCs play a significant role. Decreased number of peripheral blood EPCs has been associated with endothelial dysfunction and high cardiovascular risk. In this review, we initially report current knowledge with regard to the role of EPCs in healthy subjects and the clinical value of EPCs in different disease populations such as arterial hypertension, obstructive sleep-apnea syndrome, obesity, diabetes mellitus, peripheral arterial disease, coronary artery disease, pulmonary hypertension, and heart failure. Recent studies have introduced the novel concept that physical activity, either performed as a single exercise session or performed as part of an exercise training program, results in a significant increase of circulating EPCs. In the second part of this review we provide preliminary evidence from recent studies investigating the effects of acute and long-term exercise in healthy subjects and athletes as well as in disease populations. PMID:23272272

Hypoxia-activated chemotherapeutic TH-302 enhances the effects of VEGF-A inhibition and radiation on sarcomas.

PubMed

Yoon, C; Lee, H-J; Park, D J; Lee, Y-J; Tap, W D; Eisinger-Mathason, T S K; Hart, C P; Choy, E; Simon, M C; Yoon, S S

2015-06-30

Human sarcomas with a poor response to vascular endothelial growth factor-A (VEGF-A) inhibition and radiation therapy (RT) have upregulation of hypoxia-inducible factor 1α (HIF-1α) and HIF-1α target genes. This study examines the addition of the hypoxia-activated chemotherapy TH-302 to VEGF-A inhibition and RT (a.k.a. trimodality therapy). Trimodality therapy was examined in two xenograft models and in vitro in tumour endothelial cells and sarcoma cell lines. In both mouse models, VEGF-A inhibition and radiation showed greater efficacy than either therapy alone in slowing sarcoma growth. When TH-302 was added, this trimodality therapy completely blocked tumour growth with tumours remaining dormant for over 3 months after cessation of therapy. Trimodality therapy caused 2.6- to 6.2-fold more endothelial cell-specific apoptosis than bimodality therapies, and microvessel density and HIF-1α activity were reduced to 11-13% and 13-20% of control, respectively. When trimodality therapy was examined in vitro, increases in DNA damage and apoptosis were much more pronounced in tumour endothelial cells compared with that in sarcoma cells, especially under hypoxia. The combination of TH-302, VEGF-A inhibition, and RT is highly effective in preclinical models of sarcoma and is associated with increased DNA damage and apoptosis in endothelial cells and decreased HIF-1α activity.

The chromogranin A peptide vasostatin-I inhibits gap formation and signal transduction mediated by inflammatory agents in cultured bovine pulmonary and coronary arterial endothelial cells.

PubMed

Blois, Anna; Srebro, Boleslaw; Mandalà, Maurizio; Corti, Angelo; Helle, Karen B; Serck-Hanssen, Guldborg

2006-07-15

The proinflammatory agent tumour necrosis factor alpha (TNFalpha) is one of several agents causing vascular leakage. The N-terminal domain of CgA, vasostatin-I (CgA1-76), has recently been reported to inhibit TNFalpha induced gap formation in human umbilical venous endothelial cells. Here we report on the effect of recombinant human CgA1-78, vasostatin-I, on TNFalpha induced gap formation in two model systems of vascular leakage in arterial endothelial cells of bovine pulmonary (BPAEC) and coronary (BCAEC) origin. Vasostatin-I inhibited the TNFalpha induced gap formation in both models, being inactive in the unstimulated cells. The phosphorylation of p38MAP kinase in TNFalpha activated BPAEC was markedly attenuated in the presence of vasostatin-I and the inhibitory effect corresponded to that of the specific p38MAPK inhibitor SB203580. Vasostatin-I also inhibited the phosphorylation of p38MAPK induced by both thrombin and pertussis toxin in these cells. The results demonstrate that vasostatin-I has inhibitory effects on TNFalpha-induced disruption of confluent layers of cultured pulmonary and coronary arterial endothelial cells. This suggests that vasostatin-I may affect endothelial barrier dysfunction also in arterial vascular beds. Furthermore, the inhibitory activity of vasostatin-I may be associated with the p38MAPK signalling cascade via a pertussis toxin sensitive, presumably Galphai coupled mechanism.

Single-cell RNA sequencing highlights transcription activity of autophagy-related genes during hematopoietic stem cell formation in mouse embryos.

PubMed

Hu, Yongfei; Huang, Yan; Yi, Ying; Wang, Hongwei; Liu, Bing; Yu, Jia; Wang, Dong

2017-04-03

Accumulating evidence has demonstrated that macroautophagy/autophagy plays an essential role in self-renewal and differentiation in embryonic hematopoiesis. Here, according to the RNA sequencing data sets of 5 population cells related to hematopoietic stem cell (HSC) formation during mouse embryogenesis (endothelial cells, PTPRC/CD45 - and PTPRC/CD45 + pre-HSCs in the E11 aorta-gonad-mesonephros (AGM) region, mature HSCs in E12 and E14 fetal liver), we explored the dynamic expression of mouse autophagy-related genes in this course at the single-cell level. Our results revealed that the transcription activity of autophagy-related genes had a substantial increase when endothelial cells (ECs) specified into pre-HSCs, and the upregulation of autophagy-essential genes correlated with reduced NOTCH signaling in pre-HSCs, suggesting the autophagy activity may be greatly enhanced during pre-HSC specification from endothelial precursors. In summary, our results presented strong evidence that autophagy plays a critical role in HSC emergence during mouse midgestation.

Thrombomodulin as a marker for vascular tumors. Comparative study with factor VIII and Ulex europaeus I lectin.

PubMed

Yonezawa, S; Maruyama, I; Sakae, K; Igata, A; Majerus, P W; Sato, E

1987-10-01

Thrombomodulin (TM) is a newly described endothelial cell-associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. Various vascular tumors were characterized with immunoperoxidase staining with the use of a polyclonal anti-TM serum. The staining patterns of TM were compared with those of Factor VIII-related antigen (FVIII-RAG) and Ulex europaeus agglutinin-I (UEA-I), which have been used as markers for endothelial cells. The results showed that TM is a specific and a highly sensitive marker for angiosarcomas in comparison with FVIII-RAG or UEA-I. In contrast, UEA-I is more sensitive for benign vascular tumors than TM or FVIII-RAG. The other mesenchymal tumors of nonvascular origin showed negative staining for three endothelial markers. These results indicate that TM is a new specific and sensitive tool for the diagnosis of angiosarcomas.

Ulex europaeus I lectin as a marker for vascular endothelium in human tissues.

PubMed

Holthöfer, H; Virtanen, I; Kariniemi, A L; Hormia, M; Linder, E; Miettinen, A

1982-07-01

Ulex europaeus I agglutinin, a lectin specific for some alpha-L-fucose-containing glycocompounds, was used in fluorescence microscopy to stain cryostat sections of human tissues. The endothelium of vessels of all sizes was stained ubiquitously in all tissues studied as judged by double staining with a known endothelial marker, antibodies against human clotting factor VIII. Cultured human umbilical vein endothelial cells, but not fibroblasts, also bound Ulex lectin. The staining was not affected by the blood group type of the tissue donor. In some tissues Ulex lectin presented additional binding to epithelial structures. Also, this was independent on the blood group or the ability of the tissue donor to secrete soluble blood group substances. Lotus tetragonolobus agglutinin, another lectin specific for some alpha-L-fucose-containing moieties failed to react with endothelial cells. Our results suggest that Ulex europaeus I agglutinin is a good histologic marker for endothelium in human tissues.

Placental-Specific sFLT-1 e15a Protein Is Increased in Preeclampsia, Antagonizes Vascular Endothelial Growth Factor Signaling, and Has Antiangiogenic Activity.

PubMed

Palmer, Kirsten R; Kaitu'u-Lino, Tu'uhevaha J; Hastie, Roxanne; Hannan, Natalie J; Ye, Louie; Binder, Natalie; Cannon, Ping; Tuohey, Laura; Johns, Terrance G; Shub, Alexis; Tong, Stephen

2015-12-01

In preeclampsia, the antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFLT-1) is released from placenta into the maternal circulation, causing endothelial dysfunction and organ injury. A recently described splice variant, sFLT-1 e15a, is primate specific and the most abundant placentally derived sFLT-1. Therefore, it may be the major sFLT-1 isoform contributing to the pathophysiology of preeclampsia. sFLT-1 e15a protein remains poorly characterized: its bioactivity has not been comprehensively examined, and serum levels in normal and preeclamptic pregnancy have not been reported. We generated and validated an sFLT-1 e15a-specific ELISA to further characterize serum levels during pregnancy, and in the presence of preeclampsia. Furthermore, we performed assays to examine the bioactivity and antiangiogenic properties of sFLT-1 e15a protein. sFLT-1 e15a was expressed in the syncytiotrophoblast, and serum levels rose across pregnancy. Strikingly, serum levels were increased 10-fold in preterm preeclampsia compared with normotensive controls. We confirmed sFLT-1 e15a is bioactive and is able to inhibit vascular endothelial growth factor signaling of vascular endothelial growth factor receptor 2 and block downstream Akt phosphorylation. Furthermore, sFLT-1 e15a has antiangiogenic properties. sFLT-1 e15a decreased endothelial cell migration, invasion, and inhibited endothelial cell tube formation. Administering sFLT-1 e15a blocked vascular endothelial growth factor induced sprouts from mouse aortic rings ex vivo. We have demonstrated that sFLT-1 e15a is increased in preeclampsia, antagonizes vascular endothelial growth factor signaling, and has antiangiogenic activity. Future development of diagnostics and therapeutics for preeclampsia should consider targeting placentally derived sFLT-1 e15a. © 2015 American Heart Association, Inc.

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.

Mathematical model of depolarization mechanism of conducted vasoreactivity

NASA Astrophysics Data System (ADS)

Neganova, Anastasiia Y.; Stiukhina, Elena S.; Postnov, Dmitry E.

2015-03-01

We address the problem of conducted vasodilation, the phenomenon which is also known as functional hyperemia. Specifically, we test the mechanism of nondecremental propagation of electric signals along endothelial cell layer recently hypothesized by Figueroa et al. By means of functional modeling we focus on possible nonlinear mechanisms that can underlie such regenerative pulse transmission (RPT). Since endothelial cells (EC) are generally known as electrically inexcitable, the possible role of ECs in RPT mechanisms is not evident. By means of mathematical modeling we check the dynamical self-consistency of Figueroa's hypothesis, as well as estimate the possible contribution of specific ionic currents to the suggested RPT mechanism.

Celiac Disease–Specific TG2-Targeted Autoantibodies Inhibit Angiogenesis Ex Vivo and In Vivo in Mice by Interfering with Endothelial Cell Dynamics

PubMed Central

Kalliokoski, Suvi; Sulic, Ana-Marija; Korponay-Szabó, Ilma R.; Szondy, Zsuzsa; Frias, Rafael; Perez, Mileidys Alea; Martucciello, Stefania; Roivainen, Anne; Pelliniemi, Lauri J.; Esposito, Carla; Griffin, Martin; Sblattero, Daniele; Mäki, Markku; Kaukinen, Katri; Lindfors, Katri; Caja, Sergio

2013-01-01

A characteristic feature of celiac disease is the presence of circulating autoantibodies targeted against transglutaminase 2 (TG2), reputed to have a function in angiogenesis. In this study we investigated whether TG2-specific autoantibodies derived from celiac patients inhibit angiogenesis in both ex vivo and in vivo models and sought to clarify the mechanism behind this phenomenon. We used the ex vivo murine aorta-ring and the in vivo mouse matrigel-plug assays to address aforementioned issues. We found angiogenesis to be impaired as a result of celiac disease antibody supplementation in both systems. Our results also showed the dynamics of endothelial cells was affected in the presence of celiac antibodies. In the in vivo angiogenesis assays, the vessels formed were able to transport blood despite impairment of functionality after treatment with celiac autoantibodies, as revealed by positron emission tomography. We conclude that celiac autoantibodies inhibit angiogenesis ex vivo and in vivo and impair vascular functionality. Our data suggest that the anti-angiogenic mechanism of the celiac disease-specific autoantibodies involves extracellular TG2 and inhibited endothelial cell mobility. PMID:23824706

Endothelial Msx1 transduces hemodynamic changes into an arteriogenic remodeling response

PubMed Central

Vandersmissen, Ine; Craps, Sander; Depypere, Maarten; Coppiello, Giulia; van Gastel, Nick; Maes, Frederik; Carmeliet, Geert; Schrooten, Jan; Jones, Elizabeth A.V.; Umans, Lieve; Devlieger, Roland; Koole, Michel; Gheysens, Olivier; Zwijsen, An; Aranguren, Xabier L.

2015-01-01

Collateral remodeling is critical for blood flow restoration in peripheral arterial disease and is triggered by increasing fluid shear stress in preexisting collateral arteries. So far, no arterial-specific mediators of this mechanotransduction response have been identified. We show that muscle segment homeobox 1 (MSX1) acts exclusively in collateral arterial endothelium to transduce the extrinsic shear stimulus into an arteriogenic remodeling response. MSX1 was specifically up-regulated in remodeling collateral arteries. MSX1 induction in collateral endothelial cells (ECs) was shear stress driven and downstream of canonical bone morphogenetic protein–SMAD signaling. Flow recovery and collateral remodeling were significantly blunted in EC-specific Msx1/2 knockout mice. Mechanistically, MSX1 linked the arterial shear stimulus to arteriogenic remodeling by activating the endothelial but not medial layer to a proinflammatory state because EC but not smooth muscle cellMsx1/2 knockout mice had reduced leukocyte recruitment to remodeling collateral arteries. This reduced leukocyte infiltration in EC Msx1/2 knockout mice originated from decreased levels of intercellular adhesion molecule 1 (ICAM1)/vascular cell adhesion molecule 1 (VCAM1), whose expression was also in vitro driven by promoter binding of MSX1. PMID:26391659

A systems biology approach identified different regulatory networks targeted by KSHV miR-K12-11 in B cells and endothelial cells.

PubMed

Yang, Yajie; Boss, Isaac W; McIntyre, Lauren M; Renne, Rolf

2014-08-08

Kaposi's sarcoma associated herpes virus (KSHV) is associated with tumors of endothelial and lymphoid origin. During latent infection, KSHV expresses miR-K12-11, an ortholog of the human tumor gene hsa-miR-155. Both gene products are microRNAs (miRNAs), which are important post-transcriptional regulators that contribute to tissue specific gene expression. Advances in target identification technologies and molecular interaction databases have allowed a systems biology approach to unravel the gene regulatory networks (GRNs) triggered by miR-K12-11 in endothelial and lymphoid cells. Understanding the tissue specific function of miR-K12-11 will help to elucidate underlying mechanisms of KSHV pathogenesis. Ectopic expression of miR-K12-11 differentially affected gene expression in BJAB cells of lymphoid origin and TIVE cells of endothelial origin. Direct miRNA targeting accounted for a small fraction of the observed transcriptome changes: only 29 genes were identified as putative direct targets of miR-K12-11 in both cell types. However, a number of commonly affected biological pathways, such as carbohydrate metabolism and interferon response related signaling, were revealed by gene ontology analysis. Integration of transcriptome profiling, bioinformatic algorithms, and databases of protein-protein interactome from the ENCODE project identified different nodes of GRNs utilized by miR-K12-11 in a tissue-specific fashion. These effector genes, including cancer associated transcription factors and signaling proteins, amplified the regulatory potential of a single miRNA, from a small set of putative direct targets to a larger set of genes. This is the first comparative analysis of miRNA-K12-11's effects in endothelial and B cells, from tissues infected with KSHV in vivo. MiR-K12-11 was able to broadly modulate gene expression in both cell types. Using a systems biology approach, we inferred that miR-K12-11 establishes its GRN by both repressing master TFs and influencing signaling pathways, to counter the host anti-viral response and to promote proliferation and survival of infected cells. The targeted GRNs are more reproducible and informative than target gene identification, and our approach can be applied to other regulatory factors of interest.

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.

Small interfering RNA-mediated down-regulation of caveolin-1 differentially modulates signaling pathways in endothelial cells.

PubMed

Gonzalez, Eva; Nagiel, Aaron; Lin, Alison J; Golan, David E; Michel, Thomas

2004-09-24

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules in endothelial cells. To explore the role of this protein in receptor-modulated signaling pathways, we transfected bovine aortic endothelial cells (BAEC) with small interfering RNA (siRNA) duplexes to down-regulate caveolin-1 expression. Transfection of BAEC with duplex siRNA targeted against caveolin-1 mRNA selectively "knocked-down" the expression of caveolin-1 by approximately 90%, as demonstrated by immunoblot analyses of BAEC lysates. We used discontinuous sucrose gradients to purify caveolin-containing lipid rafts from siRNA-treated endothelial cells. Despite the near-total down-regulation of caveolin-1 expression, the lipid raft targeting of diverse signaling proteins (including the endothelial isoform of nitric-oxide synthase, Src-family tyrosine kinases, Galphaq and the insulin receptor) was unchanged. We explored the consequences of caveolin-1 knockdown on kinase pathways modulated by the agonists sphingosine-1 phosphate (S1P) and vascular endothelial growth factor (VEGF). siRNA-mediated caveolin-1 knockdown enhanced basal as well as S1P- and VEGF-induced phosphorylation of the protein kinase Akt and did not modify the basal or agonist-induced phosphorylation of extracellular signal-regulated kinases 1/2. Caveolin-1 knock-down also significantly enhanced the basal and agonist-induced activity of the small GTPase Rac. We used siRNA to down-regulate Rac expression in BAEC, and we observed that Rac knockdown significantly reduced basal, S1P-, and VEGF-induced Akt phosphorylation, suggesting a role for Rac activation in the caveolin siRNA-mediated increase in Akt phosphorylation. By using siRNA to knockdown caveolin-1 and Rac expression in cultured endothelial cells, we have found that caveolin-1 does not seem to be required for the targeting of signaling molecules to caveolae/lipid rafts and that caveolin-1 differentially modulates specific kinase pathways in endothelial cells. Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.

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.

Withaferin A protects against palmitic acid-induced endothelial insulin resistance and dysfunction through suppression of oxidative stress and inflammation

PubMed Central

Batumalaie, Kalaivani; Amin, Muhammad Arif; Murugan, Dharmani Devi; Sattar, Munavvar Zubaid Abdul; Abdullah, Nor Azizan

2016-01-01

Activation of inflammatory pathways via reactive oxygen species (ROS) by free fatty acids (FFA) in obesity gives rise to insulin resistance and endothelial dysfunction. Withaferin A (WA), possesses both antioxidant and anti-inflammatory properties and therefore would be a good strategy to suppress palmitic acid (PA)-induced oxidative stress and inflammation and hence, insulin resistance and dysfunction in the endothelium. Effect of WA on PA-induced insulin resistance in human umbilical vein endothelial cells (HUVECs) was determined by evaluating insulin signaling mechanisms whilst effect of this drug on PA-induced endothelial dysfunction was determined in acetylcholine-mediated relaxation in isolated rat aortic preparations. WA significantly inhibited ROS production and inflammation induced by PA. Furthermore, WA significantly decreased TNF-α and IL-6 production in endothelial cells by specifically suppressing IKKβ/NF-κβ phosphorylation. WA inhibited inflammation-stimulated IRS-1 serine phosphorylation and improved the impaired insulin PI3-K signaling, and restored the decreased nitric oxide (NO) production triggered by PA. WA also decreased endothelin-1 and plasminogen activator inhibitor type-1 levels, and restored the impaired endothelium-mediated vasodilation in isolated aortic preparations. These findings suggest that WA inhibited both ROS production and inflammation to restore impaired insulin resistance in cultured endothelial cells and improve endothelial dysfunction in rat aortic rings. PMID:27250532

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

PubMed

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

2017-07-01

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

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord.

PubMed

Kadam, Sachin S; Tiwari, Shubha; Bhonde, Ramesh R

2009-01-01

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child's birth; however, its importance as a "store house" of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton's jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.

Distinctive effects of CD34- and CD133-specific antibody-coated stents on re-endothelialization and in-stent restenosis at the early phase of vascular injury

PubMed Central

Wu, Xue; Yin, Tieying; Tian, Jie; Tang, Chaojun; Huang, Junli; Zhao, Yinping; Zhang, Xiaojuan; Deng, Xiaoyan; Fan, Yubo; Yu, Donghong; Wang, Guixue

2015-01-01

It is not clear what effects of CD34- and CD133-specific antibody-coated stents have on re-endothelialization and in-stent restenosis (ISR) at the early phase of vascular injury. This study aims at determining the capabilities of different coatings on stents (e.g. gelatin, anti-CD133 and anti-CD34 antibodies) to promote adhesion and proliferation of endothelial progenitor cells (EPCs). The in vitro study revealed that the adhesion force enabled the EPCs coated on glass slides to withstand flow-induced shear stress, so that allowing for the growth of the cells on the slides for 48 h. The in vivo experiment using a rabbit model in which the coated stents with different substrates were implanted showed that anti-CD34 and anti-CD133 antibody-coated stents markedly reduced the intima area and restenosis than bare mental stents (BMS) and gelatin-coated stents. Compared with the anti-CD34 antibody-coated stents, the time of cells adhesion was longer and earlier present in the anti-CD133 antibody-coated stents and anti-CD133 antibody-coated stents have superiority in re-endothelialization and inhibition of ISR. In conclusion, this study demonstrated that anti-CD133 antibody as a stent coating for capturing EPCs is better than anti-CD34 antibody in promoting endothelialization and reducing ISR. PMID:26813006

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

Joo, Hyung Joon; Seo, Ha-Rim; Jeong, Hyo Eun

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

Endothelial deletion of Ino80 disrupts coronary angiogenesis and causes congenital heart disease.

PubMed

Rhee, Siyeon; Chung, Jae I; King, Devin A; D'amato, Gaetano; Paik, David T; Duan, Anna; Chang, Andrew; Nagelberg, Danielle; Sharma, Bikram; Jeong, Youngtae; Diehn, Maximilian; Wu, Joseph C; Morrison, Ashby J; Red-Horse, Kristy

2018-01-25

During development, the formation of a mature, well-functioning heart requires transformation of the ventricular wall from a loose trabecular network into a dense compact myocardium at mid-gestation. Failure to compact is associated in humans with congenital diseases such as left ventricular non-compaction (LVNC). The mechanisms regulating myocardial compaction are however still poorly understood. Here, we show that deletion of the Ino80 chromatin remodeler in vascular endothelial cells prevents ventricular compaction in the developing mouse heart. This correlates with defective coronary vascularization, and specific deletion of Ino80 in the two major coronary progenitor tissues-sinus venosus and endocardium-causes intermediate phenotypes. In vitro, endothelial cells promote myocardial expansion independently of blood flow in an Ino80-dependent manner. Ino80 deletion increases the expression of E2F-activated genes and endothelial cell S-phase occupancy. Thus, Ino80 is essential for coronary angiogenesis and allows coronary vessels to support proper compaction of the heart wall.

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

MCPIP1-induced autophagy mediates ischemia/reperfusion injury in endothelial cells via HMGB1 and CaSR.

PubMed

Xie, Xiaolong; Zhu, Tiebing; Chen, Lulu; Ding, Shuang; Chu, Han; Wang, Jing; Yao, Honghong; Chao, Jie

2018-01-29

Monocyte chemotactic protein-1-induced protein 1 (MCPIP1) plays a important role in ischemia/reperfusion (I/R) injury. Autophagy is involved in activating endothelial cells in response to I/R. However, researchers have not clearly determined whether MCPIP1 mediates I/R injury in endothelial cells via autophagy, and its downstream mechanism remains unclear. Western blotting analyses and immunocytochemistry were applied to detect protein levels were detected in HUVECs. An in vitro scratch assay was used to detect cell migration. Cells were transfected with siRNAs to knockdown MCPIP1 and high mobility group box 1 (HMGB1) expression. The pharmacological activator of autophagy rapamycin and the specific calcium-sensing receptor (CaSR) inhibitor NPS-2143 were used to confirm the roles of autophagy and CaSR in I/R injury. I/R induced HMGB1 and CaSR expression, which subsequently upreguated the migration and apoptosis of HUVECs and coincided with the increase of autophagy. HMGB1 was involved in cell migration, whereas CaSR specifically participated in I/R-induced HUVEC apoptosis. Based on these findings, I/R-induced MCPIP1 expression regulates the migration and apoptosis of HUVECs via HMGB1 and CaSR, respectively, suggesting a new therapeutic targetof I/R injury.

Enhanced expression by the brain matrix of P-glycoprotein in brain capillary endothelial cells.

PubMed

Tatsuta, T; Naito, M; Mikami, K; Tsuruo, T

1994-10-01

P-glycoprotein (PGP), an active efflux pump of antitumor agents in multidrug-resistant tumor cells, exists in brain capillary endothelium and could be functionally involved in the blood-brain barrier. To study the regulatory mechanism of PGP expression in brain capillary endothelium, various mouse tissue matrices were tested for their abilities to enhance the expression of PGP in mouse brain capillary endothelial cells (MBEC), which express relatively small amounts of PGP. Of the four tissue matrices we examined, PGP expression in MBEC cultured on the brain matrix increased 2.0-fold. The PGP-inducing activity was similarly detected in bovine brain matrix, and the activity was enriched in the fraction of pl 9.0 by isoelectric focusing. The fraction, named PIC-fraction (PGP-inducing component), increased the PGP expression in MBEC 3.5-fold. By Northern blot analysis, a 3.3-fold enhancement of mdr gene expression was observed in MBEC cultured on the PIC-fraction. The PGP-inducing activity of the PIC-fraction was reduced by the treatment with trypsin but not with collagenase, suggesting that a proteinaceous factor distinct from type I collagen might be responsible for the PGP-inducing activity of PIC-fraction. Although the PIC-fraction increased the PGP expression in other mouse brain capillary endothelial cells, the PIC-fraction did not increase PGP expression in mouse aortic endothelial cells and KB carcinoma cell lines expressing various amounts of PGP. These observations suggest that PGP expression in brain capillary endothelium is specifically regulated by a tissue-specific factor in the brain matrix.

Endotoxin induces fibrosis in vascular endothelial cells through a mechanism dependent on transient receptor protein melastatin 7 activity.

PubMed

Echeverría, Cesar; Montorfano, Ignacio; Hermosilla, Tamara; Armisén, Ricardo; Velásquez, Luis A; Cabello-Verrugio, Claudio; Varela, Diego; Simon, Felipe

2014-01-01

The pathogenesis of systemic inflammatory diseases, including endotoxemia-derived sepsis syndrome, is characterized by endothelial dysfunction. It has been demonstrated that the endotoxin lipopolysaccharide (LPS) induces the conversion of endothelial cells (ECs) into activated fibroblasts through endothelial-to-mesenchymal transition mechanism. Fibrogenesis is highly dependent on intracellular Ca2+ concentration increases through the participation of calcium channels. However, the specific molecular identity of the calcium channel that mediates the Ca2+ influx during endotoxin-induced endothelial fibrosis is still unknown. Transient receptor potential melastatin 7 (TRPM7) is a calcium channel that is expressed in many cell types, including ECs. TRPM7 is involved in a number of crucial processes such as the conversion of fibroblasts into activated fibroblasts, or myofibroblasts, being responsible for the development of several characteristics of them. However, the role of the TRPM7 ion channel in endotoxin-induced endothelial fibrosis is unknown. Thus, our aim was to study whether the TRPM7 calcium channel participates in endotoxin-induced endothelial fibrosis. Using primary cultures of ECs, we demonstrated that TRPM7 is a crucial protein involved in endotoxin-induced endothelial fibrosis. Suppression of TRPM7 expression protected ECs from the fibrogenic process stimulated by endotoxin. Downregulation of TRPM7 prevented the endotoxin-induced endothelial markers decrease and fibrotic genes increase in ECs. In addition, TRPM7 downregulation abolished the endotoxin-induced increase in ECM proteins in ECs. Furthermore, we showed that intracellular Ca2+ levels were greatly increased upon LPS challenge in a mechanism dependent on TRPM7 expression. These results demonstrate that TRPM7 is a key protein involved in the mechanism underlying endotoxin-induced endothelial fibrosis.

Influence of elastin-derived peptides on metalloprotease production in endothelial cells.

PubMed

Siemianowicz, Krzysztof; Gminski, Jan; Goss, Malgorzata; Francuz, Tomasz; Likus, Wirginia; Jurczak, Teresa; Garczorz, Wojciech

2010-11-01

Matrix metalloproteases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. MMP-1 and MMP-2 are produced by endothelial cells and are involved in specific vascular pathologies, including atherosclerosis and aortal aneurysm. One of the most important differences between these two metalloproteases is the possibility of hydrolysis of elastin and collagen type IV by MMP-2, but not by MMP-1. Elastin-derived peptides are generated as a result of the degradation of elastin fibers. The aim of our study was to compare the production of MMP-1 and MMP-2 in cultured human arterial endothelial cells derived from vascular pathologies localized at three different sites, the coronary artery, iliac artery and aorta, measured as their concentration in cell culture medium. The second aim was to evaluate the influence of κ-elastin (at concentrations 0.1, 0.4, 1.0, 2.5 or 5.0 μg/ml) on the production of the evaluated metalloproteases in three endothelial cell lines. The production of MMP-1 was statistically significantly greater in endothelial cells derived from the aorta compared to that in the endothelium obtained from the coronary and iliac arteries. There were no statistically significant differences in the production of MMP-2 among the endothelial cell lines tested. The addition of κ-elastin at all evaluated concentrations did not statistically significantly influence the concentration of MMP-1 in the cultured coronary artery endothelium. Furthermore, no statistically significant differences were observed in the cultured iliac artery endothelium. In the cultured endothelium derived from the aorta, κ-elastin at concentrations of 0.1 and 0.4 μg/ml significantly increased the amount of MMP-1.

Influence of elastin-derived peptides on metalloprotease production in endothelial cells

PubMed Central

SIEMIANOWICZ, KRZYSZTOF; GMINSKI, JAN; GOSS, MALGORZATA; FRANCUZ, TOMASZ; LIKUS, WIRGINIA; JURCZAK, TERESA; GARCZORZ, WOJCIECH

2010-01-01

Matrix metalloproteases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. MMP-1 and MMP-2 are produced by endothelial cells and are involved in specific vascular pathologies, including atherosclerosis and aortal aneurysm. One of the most important differences between these two metalloproteases is the possibility of hydrolysis of elastin and collagen type IV by MMP-2, but not by MMP-1. Elastin-derived peptides are generated as a result of the degradation of elastin fibers. The aim of our study was to compare the production of MMP-1 and MMP-2 in cultured human arterial endothelial cells derived from vascular pathologies localized at three different sites, the coronary artery, iliac artery and aorta, measured as their concentration in cell culture medium. The second aim was to evaluate the influence of κ-elastin (at concentrations 0.1, 0.4, 1.0, 2.5 or 5.0 μg/ml) on the production of the evaluated metalloproteases in three endothelial cell lines. The production of MMP-1 was statistically significantly greater in endothelial cells derived from the aorta compared to that in the endothelium obtained from the coronary and iliac arteries. There were no statistically significant differences in the production of MMP-2 among the endothelial cell lines tested. The addition of κ-elastin at all evaluated concentrations did not statistically significantly influence the concentration of MMP-1 in the cultured coronary artery endothelium. Furthermore, no statistically significant differences were observed in the cultured iliac artery endothelium. In the cultured endothelium derived from the aorta, κ-elastin at concentrations of 0.1 and 0.4 μg/ml significantly increased the amount of MMP-1. PMID:22993640

Epigenetic and Epitranscriptomic Factors Make a Mark on Hematopoietic Stem Cell Development.

PubMed

Kasper, Dionna M; Nicoli, Stefania

2018-03-01

Blood specification is a highly dynamic process, whereby committed hemogenic endothelial cells (ECs) progressively transdifferentiate into multipotent, self-renewing hematopoietic stem cells (HSCs). Massive changes in gene expression must occur to switch cell identity, however the factors that mediate such an effect were a mystery until recently. This review summarizes the higher-order mechanisms involved in endothelial to hematopoietic reprogramming identified thus far. Accumulating evidence from mouse and zebrafish studies reveal that numerous chromatin-modifying (epigenetic) and RNA-modifying (epitranscriptomic) factors are required for the formation of HSCs from hemogenic endothelium. These genes function throughout the endothelial-hematopoietic transition, suggesting a dynamic interplay between 'epi'-machineries. Epigenetic and epitranscriptomic regulation are key mechanisms for reshaping global EC gene expression patterns to those that support HSC production. Future studies that capture modification dynamics should bring us closer to a complete understanding of how HSCs transition from hemogenic endothelium at the molecular level.

Endothelial- and Platelet-Derived Microparticles Are Generated During Liver Resection in Humans.

PubMed

Banz, Yara; Item, Gian-Marco; Vogt, Andreas; Rieben, Robert; Candinas, Daniel; Beldi, Guido

2016-01-01

Cell-derived plasma microparticles (<1.5 μm) originating from various cell types have the potential to regulate thrombogenesis and inflammatory responses. The aim of this study was to test the hypothesis that microparticles generated during hepatic surgery co-regulate postoperative procoagulant and proinflammatory events. In 30 patients undergoing liver resection, plasma microparticles were isolated, quantitated, and characterized as endothelial (CD31+, CD41-), platelet (CD41+), or leukocyte (CD11b+) origin by flow cytometry and their procoagulant and proinflammatory activity was measured by immunoassays. During liver resection, the total numbers of microparticles increased with significantly more Annexin V-positive, endothelial and platelet-derived microparticles following extended hepatectomy compared to standard and minor liver resections. After liver resection, microparticle tissue factor and procoagulant activity increased along with overall coagulation as assessed by thrombelastography. Levels of leukocyte-derived microparticles specifically increased in patients with systemic inflammation as assessed by C-reactive protein but are independent of the extent of liver resection. Endothelial and platelet-derived microparticles are specifically elevated during liver resection, accompanied by increased procoagulant activity. Leukocyte-derived microparticles are a potential marker for systemic inflammation. Plasma microparticles may represent a specific response to surgical stress and may be an important mediator of postoperative coagulation and inflammation.

Protein Kinase D-dependent Phosphorylation and Nuclear Export of Histone Deacetylase 5 Mediates Vascular Endothelial Growth Factor-induced Gene Expression and Angiogenesis*S⃞

PubMed Central

Ha, Chang Hoon; Wang, Weiye; Jhun, Bong Sook; Wong, Chelsea; Hausser, Angelika; Pfizenmaier, Klaus; McKinsey, Timothy A.; Olson, Eric N.; Jin, Zheng-Gen

2008-01-01

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cγ-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis. PMID:18332134

Overexpression of Prox1 Induces the Differentiation of Human Adipose-Derived Stem Cells into Lymphatic Endothelial-Like Cells In Vitro.

PubMed

Deng, Jingcheng; Dai, Tingting; Sun, Yiyu; Zhang, Qi; Jiang, Zhaohua; Li, Shengli; Cao, Weigang

2017-02-01

Constant levels of homeobox transcription factor Prox1 expression are required throughout the life of lymphatic endothelial cells (LECs) to maintain their differentiated identity. Recent studies have demonstrated that using human LECs for cell transplantation therapy may improve secondary lymphedema in a nude rat model. However, the application is currently limited by the low yield of LECs. In this study, Prox1 was overexpressed in human adipose tissue-derived stem cells (hADSCs) by using the transfection of lentiviral vectors to induce the differentiation of hADSCs to LECs. After 14 days of Prox1 overexpression, flow cytometry analysis found that the expression of LEC-specific markers such as Podoplanin and VEGFR3, along with the endothelial cell (EC) marker CD31, on Prox1-overexpressed hADSCs was significantly increased; however, the expression of mesenchymal stem cell markers, such as CD29, CD44, and CD90, was substantially reduced. In addition, the mRNA levels of the LEC-specific markers, such as Prox1, Podoplanin, LYVE1, and VEGFR3, in Prox1-overexpressed hADSCs were significantly increased at day 7 and maintained a continuously increased expression level for 28 observation days, according to real-time reverse transcriptase-polymerase chain reaction results. Western blotting and immunofluorescence staining results further confirmed that overexpression of Prox1 in hADSCs significantly increased the protein levels of Podoplanin, LYVE1, and VEGFR3, as well as those of the EC markers such as VWF and CD144, at day 14. Moreover, these differentiated cells were found to form tube-like structures in matrigel, measured by the tube formation assay. These findings suggested that overexpression of Prox1 in hADSCs successfully induced the differentiation of hADSCs into stable lymphatic endothelial-like cells. This study achieved a long-lasting expression of Prox1 in lymphatic endothelial-like cells, and it provided a potentially useful approach for developing novel therapies for limb lymphedema and lymphatic system-related diseases.

Endothelial differentiation of bone marrow mesenchyme stem cells applicable to hypoxia and increased migration through Akt and NFκB signals.

PubMed

Liu, Cheng; Tsai, An-Ly; Li, Ping-Chia; Huang, Chia-Wei; Wu, Chia-Ching

2017-02-07

Bone marrow mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) are used to repair hypoxic or ischemic tissue. However, the underlining mechanism of resistance in the hypoxic microenvironment and the efficacy of migration to the injured tissue are still unknown. The current study aims to understand the hypoxia resistance and migration ability of MSCs during differentiation toward endothelial lineages by biochemical and mechanical stimuli. MSCs were harvested from the bone marrow of 6-8-week-old Sprague-Dawley rats. The endothelial growth medium (EGM) was added to MSCs for 3 days to initiate endothelial differentiation. Laminar shear stress was used as the fluid mechanical stimulation. Application of EGM facilitated the early endothelial lineage cells (eELCs) to express EPC markers. When treating the hypoxic mimetic desferrioxamine, both MSCs and eELCs showed resistance to hypoxia as compared with the occurrence of apoptosis in rat fibroblasts. The eELCs under hypoxia increased the wound closure and C-X-C chemokine receptor type 4 (CXCR4) gene expression. Although the shear stress promoted eELC maturation and aligned cells parallel to the flow direction, their migration ability was not superior to that of eELCs either under normoxia or hypoxia. The eELCs showed higher protein expressions of CXCR4, phosphorylated Akt (pAkt), and endogenous NFκB and IκBα than MSCs under both normoxia and hypoxia conditions. The potential migratory signals were discovered by inhibiting either Akt or NFκB using specific inhibitors and revealed decreases of wound closure and transmigration ability in eELCs. The Akt and NFκB pathways are important to regulate the early endothelial differentiation and its migratory ability under a hypoxic microenvironment.

Prokineticin Receptor‐1 Is a New Regulator of Endothelial Insulin Uptake and Capillary Formation to Control Insulin Sensitivity and Cardiovascular and Kidney Functions

PubMed Central

Dormishian, Mojdeh; Turkeri, Gulen; Urayama, Kyoji; Nguyen, Thu Lan; Boulberdaa, Mounia; Messaddeq, Nadia; Renault, Gilles; Henrion, Daniel; Nebigil, Canan G.

2013-01-01

Background Reciprocal relationships between endothelial dysfunction and insulin resistance result in a vicious cycle of cardiovascular, renal, and metabolic disorders. The mechanisms underlying these impairments are unclear. The peptide hormones prokineticins exert their angiogenic function via prokineticin receptor‐1 (PKR1). We explored the extent to which endothelial PKR1 contributes to expansion of capillary network and the transcapillary passage of insulin into the heart, kidney, and adipose tissues, regulating organ functions and metabolism in a specific mice model. Methods and Results By combining cellular studies and studies in endothelium‐specific loss‐of‐function mouse model (ec‐PKR1−/−), we showed that a genetically induced PKR1 loss in the endothelial cells causes the impaired capillary formation and transendothelial insulin delivery, leading to insulin resistance and cardiovascular and renal disorders. Impaired insulin delivery in endothelial cells accompanied with defective expression and activation of endothelial nitric oxide synthase in the ec‐PKR1−/− aorta, consequently diminishing endothelium‐dependent relaxation. Despite having a lean body phenotype, ec‐PKR1−/− mice exhibited polyphagia, polydipsia, polyurinemia, and hyperinsulinemia, which are reminiscent of human lipodystrophy. High plasma free fatty acid levels and low leptin levels further contribute to the development of insulin resistance at the later age. Peripheral insulin resistance and ectopic lipid accumulation in mutant skeletal muscle, heart, and kidneys were accompanied by impaired insulin‐mediated Akt signaling in these organs. The ec‐PKR1−/− mice displayed myocardial fibrosis, low levels of capillary formation, and high rates of apoptosis, leading to diastolic dysfunction. Compact fibrotic glomeruli and high levels of phosphate excretion were found in mutant kidneys. PKR1 restoration in ec‐PKR1−/− mice reversed the decrease in capillary recruitment and insulin uptake and improved heart and kidney function and insulin resistance. Conclusions We show a novel role for endothelial PKR1 signaling in cardiac, renal, and metabolic functions by regulating transendothelial insulin uptake and endothelial cell proliferation. Targeting endothelial PKR1 may serve as a therapeutic strategy for ameliorating these disorders. PMID:24152983

VEGF induces differentiation of functional endothelium from human embryonic stem cells: implications for tissue engineering

PubMed Central

Nourse, Marilyn B.; Halpin, Daniel E.; Scatena, Marta; Mortisen, Derek J.; Tulloch, Nathaniel L.; Hauch, Kip D.; Torok-Storb, Beverly; Ratner, Buddy D.; Pabon, Lil; Murry, Charles E.

2010-01-01

Objective Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs. Methods and Results To enhance endothelial cell differentiation above a baseline of ∼2% in embryoid body (EB) spontaneous differentiation, three alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10-14. Continuous VEGF treatment resulted in a four- to five-fold enrichment of CD31+ cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31+ cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNFα, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells. Conclusions VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. These enrichment methods increase endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants. PMID:19875721

Astrocyte- and endothelial-targeted CCL2 conditional knockout mice: critical tools for studying the pathogenesis of neuroinflammation.

PubMed

Ge, Shujun; Murugesan, Nivetha; Pachter, Joel S

2009-09-01

While the expression of the C-C chemokine ligand 2 (CCL2) in the central nervous system (CNS) is associated with numerous neuroinflammatory conditions, the critical cellular sources of this chemokine, which is responsible for disease processes-as well as associated pathogenic mechanisms, remain unresolved. As the potential for anti-CCL2 therapeutics in treating neuroinflammatory disease is likely to be contingent upon effective drug delivery to the source(s) and/or target(s) of CCL2 action in the CNS, tools to highlight the course of CCL2 action during neuroinflammation are imperative. In response to this need, we used the Cre/loxP and FLP-FRT recombination system to develop the first two, cell-conditional CCL2 knockout mice-separately targeting CCL2 gene elimination to astrocytes and endothelial cells, both of which have been considered to play crucial though undefined roles in neuroinflammatory disease. Specifically, mice containing a floxed CCL2 allele were intercrossed with GFAP-Cre or Tie2-Cre transgenic mice to generate mice with CCL2-deficient astrocytes (astrocyte KO) or endothelial cells (endothelial KO), respectively. Polymerase chain reaction, reverse transcription polymerase chain reaction/quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay of CCL2 gene, RNA, and protein, respectively, from cultured astrocytes and brain microvascular endothelial cells (BMEC) established the efficiency and specificity of the CCL2 gene deletions and a CCL2 null phenotype in these CNS cells. Effective cell-conditional knockout of CCL2 was also confirmed in an in vivo setting, wherein astrocytes and BMEC were retrieved by immune-guided laser capture microdissection from their in situ positions in the brains of mice experiencing acute, lipopolysaccharide-mediated endotoxemia to induce CCL2 gene expression. In vivo analysis further revealed apparent cross-talk between BMEC and astrocytes regarding the regulation of astrocyte CCL2 expression. Use of astrocyte KO and endothelial KO mice should prove critical in elaborating the pathogenic mechanisms of and optimizing the treatments for neuroinflammatory disease.

Expression and subcellular localization of a novel nuclear acetylcholinesterase protein.

PubMed

Santos, Susana Constantino Rosa; Vala, Inês; Miguel, Cláudia; Barata, João T; Garção, Pedro; Agostinho, Paula; Mendes, Marta; Coelho, Ana V; Calado, Angelo; Oliveira, Catarina R; e Silva, João Martins; Saldanha, Carlota

2007-08-31

Acetylcholine is found in the nervous system and also in other cell types (endothelium, lymphocytes, and epithelial and blood cells), which are globally termed the non-neuronal cholinergic system. In this study we investigated the expression and subcellular localization of acetylcholinesterase (AChE) in endothelial cells. Our results show the expression of the 70-kDa AChE in both cytoplasmic and nuclear compartments. We also describe, for the first time, a nuclear and cytoskeleton-bound AChE isoform with approximately 55 kDa detected in endothelial cells. This novel isoform is decreased in response to vascular endothelial growth factor via the proteosomes pathway, and it is down-regulated in human leukemic T-cells as compared with normal T-cells, suggesting that the decreased expression of the 55-kDa AChE protein may contribute to an angiogenic response and associate with tumorigenesis. Importantly, we show that its nuclear expression is not endothelial cell-specific but also evidenced in non-neuronal and neuronal cells. Concerning neuronal cells, we can distinguish an exclusively nuclear expression in postnatal neurons in contrast to a cytoplasmic and nuclear expression in embryonic neurons, suggesting that the cell compartmentalization of this new AChE isoform is changed during the development of nervous system. Overall, our studies suggest that the 55-kDa AChE may be involved in different biological processes such as neural development, tumor progression, and angiogenesis.

Reprogramming of Adult Peripheral Blood Cells into Human Induced Pluripotent Stem Cells as a Safe and Accessible Source of Endothelial Cells.

PubMed

Simara, Pavel; Tesarova, Lenka; Rehakova, Daniela; Farkas, Simon; Salingova, Barbara; Kutalkova, Katerina; Vavreckova, Eva; Matula, Pavel; Matula, Petr; Veverkova, Lenka; Koutna, Irena

2018-01-01

New approaches in regenerative medicine and vasculogenesis have generated a demand for sufficient numbers of human endothelial cells (ECs). ECs and their progenitors reside on the interior surface of blood and lymphatic vessels or circulate in peripheral blood; however, their numbers are limited, and they are difficult to expand after isolation. Recent advances in human induced pluripotent stem cell (hiPSC) research have opened possible avenues to generate unlimited numbers of ECs from easily accessible cell sources, such as the peripheral blood. In this study, we reprogrammed peripheral blood mononuclear cells, human umbilical vein endothelial cells (HUVECs), and human saphenous vein endothelial cells (HSVECs) into hiPSCs and differentiated them into ECs. The phenotype profiles, functionality, and genome stability of all hiPSC-derived ECs were assessed and compared with HUVECs and HSVECs. hiPSC-derived ECs resembled their natural EC counterparts, as shown by the expression of the endothelial surface markers CD31 and CD144 and the results of the functional analysis. Higher expression of endothelial progenitor markers CD34 and kinase insert domain receptor (KDR) was measured in hiPSC-derived ECs. An analysis of phosphorylated histone H2AX (γH2AX) foci revealed that an increased number of DNA double-strand breaks upon reprogramming into pluripotent cells. However, differentiation into ECs restored a normal number of γH2AX foci. Our hiPSCs retained a normal karyotype, with the exception of the HSVEC-derived hiPSC line, which displayed mosaicism due to a gain of chromosome 1. Peripheral blood from adult donors is a suitable source for the unlimited production of patient-specific ECs through the hiPSC interstage. hiPSC-derived ECs are fully functional and comparable to natural ECs. The protocol is eligible for clinical applications in regenerative medicine, if the genomic stability of the pluripotent cell stage is closely monitored.

Glycosyltransferase-programmed stereosubstitution (GPS) to create HCELL: engineering a roadmap for cell migration.

PubMed

Sackstein, Robert

2009-07-01

During evolution of the vertebrate cardiovascular system, the vast endothelial surface area associated with branching vascular networks mandated the development of molecular processes to efficiently and specifically recruit circulating sentinel host defense cells and tissue repair cells at localized sites of inflammation/tissue injury. The forces engendered by high-velocity blood flow commensurately required the evolution of specialized cell surface molecules capable of mediating shear-resistant endothelial adhesive interactions, thus literally capturing relevant cells from the blood stream onto the target endothelial surface and permitting subsequent extravasation. The principal effectors of these shear-resistant binding interactions comprise a family of C-type lectins known as 'selectins' that bind discrete sialofucosylated glycans on their respective ligands. This review explains the 'intelligent design' of requisite reagents to convert native CD44 into the sialofucosylated glycoform known as hematopoietic cell E-/L-selectin ligand (HCELL), the most potent E-selectin counter-receptor expressed on human cells, and will describe how ex vivo glycan engineering of HCELL expression may open the 'avenues' for the efficient vascular delivery of cells for a variety of cell therapies.

Autocrine release of angiopoietin-2 mediates cerebrovascular disintegration in Moyamoya disease

PubMed Central

Blecharz, Kinga G; Frey, Dietmar; Schenkel, Tobias; Prinz, Vincent; Bedini, Gloria; Krug, Susanne M; Czabanka, Marcus; Wagner, Josephin; Fromm, Michael; Bersano, Anna

2016-01-01

Moyamoya disease is a rare steno-occlusive cerebrovascular disorder often resulting in hemorrhagic and ischemic strokes. Although sharing the same ischemic stimulus with atherosclerotic cerebrovascular disease, Moyamoya disease is characterized by a highly instable cerebrovascular system which is prone to rupture due to pathological neovascularization. To understand the molecular mechanisms underlying this instability, angiopoietin-2 gene expression was analyzed in middle cerebral artery lesions obtained from Moyamoya disease and atherosclerotic cerebrovascular disease patients. Angiopoietin-2 was significantly up-regulated in Moyamoya vessels, while serum concentrations of soluble angiopoietins were not changed. For further evaluations, cerebral endothelial cells incubated with serum from these patients in vitro were applied. In contrast to atherosclerotic cerebrovascular disease serum, Moyamoya disease serum induced an angiopoietin-2 overexpression and secretion, accompanied by loss of endothelial integrity. These effects were absent or inverse in endothelial cells of non-brain origin suggesting brain endothelium specificity. The destabilizing effects on brain endothelial cells to Moyamoya disease serum were partially suppressed by the inhibition of angiopoietin-2. Our findings define brain endothelial cells as the potential source of vessel-destabilizing factors inducing the high plasticity state and disintegration in Moyamoya disease in an autocrine manner. We also provide new insights into Moyamoya disease pathophysiology that may be helpful for preventive treatment strategies in future. PMID:27381827

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

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

The control of vascular endothelial cell injury.

PubMed

Murota, S; Morita, I; Suda, N

1990-01-01

The mechanism by which MCI-186 showed a potent cytoprotective effect on the in vitro endothelial cell injury due to 15-HPETE was studied. Stimulation of human leukocytes with various chemical mediators such as TPA, f-Met-Leu-Phe, LTB4, etc. elicited the production of active oxygens, which could be detected by luminol-dependent chemiluminescence. Among the chemical mediators tested, TPA elicited the chemiluminescence the most, and f-Met-Leu-Phe and LTB4 came next. When the leukocytes were directly placed on a monolayer of cultured endothelial cells, followed by stimulating the leukocytes with TPA, severe endothelial cell injury was observed. The effect of TPA was dose dependent. There was good correlation between the active oxygen releasing activity and the cytotoxic activity. When the leukocytes were placed on a filter which was set apart from the monolayer of endothelial cell in a culture dish, and stimulated the leukocytes with TPA, no cytotoxicity was observed. These data strongly suggest that the substance responsible for the cytotoxicity must be a very labile and short-lived substance, presumably active oxygens. On the other hand, MCI-186 was found to have a complete quenching activity to the chemiluminescence due to active oxygens in the TPA-leukocyte system. Taken together, these factors indicate that the potent cytoprotective effect of MCI-186 may be due to its specific radical scavenging activity.

Endothelial Mineralocorticoid Receptor Mediates Parenchymal Arteriole and Posterior Cerebral Artery Remodeling During Angiotensin II-Induced Hypertension.

PubMed

Diaz-Otero, Janice M; Fisher, Courtney; Downs, Kelsey; Moss, M Elizabeth; Jaffe, Iris Z; Jackson, William F; Dorrance, Anne M

2017-12-01

The brain is highly susceptible to injury caused by hypertension because the increased blood pressure causes artery remodeling that can limit cerebral perfusion. Mineralocorticoid receptor (MR) antagonism prevents hypertensive cerebral artery remodeling, but the vascular cell types involved have not been defined. In the periphery, the endothelial MR mediates hypertension-induced vascular injury, but cerebral and peripheral arteries are anatomically distinct; thus, these findings cannot be extrapolated to the brain. The parenchymal arterioles determine cerebrovascular resistance. Determining the effects of hypertension and MR signaling on these arterioles could lead to a better understanding of cerebral small vessel disease. We hypothesized that endothelial MR signaling mediates inward cerebral artery remodeling and reduced cerebral perfusion during angiotensin II (AngII) hypertension. The biomechanics of the parenchymal arterioles and posterior cerebral arteries were studied in male C57Bl/6 and endothelial cell-specific MR knockout mice and their appropriate controls using pressure myography. AngII increased plasma aldosterone and decreased cerebral perfusion in C57Bl/6 and MR-intact littermates. Endothelial cell MR deletion improved cerebral perfusion in AngII-treated mice. AngII hypertension resulted in inward hypotrophic remodeling; this was prevented by MR antagonism and endothelial MR deletion. Our studies suggest that endothelial cell MR mediates hypertensive remodeling in the cerebral microcirculation and large pial arteries. AngII-induced inward remodeling of cerebral arteries and arterioles was associated with a reduction in cerebral perfusion that could worsen the outcome of stroke or contribute to vascular dementia. © 2017 American Heart Association, Inc.

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.

Establishing Clonal Cell Lines with Endothelial-Like Potential from CD9hi, SSEA-1− Cells in Embryonic Stem Cell-Derived Embryoid Bodies

PubMed Central

Lian, Qizhou; Yeo, KengSuan; Que, Jianwen; Tan, EileenKhiaWay; Yu, Fenggang; Yin, Yijun; Salto-Tellez, Manuel; Oakley, Reida Menshawe El; Lim, Sai-Kiang

2006-01-01

Background Differentiation of embryonic stem cells (ESCs) into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. Methodology/Principal Findings We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs). Despite originating from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r2 = 0.93) while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9hi, SSEA-1− while ESCs are CD9lo, SSEA-1+. Isolation of CD9hi, SSEA-1− cells that constituted 1%–10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r2 = 0.95) and a propensity to differentiate into endothelial-like cells. Conclusions By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells with endothelial-like potential from mouse ESCs. PMID:17183690

Rat liver endothelial and Kupffer cell-mediated mutagenicity and polycyclic aromatic hydrocarbons and aflatoxin B sub 1

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

Steinberg, P.; Schlemper, B.; Molitor, E.

The ability of isolated rat liver endothelial and Kupffer cells to activate benzo(a)pyrene (BP), trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (DDBP), trans-1,2-dihydroxy-1,2-dihydrochrysene (DDCH), and aflatoxin B{sub 1} (AFB{sub 1}) to mutagenic metabolites was assessed by means of a cell-mediated bacterial mutagenicity assay and compared with the ability of parenchymal cells to activate these compounds. Endothelial and Kupffer cells from untreated rats were able to activate AFB{sub 1} and DDBP; DDBP was activated even in the absence of an NADPH-generating system. Pretreating the animals with Aroclor 1254 strongly enhanced the mutagenicity of the dihydrodiol, whereas the mutagenicity of AFB{sub 1} showed a slight increase. BP andmore » DDCH were only activated by endothelial and Kupffer cells isolated from Aroclor 1254-pretreated rats. Parenchymal cells form untreated animals activated all four carcinogens tested; Aroclor 1254 enhanced the parenchymal cell-mediated mutagenicity of BP and DDCH but did not affect that of DDBP and clearly reduced that of AFB{sub 1}. The reduced mutagenicity of AFB{sub 1} correlates with the decrease in the amount of 2{alpha}-hydroxytestosterone formed when testosterone was incubated with parenchymal cell microsomes from Aroclor 1254-pretreated rats (compared with microsomes from untreated animals): the formation of 2{alpha}-hydroxytestosterone is specifically catalyzed by cytochrome P-450h, a hemoprotein thought to be involved in the activation of AFB{sub 1}. These results show that not only rat liver parenchymal cells, but also endothelial and Kupffer cells, activated several carcinogens to mutagenic metabolites.« less

Minoxidil-induced hair growth is mediated by adenosine in cultured dermal papilla cells: possible involvement of sulfonylurea receptor 2B as a target of minoxidil.

PubMed

Li, M; Marubayashi, A; Nakaya, Y; Fukui, K; Arase, S

2001-12-01

The mechanism by which minoxidil, an adenosine-triphosphate-sensitive potassium channel opener, induces hypertrichosis remains to be elucidated. Minoxidil has been reported to stimulate the production of vascular endothelial growth factor, a possible promoter of hair growth, in cultured dermal papilla cells. The mechanism of production of vascular endothelial growth factor remains unclear, however. We hypothesize that adenosine serves as a mediator of vascular endothelial growth factor production. Minoxidil-induced increases in levels of intracellular Ca(2+) and vascular endothelial growth factor production in cultured dermal papilla cells were found to be inhibited by 8-sulfophenyl theophylline, a specific antagonist for adenosine receptors, suggesting that dermal papilla cells possess adenosine receptors and sulfonylurea receptors, the latter of which is a well-known target receptor for adenosine-triphosphate-sensitive potassium channel openers. The expression of sulfonylurea receptor 2B and of the adenosine A1, A2A, and A2B receptors was detected in dermal papilla cells by means of reverse transcription polymerase chain reaction analysis. In order to determine which of the adenosine receptor subtypes contribute to minoxidil-induced hair growth, the effects of subtype-specific antagonists for adenosine receptors were investigated. Significant inhibition in increase in intracellular calcium level by minoxidil or adenosine was observed as the result of pretreatment with 8-cyclopentyl-1,3-dipropylxanthine, an antagonist for adenosine A1 receptor, but not by 3,7-dimethyl-1-propargyl-xanthine, an antagonist for adenosine A2 receptor, whereas vascular endothelial growth factor production was blocked by both adenosine A1 and A2 receptor antagonists. These results indicate that the effect of minoxidil is mediated by adenosine, which triggers intracellular signal transduction via both adenosine A1 and A2 receptors, and that the expression of sulfonylurea receptor 2B in dermal papilla cells might play a role in the production of adenosine.

Stabilization of VEGFR2 signaling by cerebral cavernous malformation 3 is critical for vascular development.

PubMed

He, Yun; Zhang, Haifeng; Yu, Luyang; Gunel, Murat; Boggon, Titus J; Chen, Hong; Min, Wang

2010-04-06

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: CCM1, CCM2, and CCM3. CCM3, also known as PDCD10 (programmed cell death 10), was initially identified as a messenger RNA whose abundance was induced by apoptotic stimuli in vitro. However, the in vivo function of CCM3 has not been determined. Here, we describe mice with a deletion of the CCM3 gene either ubiquitously or specifically in the vascular endothelium, smooth muscle cells, or neurons. Mice with global or endothelial cell-specific deletion of CCM3 exhibited defects in embryonic angiogenesis and died at an early embryonic stage. CCM3 deletion reduced vascular endothelial growth factor receptor 2 (VEGFR2) signaling in embryos and endothelial cells. In response to VEGF stimulation, CCM3 was recruited to and stabilized VEGFR2, and the carboxyl-terminal domain of CCM3 was required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients lacking the carboxyl-terminal domain were labile and were unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 promotes VEGFR2 signaling during vascular development.

Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates HUVEC proteins related to inflammatory and stress responses.

PubMed

Neves, Gabriela Westerlund Peixoto; Curty, Nathália de Andrade; Kubitschek-Barreira, Paula Helena; Fontaine, Thierry; Souza, Gustavo Henrique Martins Ferreira; Cunha, Marcel Lyra; Goldman, Gustavo H; Beauvais, Anne; Latgé, Jean-Paul; Lopes-Bezerra, Leila M

2017-01-16

Aspergillus fumigatus, the main etiologic agent causing invasive aspergillosis, can induce an inflammatory response and a prothrombotic phenotype upon contact with human umbilical vein endothelial cells (HUVECs). However, the fungal molecules involved in this endothelial response remain unknown. A. fumigatus hyphae produce an extracellular matrix composed of galactomannan, galactosaminogalactan and α-(1,3)-glucan. In this study, we investigated the consequences of UGM1 gene deletion in A. fumigatus, which produces a mutant with increased galactosaminogalactan production. The ∆ugm1 mutant exhibited an HUVEC-hyperadhesive phenotype and induced increased endothelial TNF-α secretion and tissue factor mRNA overexpression in this "semi-professional" immune host cell. Using a shotgun proteomics approach, we show that the A. fumigatus ∆ugm1 strain can modulate the levels of proteins in important endothelial pathways related to the inflammatory response mediated by TNF-α and to stress response pathways. Furthermore, a purified galactosaminogalactan fraction was also able to induce TNF-α secretion and the coincident HUVEC pathways regulated by the ∆ugm1 mutant, which overexpresses this component, as demonstrated by fluorescence microscopy. This work contributes new data regarding endothelial mechanisms in response to A. fumigatus infection. Invasive aspergillosis is the main opportunistic fungal infection described in neutropenic hematologic patients. One important clinical aspect of this invasive fungal infection is vascular thrombosis, which could be related, at least in part, to the activation of endothelial cells, as shown in previous reports from our group. It is known that direct contact between the A. fumigatus hyphal cell wall and the HUVEC cell surface is necessary to induce an endothelial prothrombotic phenotype and secretion of pro-inflammatory cytokines, though the cell surface components of this angioinvasive fungus that trigger this endothelial response are unknown. The present work employs a discovery-driven proteomics approach to reveal the role of one important cell wall polysaccharide of A. fumigatus, galactosaminogalactan, in the HUVEC interaction and the consequent mechanisms of endothelial activation. This is the first report of the overall panel of proteins related to the HUVEC response to a specific and purified cell wall component of the angioinvasive fungus A. fumigatus. Copyright © 2016 Elsevier B.V. All rights reserved.

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

PubMed Central

Shetty, Shishir; Weston, Christopher J.; Adams, David H.; Lalor, Patricia F.

2014-01-01

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this 'adhesion cascade' can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process. PMID:24686418

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.

Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice

PubMed Central

McKenzie, Jennifer A.; Buettmann, Evan G.; Gardner, Michael J.; Silva, Matthew J.

2015-01-01

Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10–24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2. PMID:26344756

Development and characterization of monoclonal antibodies specific for chicken IL-8

USDA-ARS?s Scientific Manuscript database

Limited information on chicken cytokines and chemokines hinders progress in understaidng the role of cell-mediated immunity in infections. Interleukin-8/CXCL8 (IL-8) is a CXC-family chemokine produced by fibroblasts and other cell types, including epithelial cells, endothelial cells, neutrophils, a...

Studies of the endothelial origin of cells in systemic angioendotheliomatosis and other vascular lesions of the brain and meninges using ulex europaeus lectin stains.

PubMed

Schelper, R L; Olson, S P; Carroll, T J; Hart, M N; Witters, E

1986-01-01

Ulex europaeus agglutinin I (UEA-I) is a plant lectin which binds specifically to alpha-L-fucose moieties on the surface glycoproteins of human endothelial cells. The binding is completely inhibited by preincubation of the lectin with fucose. UEA-I can be conjugated directly to fluorescein or peroxidase and can be used to stain endothelium of paraffin embedded tissues. UEA-I staining was evaluated on normal and infarcted brain, systemic angioendotheliomatosis, metastatic epidural angiosarcoma, hemangioendothelioma, hemangioblastoma, angioblastic meningioma of both the hemangioblastic and hemangiopericytic types, and vascular meningioma. The endothelium, but not neuropil of normal and infarcted brain was positive for UEA-I. The tumor cells of hemangioendothelioma and angiosarcoma also stained. However, no staining was seen in malignant intravascular cells of angioendotheliomatosis, the stromal cells of hemangioblastoma, or pericytes of angioblastic meningioma. It is concluded that the malignant cells in angioendotheliomatosis, the stromal cells of hemangioblastoma and the pericytes of angioblastic meningioma do not produce surface glycoproteins characteristic of endothelial cells.

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.

Cell line specific modulation of connexin43 expression after exposure to ionizing radiation.

PubMed

Banaz-Yaşar, Ferya; Tischka, Rabea; Iliakis, George; Winterhager, Elke; Gellhaus, Alexandra

2005-01-01

Gap junctional intercellular communication plays a significant role in mediating radiation-induced bystander effects. However, the level of Cx43 itself is influenced by ionizing radiation, which could modify the bystander effect. Here we have investigated several cell lines for the modulation of Cx43 expression 24 h after irradiation with 5 Gy X-rays. The mouse endothelial cell line bEnd3 revealed a significantly elevated level of Cx43 already 15 min after exposure to X-rays, whereas human hybrid endothelial cells (EA.hy926) exhibited a transient downregulation of Cx43 mRNA. No obvious changes in the communication properties of the different cell lines could be observed after irradiation. The communication-deficient malignant human trophoblast cell line Jeg3 stably transfected with Cx43 did not reveal any induction of endogenous nor alteration in the exogenous Cx43 transcript level upon exposure to 5 Gy. Taken together, our data show a cell line specific modulation of Cx43 expression after exposure to X-rays.

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.

Molecular Signatures of Tissue-Specific Microvascular Endothelial Cell Heterogeneity in Organ Maintenance and Regeneration

PubMed Central

Nolan, Daniel J.; Ginsberg, Michael; Israely, Edo; Palikuqi, Brisa; Poulos, Michael G.; James, Daylon; Ding, Bi-Sen; Schachterle, William; Liu, Ying; Rosenwaks, Zev; Butler, Jason M.; Xiang, Jenny; Rafii, Arash; Shido, Koji; Rabbany, Sina Y.; Elemento, Olivier; Rafii, Shahin

2013-01-01

SUMMARY Microvascular endothelial cells (ECs) within different tissues are endowed with distinct but as yet unrecognized structural, phenotypic, and functional attributes. We devised EC purification, cultivation, profiling, and transplantation models that establish tissue-specific molecular libraries of ECs devoid of lymphatic ECs or parenchymal cells. These libraries identify attributes that confer ECs with their organotypic features. We show that clusters of transcription factors, angiocrine growth factors, adhesion molecules, and chemokines are expressed in unique combinations by ECs of each organ. Furthermore, ECs respond distinctly in tissue regeneration models, hepatectomy, and myeloablation. To test the data set, we developed a transplantation model that employs generic ECs differentiated from embryonic stem cells. Transplanted generic ECs engraft into regenerating tissues and acquire features of organotypic ECs. Collectively, we demonstrate the utility of informational databases of ECs toward uncovering the extravascular and intrinsic signals that define EC heterogeneity. These factors could be exploited therapeutically to engineer tissue-specific ECs for regeneration. PMID:23871589

Gene regulation by mechanical forces

NASA Technical Reports Server (NTRS)

Oluwole, B. O.; Du, W.; Mills, I.; Sumpio, B. E.

1997-01-01

Endothelial cells are subjected to various mechanical forces in vivo from the flow of blood across the luminal surface of the blood vessel. The purpose of this review was to examine the data available on how these mechanical forces, in particular cyclic strain, affect the expression and regulation of endothelial cell function. Studies from various investigators using models of cyclic strain in vitro have shown that various vasoactive mediators such as nitric oxide and prostacyclin are induced by the effect of mechanical deformation, and that the expression of these mediators may be regulated at the transcription level by mechanical forces. There also seems to be emerging evidence that endothelial cells may also act as mechanotransducers, whereby the transmission of external forces induces various cytoskeletal changes and second messenger cascades. Furthermore, it seems these forces may act on specific response elements of promoter genes.

Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium.

PubMed

Gnecco, Juan S; Pensabene, Virginia; Li, David J; Ding, Tianbing; Hui, Elliot E; Bruner-Tran, Kaylon L; Osteen, Kevin G

2017-07-01

The endometrium is the inner lining of the uterus. Following specific cyclic hormonal stimulation, endometrial stromal fibroblasts (stroma) and vascular endothelial cells exhibit morphological and biochemical changes to support embryo implantation and regulate vascular function, respectively. Herein, we integrated a resin-based porous membrane in a dual chamber microfluidic device in polydimethylsiloxane that allows long term in vitro co-culture of human endometrial stromal and endothelial cells. This transparent, 2-μm porous membrane separates the two chambers, allows for the diffusion of small molecules and enables high resolution bright field and fluorescent imaging. Within our primary human co-culture model of stromal and endothelial cells, we simulated the temporal hormone changes occurring during an idealized 28-day menstrual cycle. We observed the successful differentiation of stroma into functional decidual cells, determined by morphology as well as biochemically as measured by increased production of prolactin. By controlling the microfluidic properties of the device, we additionally found that shear stress forces promoted cytoskeleton alignment and tight junction formation in the endothelial layer. Finally, we demonstrated that the endometrial perivascular stroma model was sustainable for up to 4 weeks, remained sensitive to steroids and is suitable for quantitative biochemical analysis. Future utilization of this device will allow the direct evaluation of paracrine and endocrine crosstalk between these two cell types as well as studies of immunological events associated with normal vs. disease-related endometrial microenvironments.

Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

NASA Astrophysics Data System (ADS)

Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

1999-07-01

Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

Radiation-induced interleukin-6 expression through MAPK/p38/NF-kappaB signaling pathway and the resultant antiapoptotic effect on endothelial cells through Mcl-1 expression with sIL6-Ralpha.

PubMed

Chou, Chia-Hung; Chen, Shee-Uan; Cheng, Jason Chia-Hsien

2009-12-01

To investigate the mechanism of interleukin-6 (IL-6) activity induced by ionizing radiation. Human umbilical vascular endothelial cells (HUVECs) were irradiated with different doses to induce IL-6. The IL-6 promoter assay and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to examine transcriptional regulation. Specific chemical inhibitors, decoy double-stranded oligodeoxynucleotides, and Western blotting were conducted to investigate the signal transduction pathway. Recombinant soluble human IL-6 receptor alpha-chain (sIL6-Ralpha) and specific small interfering RNA were used to evaluate the biologic function of radiation-induced IL-6. Four grays of radiation induced the highest level of IL-6 protein. The promoter assay and RT-PCR data revealed that the induction of IL-6 was mediated through transcriptional regulation. The p38 inhibitor SB203580, by blocking nuclear factor-kappaB (NF-kappaB) activation, prevented both the transcriptional and translational regulation of radiation-induced IL-6 expression. The addition of sIL6-Ralpha rescued HUVECs from radiation-induced death in an IL-6 concentratio-dependent manner. The antiapoptotic effect of combined sIL6-Ralpha and radiation-induced IL-6 was inhibited by mcl-1-specific small interfering RNA. Radiation transcriptionally induces IL-6 expression in endothelial cells through mitogen-activated protein kinase/p38-mediated NF-kappaB/IkappaB (inhibitor of NF-kappaB) complex activation. In the presence of sIL6-Ralpha, radiation-induced IL-6 expression acts through Mcl-1 expression to rescue endothelial cells from radiation-induced death.

HMGB1 induces an inflammatory response in endothelial cells via the RAGE-dependent endoplasmic reticulum stress pathway

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

Luo, Ying; Li, Shu-Jun; Yang, Jian

Highlights: •Mechanisms of inflammatory response induced by HMGB1 are incompletely understood. •We found that endoplasmic reticulum stress mediate the inflammatory response induced by HMGB1. •RAGE-mediated ERS pathways are involved in those processes. •We reported a new mechanism for HMGB1 induced inflammatory response. -- Abstract: The high mobility group 1B protein (HMGB1) mediates chronic inflammatory responses in endothelial cells, which play a critical role in atherosclerosis. However, the underlying mechanism is unknown. The goal of our study was to identify the effects of HMGB1 on the RAGE-induced inflammatory response in endothelial cells and test the possible involvement of the endoplasmic reticulummore » stress pathway. Our results showed that incubation of endothelial cells with HMGB1 (0.01–1 μg/ml) for 24 h induced a dose-dependent activation of endoplasmic reticulum stress transducers, as assessed by PERK and IRE1 protein expression. Moreover, HMGB1 also promoted nuclear translocation of ATF6. HMGB1-mediated ICAM-1 and P-selectin production was dramatically suppressed by PERK siRNA or IRE1 siRNA. However, non-targeting siRNA had no such effects. HMGB1-induced increases in ICAM-1 and P-selectin expression were also inhibited by a specific eIF2α inhibitor (salubrinal) and a specific JNK inhibitor (SP600125). Importantly, a blocking antibody specifically targeted against RAGE (anti-RAGE antibody) decreased ICAM-1, P-selectin and endoplasmic reticulum stress molecule (PERK, eIF2α, IRE1 and JNK) protein expression levels. Collectively, these novel findings suggest that HMGB1 promotes an inflammatory response by inducing the expression of ICAM-1 and P-selectin via RAGE-mediated stimulation of the endoplasmic reticulum stress pathway.« less

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

Lu Jiawei; Division of Molecular Medicine, Harbor-UCLA Medical Center, David Geffen School of Medicine, University of California Los Angeles, Torrance, CA 90502; Lu Zhenyu

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

Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation.

PubMed

Krawczenko, Agnieszka; Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

2017-01-01

Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.

The skeletal vascular system - Breathing life into bone tissue.

PubMed

Stegen, Steve; Carmeliet, Geert

2017-08-26

During bone development, homeostasis and repair, a dense vascular system provides oxygen and nutrients to highly anabolic skeletal cells. Characteristic for the vascular system in bone is the serial organization of two capillary systems, each typified by specific morphological and physiological features. Especially the arterial capillaries mediate the growth of the bone vascular system, serve as a niche for skeletal and hematopoietic progenitors and couple angiogenesis to osteogenesis. Endothelial cells and osteoprogenitor cells interact not only physically, but also communicate to each other by secretion of growth factors. A vital angiogenic growth factor is vascular endothelial growth factor and its expression in skeletal cells is controlled by osteogenic transcription factors and hypoxia signaling, whereas the secretion of angiocrine factors by endothelial cells is regulated by Notch signaling, blood flow and possibly hypoxia. Bone loss and impaired fracture repair are often associated with reduced and disorganized blood vessel network and therapeutic targeting of the angiogenic response may contribute to enhanced bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo

NASA Astrophysics Data System (ADS)

Kim, K. Jin; Li, Bing; Winer, Jane; Armanini, Mark; Gillett, Nancy; Phillips, Heidi S.; Ferrara, Napoleone

1993-04-01

THE development of new blood vessels (angiogenesis) is required for many physiological processes including embryogenesis, wound healing and corpus luteum formation1,2. Blood vessel neoformation is also important in the pathogenesis of many disorders1-5, particularly rapid growth and metastasis of solid tumours3-5. There are several potential mediators of tumour angiogenesis, including basic and acidic fibroblast growth factors, tumour necrosis factor-α and transforming factors-α and -β 1,2. But it is unclear whether any of these agents actually mediates angiogenesis and tumour growth in vivo. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and an angiogenesis inducer released by a variety of tumour cells and expressed in human tumours in situ. To test whether VEGF may be a tumour angiogenesis factor in vivo, we injected human rhabdomyosar-coma, glioblastoma multiforme or leiomyosarcoma cell lines into nude mice. We report here that treatment with a monoclonal antibody specific for VEGF inhibited the growth of the tumours, but had no effect on the growth rate of the tumour cells In vitro. The density of vessels was decreased in the antibody-treated tumours. These findings demonstrate that inhibition of the action of an angiogenic factor spontaneously produced by tumour cells may suppress tumour growth in vivo.

Patterning vascular networks in vivo for tissue engineering applications.

PubMed

Chaturvedi, Ritika R; Stevens, Kelly R; Solorzano, Ricardo D; Schwartz, Robert E; Eyckmans, Jeroen; Baranski, Jan D; Stapleton, Sarah Chase; Bhatia, Sangeeta N; Chen, Christopher S

2015-05-01

The ultimate design of functionally therapeutic engineered tissues and organs will rely on our ability to engineer vasculature that can meet tissue-specific metabolic needs. We recently introduced an approach for patterning the formation of functional spatially organized vascular architectures within engineered tissues in vivo. Here, we now explore the design parameters of this approach and how they impact the vascularization of an engineered tissue construct after implantation. We used micropatterning techniques to organize endothelial cells (ECs) into geometrically defined "cords," which in turn acted as a template after implantation for the guided formation of patterned capillaries integrated with the host tissue. We demonstrated that the diameter of the cords before implantation impacts the location and density of the resultant capillary network. Inclusion of mural cells to the vascularization response appears primarily to impact the dynamics of vascularization. We established that clinically relevant endothelial sources such as induced pluripotent stem cell-derived ECs and human microvascular endothelial cells can drive vascularization within this system. Finally, we demonstrated the ability to control the juxtaposition of parenchyma with perfused vasculature by implanting cords containing a mixture of both a parenchymal cell type (hepatocytes) and ECs. These findings define important characteristics that will ultimately impact the design of vasculature structures that meet tissue-specific needs.

Serum-deprived human multipotent mesenchymal stromal cells (MSCs) are highly angiogenic

PubMed Central

Oskowitz, Adam; McFerrin, Harris; Gutschow, Miriam; Carter, Mary Leita; Pochampally, Radhika

2016-01-01

Recent reports have indicated that mesenchymal stromal cells (MSCs) from bone marrow have a potential in vascular remodeling and angiogenesis. Here, we report a unique phenomenon that under serum-deprived conditions MSCs survive and replicate. Secretome analysis of MSCs grown under serum-deprived conditions (SD-MSCs) identified a significant upregulation of prosurvival and angiogenic factors including VEGF-A, ANGPTs, IGF-1, and HGF. An ex vivo rat aortic assay demonstrated longer neovascular sprouts generated from rat aortic rings cultured in SD-MSC-conditioned media compared to neovascular sprouts from aortas grown in MSC-conditioned media. With prolonged serum deprivation, a subpopulation of SD-MSCs began to exhibit an endothelial phenotype. This population expressed endothelial-specific proteins including VEGFR2, Tie2/TEK, PECAM/CD31, and eNOS and also demonstrated the ability to uptake acetylated LDL. SD-MSCs also exhibited enhanced microtubule formation in an in vitro angiogenesis assay. Modified chick chorioallantoic membrane (CAM) angiogenesis assays showed significantly higher angiogenic potential for SD-MSCs compared to MSCs. Analysis of CAMs grown with SD-MSCs identified human-specific CD31-positive cells in vascular structures. We conclude that under the stress of serum deprivation MSCs are highly angiogenic and a population of these cells has the potential to differentiate into endothelial-like cells. PMID:21421339

Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors.

PubMed

Rehman, Jalees; Li, Jingling; Orschell, Christie M; March, Keith L

2003-03-04

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood and can enhance angiogenesis after infusion into host animals. It is not known whether the proangiogenic effects are a result of such events as endothelial differentiation and subsequent proliferation of EPCs or secondary to secretion of angiogenic growth factors. Human EPCs were isolated as previously described, and their phenotypes were confirmed by uptake of acetylated LDL and binding of ulex-lectin. EPC proliferation and surface marker expression were analyzed by flow cytometry, and conditioned medium was assayed for growth factors. The majority of EPCs expressed monocyte/macrophage markers such as CD14 (95.7+/-0.3%), Mac-1 (57.6+/-13.5%), and CD11c (90.8+/-4.9%). A much lower percentage of cells expressed the specific endothelial marker VE-cadherin (5.2+/-0.7%) or stem/progenitor-cell markers AC133 (0.16+/-0.05%) and c-kit (1.3+/-0.7%). Compared with circulating monocytes, cultured EPCs showed upregulation of monocyte activation and macrophage differentiation markers. EPCs did not demonstrate any significant proliferation but did secrete the angiogenic growth factors vascular endothelial growth factor, hepatocyte growth factor, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Our findings suggest that acetylated LDL(+)ulex-lectin(+) cells, commonly referred to as EPCs, do not proliferate but release potent proangiogenic growth factors. The majority of acetylated LDL(+)ulex-lectin(+) cells are derived from monocyte/macrophages. The findings of low proliferation and endothelial differentiation suggest that their angiogenic effects are most likely mediated by growth factor secretion. These findings may allow for development of novel angiogenic therapies relying on secreted growth factors or on recruitment of endogenous monocytes/macrophages to sites of ischemia.

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

PubMed Central

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

2013-01-01

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

Aberrant pulmonary lymphatic development in the nitrofen mouse model of congenital diaphragmatic hernia

PubMed Central

Shue, Eveline; Wu, Jianfeng; Schecter, Samuel; Miniati, Doug

2013-01-01

Purpose Many infants develop a postsurgical chylothorax after diaphragmatic hernia repair. The pathogenesis remains elusive but may be due to dysfunctional lymphatic development. This study characterizes pulmonary lymphatic development in the nitrofen mouse model of CDH. Methods CD1 pregnant mice were fed nitrofen/bisdiamine (N/B) or olive oil at E8.5. At E14.5 and E15.5, lung buds were categorized by phenotype: normal, N/B without CDH (N/B−CDH), or N/B with CDH (N/B+CDH). Anti-CD31 was used to localize all endothelial cells, while anti-LYVE-1 was used to identify lymphatic endothelial cells in lung buds using immunofluorescence. Differential protein expression of lymphatic-specific markers was analyzed. Results Lymphatic endothelial cells localized to the mesenchyme surrounding the airway epithelium at E15.5. CD31 and LYVE-1 colocalization identified lymphatic endothelial cells. LYVE-1 expression was upregulated in N/B+CDH lung buds in comparison to N/B−CDH and normal lung buds by immunofluorescence. Western blotting shows that VEGF-D, LYVE-1, Prox-1, and VEGFR-3 expression was upregulated in N/B+CDH lung buds in comparison to N/B−CDH or control lung buds at E14.5. Conclusions Lung lymphatics are hyperplastic in N/B+CDH. Upregulation of lymphatic-specific genes suggest that lymphatic hyperplasia plays an important role in dysfunctional lung lymphatic development in the nitrofen mouse model of CDH. PMID:23845607

Aberrant pulmonary lymphatic development in the nitrofen mouse model of congenital diaphragmatic hernia.

PubMed

Shue, Eveline; Wu, Jianfeng; Schecter, Samuel; Miniati, Doug

2013-06-01

Many infants develop a postsurgical chylothorax after diaphragmatic hernia repair. The pathogenesis remains elusive but may be owing to dysfunctional lymphatic development. This study characterizes pulmonary lymphatic development in the nitrofen mouse model of CDH. CD1 pregnant mice were fed nitrofen/bisdiamine (N/B) or olive oil at E8.5. At E14.5 and E15.5, lung buds were categorized by phenotype: normal, N/B without CDH (N/B - CDH), or N/B with CDH (N/B+CDH). Anti-CD31 was used to localize all endothelial cells, while anti-LYVE-1 was used to identify lymphatic endothelial cells in lung buds using immunofluorescence. Differential protein expression of lymphatic-specific markers was analyzed. Lymphatic endothelial cells localized to the mesenchyme surrounding the airway epithelium at E15.5. CD31 and LYVE-1 colocalization identified lymphatic endothelial cells. LYVE-1 expression was upregulated in N/B+CDH lung buds in comparison to N/B - CDH and normal lung buds by immunofluorescence. Western blotting shows that VEGF-D, LYVE-1, Prox-1, and VEGFR-3 expression was upregulated in N/B+CDH lung buds in comparison to N/B - CDH or control lung buds at E14.5. Lung lymphatics are hyperplastic in N/B+CDH. Upregulation of lymphatic-specific genes suggests that lymphatic hyperplasia plays an important role in dysfunctional lung lymphatic development in the nitrofen mouse model of CDH. Copyright © 2013 Elsevier Inc. All rights reserved.

A Unique Role for Endothelial Cell Kinesin Light Chain 1, Variant 1 in Leukocyte Transendothelial Migration

PubMed Central

Cyrus, Bita F.; Muller, William A.

2017-01-01

A reservoir of parajunctional membrane in endothelial cells, the lateral border recycling compartment (LBRC), is critical for transendothelial migration (TEM). We have previously shown that targeted recycling of the LBRC to the site of TEM requires microtubules and a kinesin molecular motor. However, the identity of the kinesin and mechanism of cargo binding were not known. We show that microinjection of endothelial cells with a monoclonal antibody specific for kinesin-1 significantly blocked LBRC-targeted recycling and TEM. In complementary experiments, knocking down KIF5B, a ubiquitous kinesin-1 isoform, in endothelial cells significantly decreased targeted recycling of the LBRC and leukocyte TEM. Kinesin heavy chains move cargo along microtubules by one of many kinesin light chains (KLCs), which directly bind the cargo. Knocking down KLC 1 isoform variant 1 (KLC1C) significantly decreased LBRC-targeted recycling and TEM, whereas knocking down other isoforms of KLC1 had no effect. Re-expression of KLC1C resistant to the knockdown shRNA restored targeted recycling and TEM. Thus kinesin-1 and KLC1C are specifically required for targeted recycling and TEM. These data suggest that of the many potential combinations of the 45 kinesin family members and multiple associated light chains, KLC1C links the LBRC to kinesin-1 (KIF5B) during targeted recycling and TEM. Thus, KLC1C can potentially be used as a target for anti-inflammatory therapy. PMID:26994343

A critical role for phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac exchanger 1 in endothelial junction disruption and vascular hyperpermeability

PubMed Central

Naikawadi, Ram P.; Cheng, Ni; Vogel, Stephen M.; Qian, Feng; Wu, Dianqing; Malik, Asrar B.; Ye, Richard D.

2013-01-01

Rationale The small GTPase Rac is critical to vascular endothelial functions, yet its regulation in endothelial cells remains unclear. Understanding the upstream pathway may delineate Rac activation mechanisms and its role in maintaining vascular endothelial barrier integrity. Objective By investigating P-Rex1, one of the Rac-specific guanine nucleotide exchange factors (GEFs) previously known for G protein-coupled receptor (GPCR) signaling, we sought to determine whether Rac-GEF is a nodal for signal integration and potential target for drug intervention. Methods and Results Using gene deletion and siRNA silencing approach, we investigated the role of P-Rex1 in lung microvascular endothelial cells (HLMVECs). TNF-α exposure led to disruption of endothelial junctions, and silencing P-Rex1 protected junction integrity. TNF-α stimulated Rac activation and ROS production in a P-Rex1-dependent manner. Removal of P-Rex1 significantly reduced ICAM-1 expression, PMN transendothelial migration and leukocyte sequestration in TNF-α challenged mouse lungs. The P-Rex1 knockout mice were also refractory to lung vascular hyper-permeability and edema in a LPS-induced sepsis model. Conclusions These results demonstrate for the first time that P-Rex1 expressed in endothelial cells is activated downstream of TNF-α, which is not a GPCR agonist. Our data identify P-Rex1 as a critical mediator of vascular barrier disruption. Targeting P-Rex1 may effectively protect against TNF-α and LPS-induced endothelial junction disruption and vascular hyper-permeability. PMID:22965143

Specific gene delivery to liver sinusoidal and artery endothelial cells.

PubMed

Abel, Tobias; El Filali, Ebtisam; Waern, Johan; Schneider, Irene C; Yuan, Qinggong; Münch, Robert C; Hick, Meike; Warnecke, Gregor; Madrahimov, Nodir; Kontermann, Roland E; Schüttrumpf, Jörg; Müller, Ulrike C; Seppen, Jurgen; Ott, Michael; Buchholz, Christian J

2013-09-19

Different types of endothelial cells (EC) fulfill distinct tasks depending on their microenvironment. ECs are therefore difficult to genetically manipulate ex vivo for functional studies or gene therapy. We assessed lentiviral vectors (LVs) targeted to the EC surface marker CD105 for in vivo gene delivery. The mouse CD105-specific vector, mCD105-LV, transduced only CD105-positive cells in primary liver cell cultures. Upon systemic injection, strong reporter gene expression was detected in liver where mCD105-LV specifically transduced liver sinusoidal ECs (LSECs) but not Kupffer cells, which were mainly transduced by nontargeted LVs. Tumor ECs were specifically targeted upon intratumoral vector injection. Delivery of the erythropoietin gene with mCD105-LV resulted in substantially increased erythropoietin and hematocrit levels. The human CD105-specific vector (huCD105-LV) transduced exclusively human LSECs in mice transplanted with human liver ECs. Interestingly, when applied at higher dose and in absence of target cells in the liver, huCD105-LV transduced ECs of a human artery transplanted into the descending mouse aorta. The data demonstrate for the first time targeted gene delivery to specialized ECs upon systemic vector administration. This strategy offers novel options to better understand the physiological functions of ECs and to treat genetic diseases such as those affecting blood factors.

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

PubMed Central

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

2013-01-01

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

In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor ▿

PubMed Central

Wamsley, Heather L.; Barbet, Anthony F.

2008-01-01

Endothelial cell culture and preliminary immunofluorescent staining of Anaplasma-infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells and other potentially cryptic sites of Anaplasma sp. infection in mammalian tissues, a sensitive and specific isothermal in situ technique to detect localized Anaplasma gene sequences by using rolling-circle amplification of circularizable, linear, oligonucleotide probes (padlock probes) was developed. Cytospin preparations of uninfected or Anaplasma-infected cell cultures were examined using this technique. Via fluorescence microscopy, the technique described here, and a combination of differential interference contrast microscopy and von Willebrand factor immunofluorescence, Anaplasma phagocytophilum and Anaplasma marginale were successfully localized in situ within intact cultured mammalian cells. This work represents the first application of this in situ method for the detection of a microorganism and forms the foundation for future applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected mammalian and arthropod hosts and in cell cultures. PMID:18495855

Expression of vascular endothelial growth factor does not promote transformation but confers a growth advantage in vivo to Chinese hamster ovary cells.

PubMed Central

Ferrara, N; Winer, J; Burton, T; Rowland, A; Siegel, M; Phillips, H S; Terrell, T; Keller, G A; Levinson, A D

1993-01-01

Vascular endothelial growth factor (VEGF) is a mitogen with a specificity for endothelial cells in vitro and an angiogenic inducer in vivo. We tested the hypothesis that VEGF may confer on expressing cells a growth advantage in vivo. Dihydrofolatereductase--Chinese hamster ovary cells were transfected with expression vectors which direct the constitutive synthesis of VEGF. Neither the expression nor the exogenous administration of VEGF stimulated anchorage-dependent or anchorage-independent growth of Chinese hamster ovary cells in vitro. However, VEGF-expressing clones, unlike control cells, demonstrated an ability to proliferate in nude mice. Histologic examination revealed that the proliferative lesions were compact, well vascularized, and nonedematous. Ultrastructural analysis revealed that capillaries within the lesions were of the continuous type. These findings indicate that the expression of VEGF may confer on cells the ability to grow in vivo in the absence of transformation by purely paracrine mechanisms. Since VEGF is a widely distributed protein, this property may have relevance for a variety of physiological and pathological proliferative processes. Images PMID:8423215

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.

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.

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

The role of platelet and endothelial GARP in thrombosis and hemostasis.

PubMed

Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

The role of platelet and endothelial GARP in thrombosis and hemostasis

PubMed Central

Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F.; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M.; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Background Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. Objectives To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Methods Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Results Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Conclusions Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice. PMID:28278197

[Extracellular signal-regulated kinase signaling pathway regulates the endothelial differentiation of periodontal ligament stem cells].

PubMed

Zhu, Hong; Luo, Lankun; Wang, Ying; Tan, Jun; Xue, Peng; Wang, Qintao

2016-03-01

To investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC). Human PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed. Phosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05). The endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.

Embryonic vasculogenesis and hematopoietic specification

USDA-ARS?s Scientific Manuscript database

Vasculogenesis is the process by which blood vessels are formed de novo. In mammals, vasculogenesis occurs in parallel with hematopoiesis, the formation of blood cells. Thus, it is debated whether vascular endothelial cells and blood cells are derived from a common progenitor. Whether or not this is...

Fucoidan-induced osteogenic differentiation promotes angiogenesis by inducing vascular endothelial growth factor secretion and accelerates bone repair.

PubMed

Kim, Beom-Su; Yang, Sun-Sik; You, Hyung-Keun; Shin, Hong-In; Lee, Jun

2018-03-01

Osteogenesis and angiogenesis, including cell-cell communication between blood vessel cells and bone cells, are essential for bone repair. Fucoidan is a chemical compound that has a variety of biological activities. It stimulates osteoblast differentiation in human mesenchymal stem cells (MSCs), which in turn induces angiogenesis. However, the mechanism by which this communication between osteoblasts and endothelial cells is mediated remains unclear. Thus, the aim of this study was to clarify the relationship between fucoidan-induced osteoblastic differentiation in MSCs and angiogenesis in endothelial cells. First, the effect was confirmed of fucoidan on osteoblast differentiation in MSCs and obtained conditioned media from these cells (Fucoidan-MSC-CM). Next, the angiogenic activity of Fucoidan-MSC-CM was investigated and it was found that it stimulated angiogenesis, demonstrated by proliferation, tube formation, migration and sprout capillary formation in human umbilical vein endothelial cells. Messenger ribonucleic acid expression and protein secretion of vascular endothelial growth factor (VEGF) were dramatically increased during fucoidan-induced osteoblast differentiation and that its angiogenic activities were reduced by a VEGF/VEGF receptor-specific binding inhibitor. Furthermore, Fucoidan-MSC-CM increased the phosphorylation of mitogen-activated protein kinase and PI3K/AKT/eNOS signalling pathway, and that its angiogenic effects were markedly suppressed by SB203580 and AKT 1/2 inhibitor. Finally, an in vivo study was conducted and it was found that fucoidan accelerated new blood vessel formation and partially promoted bone formation in a rabbit model of a calvarial bone defect. This is the first study to investigate the angiogenic effect of fucoidan-induced osteoblastic differentiation through VEGF secretion, suggesting the therapeutic potential of fucoidan for enhancing bone repair. Copyright © 2017 John Wiley & Sons, Ltd.

Agmatine promotes the migration of murine brain endothelial cells via multiple signaling pathways.

PubMed

Jung, Hyun-Joo; Jeon, Yong-Heui; Bokara, Kiran Kumar; Koo, Bon-Nyeo; Lee, Won Taek; Park, Kyung Ah; Lee, Jong-Eun

2013-01-17

The combination of adhesion and migration of endothelial cells (ECs) is an integral process for evolution, organization, repair and vessel formation in living organisms. Agmatine, a polycationic amine existing in brain, has been investigated to exert neuroprotective effects. Up to date, there are no studies reporting that agmatine modulates murine brain endothelial (bEnd.3) cells migration. In the present study, we intend to investigate the role of agmatine in bEnd.3 cells migration and the molecular mechanism mediating this action. The effect of agmatine on the bEnd.3 cells migration was examined by migration assay, and the mechanism involved for this effect was investigated by western blot analysis and NO contents measurements. Agmatine treatment (50, 100 and 200 μM) significantly accelerated bEnd.3 cells migration in a concentration-dependent manner. Western blotting revealed that agmatine treatment significantly induced vascular endothelial growth factor (VEGF), VEGF receptor 2 (Flk-1/KDR or VEGFR2), phosphatidylinositol 3-kinase (PI3K), Akt/protein kinase B (also known as PKB, PI3K downstream effector protein), endothelial nitric oxide synthase (eNOS) nitric oxide (NO; product by eNOS) and intercellular adhesion molecule 1 (ICAM-1) expressions during bEnd.3 cells migration. The expression of ICAM-1 and migration of bEnd.3 cells, induced by agmatine, were significantly attenuated by treatment of wortmannin, a specific PI3K inhibitor. Taken together, we provide the first evidence that activation of VEGF/VEGFR2 and the consequential PI3K/Akt/eNOS/NO/ICAM-1 signaling pathways are serial events, through which the treatment of agmatine could lead to bEnd.3 cells migration. Copyright © 2012 Elsevier Inc. All rights reserved.

Double-chimera proteins to enhance recruitment of endothelial cells and their progenitor cells.

PubMed

Behjati, M; Kazemi, M; Hashemi, M; Zarkesh-Esfahanai, S H; Bahrami, E; Hashemi-Beni, B; Ahmadi, R

2013-08-20

Enhanced attraction of selective vascular reparative cells is of great importance in order to increase vascular patency after endovascular treatments. We aimed to evaluate efficient attachment of endothelial cells and their progenitors on surfaces coated with mixture of specific antibodies, L-selectin and VE-cadherin, with prohibited platelet attachment. The most efficient conditions for coating of L-selectin-Fc chimera and VE-cadherin-Fc chimera proteins were first determined by protein coating on ELISA plates. The whole processes were repeated on titanium substrates, which are commonly used to coat stents. Endothelial progenitor cells (EPCs) and human umbilical vein endothelial cells (HUVECs) were isolated and characterized by flow cytometry. Cell attachment, growth, proliferation, viability and surface cytotoxicity were evaluated using nuclear staining and MTT assay. Platelet and cell attachment were evaluated using scanning electron microscopy. Optimal concentration of each protein for surface coating was 50 ng/ml. The efficacy of protein coating was both heat and pH independent. Calcium ions had significant impact on simultaneous dual-protein coating (P<0.05). Coating stability data revealed more than one year stability for these coated proteins at 4°C. L-selectin and VE-cadherin (ratio of 50:50) coated surface showed highest EPC and HUVEC attachment, viability and proliferation compared to single protein coated and non-coated titanium surfaces (P<0.05). This double coated surface did not show any cytotoxic effect. Surfaces coated with L-selectin and VE-cadherin are friendly surface for EPC and endothelial cell attachment with less platelet attachment. These desirable factors make the L-selectin and VE-cadherin coated surfaces perfect candidate endovascular device. Copyright © 2012 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

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.

Targeting Therapy Resistant Tumor Vessels

DTIC Science & Technology

2007-05-01

Porkka K, Laakko- nen P, Ruoslahti E. Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels. J Cell...anti-angiogenic therapy. Markers of such vessels will be useful in developing strategies for complete destruction of breast cancer vasculature, and in...express specific markers , and that these lymphatic markers are tumor type specific and distinct from blood vessel markers in the same tumors. The

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

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

Effect of Leptospira interrogans outer membrane proteins LipL32 on HUVEC.

PubMed

Sun, Zhan; Bao, Lang; Li, DaoKun; Huang, Bi; Wu, Bingting

2010-09-01

Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)-LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx-LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions. Copyright 2010 Elsevier Ltd. All rights reserved.

A senescence secretory switch mediated by PI3K/AKT/mTOR activation controls chemoprotective endothelial secretory responses

PubMed Central

Bent, Eric H.; Gilbert, Luke A.; Hemann, Michael T.

2016-01-01

Cancer therapy targets malignant cells that are surrounded by a diverse complement of nonmalignant stromal cells. Therapy-induced damage of normal cells can alter the tumor microenvironment, causing cellular senescence and activating cancer-promoting inflammation. However, how these damage responses are regulated (both induced and resolved) to preserve tissue homeostasis and prevent chronic inflammation is poorly understood. Here, we detail an acute chemotherapy-induced secretory response that is self-limiting in vitro and in vivo despite the induction of cellular senescence. We used tissue-specific knockout mice to demonstrate that endothelial production of the proinflammatory cytokine IL-6 promotes chemoresistance and show that the chemotherapeutic doxorubicin induces acute IL-6 release through reactive oxygen species-mediated p38 activation in vitro. Doxorubicin causes endothelial senescence but, surprisingly, without a typical senescence secretory response. We found that endothelial cells repress senescence-associated inflammation through the down-regulation of PI3K/AKT/mTOR signaling and that reactivation of this pathway restores senescence-associated inflammation. Thus, we describe a mechanism by which damage-associated paracrine secretory responses are restrained to preserve tissue homeostasis and prevent chronic inflammation. PMID:27566778

VEGF isoforms have differential effects on permeability of human pulmonary microvascular endothelial cells.

PubMed

Ourradi, Khadija; Blythe, Thomas; Jarrett, Caroline; Barratt, Shaney L; Welsh, Gavin I; Millar, Ann B

2017-06-02

Alternative splicing of Vascular endothelial growth factor-A mRNA transcripts (commonly referred as VEGF) leads to the generation of functionally differing isoforms, the relative amounts of which have potentially significant physiological outcomes in conditions such as acute respiratory distress syndrome (ARDS). The effect of such isoforms on pulmonary vascular permeability is unknown. We hypothesised that VEGF 165 a and VEGF 165 b isoforms would have differing effects on pulmonary vascular permeability caused by differential activation of intercellular signal transduction pathways. To test this hypothesis we investigated the physiological effect of VEGF 165 a and VEGF 165 b on Human Pulmonary Microvascular Endothelial Cell (HPMEC) permeability using three different methods: trans-endothelial electrical resistance (TEER), Electric cell-substrate impedance sensing (ECIS) and FITC-BSA passage. In addition, potential downstream signalling pathways of the VEGF isoforms were investigated by Western blotting and the use of specific signalling inhibitors. VEGF 165 a increased HPMEC permeability using all three methods (paracellular and transcellular) and led to associated VE-cadherin and actin stress fibre changes. In contrast, VEGF 165 b decreased paracellular permeability and did not induce changes in VE-cadherin cell distribution. Furthermore, VEGF 165 a and VEGF 165 b had differing effects on both the phosphorylation of VEGF receptors and downstream signalling proteins pMEK, p42/44MAPK, p38 MAPK, pAKT and peNOS. Interestingly specific inhibition of the pMEK, p38 MAPK, PI3 kinase and eNOS pathways blocked the effects of both VEGF 165 a and VEGF 165 b on paracellular permeability and the effect of VEGF 165 a on proliferation/migration, suggesting that this difference in cellular response is mediated by an as yet unidentified signalling pathway(s). This study demonstrates that the novel isoform VEGF 165 a and VEGF 165 b induce differing effects on permeability in pulmonary microvascular endothelial cells.

Intact mitochondrial Ca2+ uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS).

PubMed

Charoensin, Suphachai; Eroglu, Emrah; Opelt, Marissa; Bischof, Helmut; Madreiter-Sokolowski, Corina T; Kirsch, Andrijana; Depaoli, Maria R; Frank, Saša; Schrammel, Astrid; Mayer, Bernd; Waldeck-Weiermair, Markus; Graier, Wolfgang F; Malli, Roland

2017-01-01

Mitochondrial Ca 2+ uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO • ) production. However, it is not entirely clear if the organelles support or counteract NO • biosynthesis by taking up Ca 2+ . The objective of this study was to verify whether or not mitochondrial Ca 2+ uptake influences Ca 2+ -triggered NO • generation by endothelial NO • synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO • probes, the geNOps, and Ca 2+ sensors to monitor single cell NO • and Ca 2+ dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP 3 )-generating agonist. Mitochondrial Ca 2+ uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca 2+ uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca 2+ uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca 2+ -triggered NO • increase independently of global cytosolic Ca 2+ signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca 2+ sequestration and Ca 2+ -induced NO • signals. The positive correlation between mitochondrial Ca 2+ elevation and NO • production was independent of eNOS phosphorylation at serine 1177 . Our findings emphasize that manipulating mitochondrial Ca 2+ uptake may represent a novel strategy to control eNOS-mediated NO • production. Copyright © 2016. Published by Elsevier Inc.

The Phosphatase PTP-PEST/PTPN12 Regulates Endothelial Cell Migration and Adhesion, but Not Permeability, and Controls Vascular Development and Embryonic Viability*

PubMed Central

Souza, Cleiton Martins; Davidson, Dominique; Rhee, Inmoo; Gratton, Jean-Philippe; Davis, Elaine C.; Veillette, André

2012-01-01

Protein-tyrosine phosphatase (PTP)-PEST (PTPN12) is ubiquitously expressed. It is essential for normal embryonic development and embryonic viability in mice. Herein we addressed the involvement of PTP-PEST in endothelial cell functions using a combination of genetic and biochemical approaches. By generating primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is not needed for endothelial cell differentiation and proliferation or for the control of endothelial cell permeability. Nevertheless, it is required for integrin-mediated adhesion and migration of endothelial cells. PTP-PEST-deficient endothelial cells displayed increased tyrosine phosphorylation of Cas, paxillin, and Pyk2, which were previously also implicated in integrin functions. By eliminating PTP-PEST in endothelial cells in vivo, we obtained evidence that expression of PTP-PEST in endothelial cells is required for normal vascular development and embryonic viability. Therefore, PTP-PEST is a key regulator of integrin-mediated functions in endothelial cells seemingly through its capacity to control Cas, paxillin, and Pyk2. This function explains at least in part the essential role of PTP-PEST in embryonic development and viability. PMID:23105101

A small molecule targeting ALK1 prevents Notch cooperativity and inhibits functional angiogenesis.

PubMed

Kerr, Georgina; Sheldon, Helen; Chaikuad, Apirat; Alfano, Ivan; von Delft, Frank; Bullock, Alex N; Harris, Adrian L

2015-04-01

Activin receptor-like kinase 1 (ALK1, encoded by the gene ACVRL1) is a type I BMP/TGF-β receptor that mediates signalling in endothelial cells via phosphorylation of SMAD1/5/8. During angiogenesis, sprouting endothelial cells specialise into tip cells and stalk cells. ALK1 synergises with Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2 and thereby represses tip cell formation and angiogenic sprouting. The ALK1-Fc soluble protein fusion has entered clinic trials as a therapeutic strategy to sequester the high-affinity extracellular ligand BMP9. Here, we determined the crystal structure of the ALK1 intracellular kinase domain and explored the effects of a small molecule kinase inhibitor K02288 on angiogenesis. K02288 inhibited BMP9-induced phosphorylation of SMAD1/5/8 in human umbilical vein endothelial cells to reduce both the SMAD and the Notch-dependent transcriptional responses. In endothelial sprouting assays, K02288 treatment induced a hypersprouting phenotype reminiscent of Notch inhibition. Furthermore, K02288 caused dysfunctional vessel formation in a chick chorioallantoic membrane assay of angiogenesis. Such activity may be advantageous for small molecule inhibitors currently in preclinical development for specific BMP gain of function conditions, including diffuse intrinsic pontine glioma and fibrodysplasia ossificans progressiva, as well as more generally for other applications in tumour biology.

Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue

PubMed Central

Rouaud, Florian; Romero-Perez, Miguel; Wang, Huan; Lobysheva, Irina; Ramassamy, Booma; Henry, Etienne; Tauc, Patrick; Giacchero, Damien; Boucher, Jean-Luc; Deprez, Eric; Rocchi, Stéphane; Slama-Schwok, Anny

2014-01-01

Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 first induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX4 and ROS levels that could lead to a specific proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX4 –associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis. PMID:25296975

Radiation therapy affects the mechanical behavior of human umbilical vein endothelial cells.

PubMed

Mohammadkarim, Alireza; Tabatabaei, Mohammad; Parandakh, Azim; Mokhtari-Dizaji, Manijhe; Tafazzoli-Shadpour, Mohammad; Khani, Mohammad-Mehdi

2018-06-06

Radiation therapy has been widely utilized as an effective method to eliminate malignant tumors and cancerous cells. However, subjection of healthy tissues and the related networks of blood vessels adjacent to the tumor area to irradiation is inevitable. The aim of this study was to investigate the consequent effects of fractionation radiotherapy on the mechanical characteristics of human umbilical vein endothelial cells (HUVECs) through alterations in cytoskeleton organization and cell and nucleus morphology. In order to simulate the clinical condition of radiotherapy, the HUVECs were exposed to the specific dose of 2 Gy for 1-4 times among four groups with incremental total dose from 2 Gy up to 8 Gy. Fluorescence staining was performed to label F-actin filaments and nuclei. Micropipette aspiration and standard linear solid model were employed to evaluate the elastic and viscoelastic characteristics of the HUVECs. Radiotherapy significantly increased cell elastic moduli. Due to irradiation, instantaneous and equilibrium Young's modulus were also increased. Radiotherapy diminished HUVECs viscoelastic behavior and shifted their creep compliance curves downward. Furthermore, gamma irradiation elevated the nuclei sizes and to a lesser extent the cells sizes resulting in the accumulation of F-actin filaments within the rest of cell body. Endothelial stiffening correlates with endothelial dysfunction, hence the results may be helpful when the consequent effects of radiotherapy are the focus of concern. Copyright © 2018. Published by Elsevier Ltd.

Association of Abacavir and Impaired Endothelial Function in Treated and Suppressed HIV-Infected Patients

PubMed Central

Hsue, Priscilla Y.; Hunt, Peter W.; Wu, Yuaner; Schnell, Amanda; Ho, Jennifer E.; Hatano, Hiroyu; Xie, Yu; Martin, Jeffrey N.; Ganz, Peter; Deeks, Steven G.

2009-01-01

Background HIV-infected patients have accelerated atherosclerosis. Abacavir has been associated with increased risk of cardiovascular events, for reasons that remain to be elucidated. As endothelial dysfunction is central to the pathogenesis of atherosclerosis, we tested the hypothesis that current treatment with abacavir is associated with impaired endothelial function. Methods We studied a cohort of 61 antiretroviral-treated patients who had undetectable plasma HIV RNA levels. Endothelial function was assessed by measuring flow-mediated vasodilation (FMD) of the brachial artery. We compared FMD in patients treated with or without abacavir, while adjusting for traditional risk factors and HIV-specific characteristics. Results The median age was 50 years (IQR 45–57). The median duration of HIV infection was 18 years, and the median CD4 cell count was 369 cells/mm3. Thirty subjects (49%) were receiving abacavir. Overall, the median FMD in the HIV-infected patients was low (3.5%; IQR 2.3–5.6%). The FMD was lower in the abacavir-treated patients than those not on abacavir (2.8% vs. 4.9%, p=0.01). After adjustment for traditional risk factors, HIV specific factors, and baseline brachial artery diameter, current abacavir use was independently associated with lower FMD (p=0.017). Duration of therapy and CD4 count were not associated with reduced FMD. Conclusions Endothelial function, a central mechanism in atherosclerosis and a marker of cardiovascular risk, is impaired among antiretroviral-treated patients with undetectable viral loads. Current use of abacavir was independently associated with impaired endothelial function. This finding suggests that abnormal endothelial function may underlie the clinically observed increased risk in myocardial infarction among abacavir-treated patients. PMID:19542863

MicroRNA-101 mediates the suppressive effect of laminar shear stress on mTOR expression in vascular endothelial cells

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

Chen, Kui; Fan, Wendong; Wang, Xing

Highlights: Black-Right-Pointing-Pointer Laminar shear stress upregulates miR-101 expression in vascular endothelial cells. Black-Right-Pointing-Pointer miR-101 represses mTOR expression through a specific 3 Prime UTR binding site. Black-Right-Pointing-Pointer Overexpression of miR-101 inhibits G1/S transition and endothelial cell proliferation. Black-Right-Pointing-Pointer Blockade of miR-101 attenuates the suppressive effect of laminar flow on mTOR expression. -- Abstract: Shear stress associated with blood flow plays an important role in regulating gene expression and cell function in endothelial cells (ECs). MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that negatively regulate the expression of target genes by binding to the mRNA 3 Prime -untranslated region (3 Primemore » UTR) at the posttranscriptional level involved in diverse cellular processes. This study demonstrates that microRNA-101 in response to laminar shear stress (LSS) is involved in the flow regulation of gene expression in ECs. qRT-PCR analysis showed that miR-101 expression was significantly upregulated in human umbilical vein endothelial cells (HUVECs) exposed to 12 dyn/cm{sup 2} laminar shear stress for 12 h. We found that transfection of miR-101 significantly decreased the luciferase activity of plasmid reporter containing the 3 Prime UTR of mammalian target of rapamycin (mTOR) gene. Western analysis revealed that the protein level of mTOR was significantly reduced in ECs transfected with miR-101. Furthermore, miR-101 overexpression induced cell cycle arrest at the G1/S transition and suppressed endothelial cell proliferation. Finally, transfection of miR-101 inhibitors attenuated the suppressive effects of LSS on mTOR expression, which identified the efficacy of loss-of-function of miR-101 in laminar flow-treated ECs. In conclusion, we have demonstrated that upregulation of miR-101 in response to LSS contributes to the suppressive effects of LSS on mTOR expression and EC proliferation. These studies advance our understanding of the posttranscriptional mechanisms by which shear stress modulates endothelial homeostasis.« less

Nitric oxide-induced changes in endothelial expression of phosphodiesterases 2, 3, and 5.

PubMed

Schankin, Christoph J; Kruse, Lars S; Reinisch, Veronika M; Jungmann, Steffen; Kristensen, Julie C; Grau, Stefan; Ferrari, Uta; Sinicina, Inga; Goldbrunner, Roland; Straube, Andreas; Kruuse, Christina

2010-03-01

To investigate nitric oxide (NO)-mediated changes in expression of cyclic nucleotide degrading phosphodiesterases 2A (PDE2A), PDE3B, and PDE5A in human endothelial cells. Nitric oxide induces production of cyclic guanosine monophosphate (cGMP), which along with cyclic adenosine monophosphate (cAMP) is degraded by PDEs. NO donors and selective inhibitors of PDE3 and PDE5 induce migraine-like headache and play a role in endothelial dysfunction during stroke. The current study investigates possible NO modulation of cGMP-related PDEs relevant to headache induction in a cell line containing such PDEs. Real time polymerase chain reaction and Western blots were used to show expression of PDE2A, PDE3B, and PDE5A in a stable cell line of human brain microvascular endothelial cells. Effects of NO on PDE expression were analyzed at specific time intervals after continued DETA NONOate administration. This study shows the expression of PDE2A, PDE3B, and PDE5A mRNA and PDE3B and PDE5A protein in human cerebral endothelial cells. Long-term DETA NONOate administration induced an immediate mRNA up-regulation of PDE5A (1.9-fold, 0.5 hour), an early peak of PDE2A (1.4-fold, 1 and 2 hours) and later up-regulation of both PDE3B (1.6-fold, 4 hours) and PDE2A (1.7-fold, 8 hours and 1.2-fold after 24 hours). Such changes were, however, not translated into significant changes in protein expression indicating few, if any, functional effects. Long-term NO stimulation modulated PDE3 and PDE5 mRNA expression in endothelial cells. However, PDE3 and PDE5 protein levels were unaffected by NO. The presence of PDE3 or PDE5 in endothelial cells indicates that selective inhibitors may have functional effects in such cells. A complex interaction of cGMP and cAMP in response to NO administration may take place if the mRNA translates into active protein. Whether or not this plays a role in the headache mechanisms remains to be investigated.

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.

circHECTD1 promotes the silica-induced pulmonary endothelial-mesenchymal transition via HECTD1.

PubMed

Fang, Shencun; Guo, Huifang; Cheng, Yusi; Zhou, Zewei; Zhang, Wei; Han, Bing; Luo, Wei; Wang, Jing; Xie, Weiping; Chao, Jie

2018-03-14

Excessive proliferation and migration of fibroblasts contribute to pulmonary fibrosis in silicosis, and both epithelial cells and endothelial cells participate in the accumulation of fibroblasts via the epithelial-mesenchymal transition (EMT) and the endothelial-mesenchymal transition (EndMT), respectively. A mouse endothelial cell line (MML1) was exposed to silicon dioxide (SiO 2 , 50 μg/cm 2 ), and immunofluorescence and western blot analyses were performed to evaluate levels of specific endothelial and mesenchymal markers and to elucidate the mechanisms by which SiO 2 induces the EndMT. Functional changes were evaluated by analyzing cell migration and proliferation. The mRNA and circular RNA (circRNA) levels were measured using qPCR and fluorescent in situ hybridization (FISH). Lung tissue samples from both Tie2-GFP mice exposed to SiO 2 and silicosis patients were applied to confirm the observations from in vitro experiments. Based on the results from the current study, SiO 2 increased the expression of mesenchymal markers (type I collagen (COL1A1), type III collagen (COL3A1) and alpha smooth muscle actin (α-SMA/Acta2)) and decreased the expression of endothelial markers (vascular endothelial cadherin (VE-Cad/Cdh 5) and platelet endothelial cell adhesion molecule-1 (PECAM1)), indicating the occurrence of the EndMT in response to SiO 2 exposure both in vivo and in vitro. SiO 2 concomitantly increased circHECTD1 expression, which, in turn, inhibited HECTD1 protein expression. SiO 2 -induced increases in cell proliferation, migration, and changes in marker levels were restored by either a small interfering RNA (siRNA) targeting circHECTD1 or overexpression of HECTD1 via the CRISPR/Cas9 system, confirming the involvement of the circHECTD1/HECTD1 pathway in the EndMT. Moreover, tissue samples from SiO 2 -exposed mice and silicosis patients confirmed the EndMT and change in HECTD1 expression. Our findings reveal a potentially new function for the circHECTD1/HECTD1 pathway and suggest a possible mechanism of fibrosis in patients with pulmonary silicosis.

Strength of Neisseria meningitidis binding to endothelial cells requires highly-ordered CD147/β2-adrenoceptor clusters assembled by alpha-actinin-4

PubMed Central

Maïssa, Nawal; Covarelli, Valentina; Janel, Sébastien; Durel, Beatrice; Simpson, Nandi; Bernard, Sandra C.; Pardo-Lopez, Liliana; Bouzinba-Ségard, Haniaa; Faure, Camille; Scott, Mark G.H.; Coureuil, Mathieu; Morand, Philippe C.; Lafont, Frank; Nassif, Xavier; Marullo, Stefano; Bourdoulous, Sandrine

2017-01-01

Neisseria meningitidis (meningococcus) is an invasive bacterial pathogen that colonizes human vessels, causing thrombotic lesions and meningitis. Establishment of tight interactions with endothelial cells is crucial for meningococci to resist haemodynamic forces. Two endothelial receptors, CD147 and the β2-adrenergic receptor (β2AR), are sequentially engaged by meningococci to adhere and promote signalling events leading to vascular colonization, but their spatiotemporal coordination is unknown. Here we report that CD147 and β2AR form constitutive hetero-oligomeric complexes. The scaffolding protein α-actinin-4 directly binds to the cytosolic tail of CD147 and governs the assembly of CD147–β2AR complexes in highly ordered clusters at bacterial adhesion sites. This multimolecular assembly process increases the binding strength of meningococci to endothelial cells under shear stress, and creates molecular platforms for the elongation of membrane protrusions surrounding adherent bacteria. Thus, the specific organization of cellular receptors has major impacts on host–pathogen interaction. PMID:28569760

The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor β-Stimulated Aortic Endothelial Cells ▿

PubMed Central

Daubon, Thomas; Buccione, Roberto; Génot, Elisabeth

2011-01-01

Podosomes are dynamic actin-rich adhesion plasma membrane microdomains endowed with extracellular matrix-degrading activities. In aortic endothelial cells, podosomes are induced by transforming growth factor β (TGF-β), but how this occurs is largely unknown. It is thought that, in endothelial cells, podosomes play a role in vessel remodeling and/or in breaching anatomical barriers. We demonstrate here that, in bovine aortic endothelial cells, that the Cdc42-specific guanine exchange factor (GEF) Fgd1 is expressed and regulated by TGF-β to induce Cdc42-dependent podosome assembly. Within 15 min of TGF-β stimulation, Fgd1, but none of the other tested Cdc42 GEFs, undergoes tyrosine phosphorylation, associates with Cdc42, and translocates to the subcortical cytoskeleton via a cortactin-dependent mechanism. Small interfering RNA-mediated Fgd1 knockdown inhibits TGF-β-induced Cdc42 activation. Fgd1 depletion also reduces podosome formation and associated matrix degradation and these defects are rescued by reexpression of Fgd1. Although overexpression of Fgd1 does not promote podosome formation per se, it enhances TGF-β-induced matrix degradation. Our results identify Fgd1 as a TGF-β-regulated GEF and, as such, the first GEF to be involved in the process of cytokine-induced podosome formation. Our findings reveal the involvement of Fgd1 in endothelial cell biology and open up new avenues to study its role in vascular pathophysiology. PMID:21911474

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

Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

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

Chen Haibing; Department of Ophthalmology, Anhui Provincial Hospital, Hefei; Jia Weiping

2008-05-02

Poly(ADP-ribose)polymerase (PARP) inhibitors decrease angiogenesis through reducing vascular endothelium growth factor (VEGF) induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). In contrast to VEGF, pigment epithelium-derived factor (PEDF) has been demonstrated to act as a strong endogenous inhibitor of angiogenesis. Here, we show that PARP inhibition with a specific inhibitor PJ-34 or specific PARP antisense oligonucleotide upregulates hyperglycemia-induced PEDF expression in HUVECs in a dose-dependent manner. This results in the retard of activation of p38 MAP kinase and the concomitant decrease in cell apoptosis. These results give the first direct demonstration that PEDF might representmore » a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent.« less

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.

High Mobility Group Box 1 Promotes Angiogenesis from Bone Marrow-derived Endothelial Progenitor Cells after Myocardial Infarction.

PubMed

Nakamura, Yuichi; Suzuki, Satoshi; Shimizu, Takeshi; Miyata, Makiko; Shishido, Tetsuro; Ikeda, Kazuhiko; Saitoh, Shu-Ichi; Kubota, Isao; Takeishi, Yasuchika

2015-01-01

High mobility group box 1 (HMGB1) is a DNA-binding protein secreted into the extracellular space from necrotic cells that acts as a cytokine. We examined the role of HMGB1 in angiogenesis from bone marrow-derived cells in the heart using transgenic mice exhibiting the cardiac-specific overexpression of HMGB1 (HMGB1-TG). HMGB1-TG mice and wild-type littermate (WT) mice were lethally irradiated and injected with bone marrow cells from green fluorescent protein mice through the tail vein. After bone marrow transplantation, the left anterior descending artery was ligated to induce myocardial infarction (MI). Flow cytometry revealed that the levels of circulating endothelial progenitor cells (EPCs) mobilized from the bone marrow increased after MI in the HMGB-TG mice versus the WT mice. In addition, the size of MI was smaller in the HMGB1-TG mice than in the WT mice, and immunofluorescence staining demonstrated that the number of engrafted vascular endothelial cells derived from bone marrow in the border zones of the MI areas was increased in the HMGB1-TG mice compared to that observed in the WT mice. Moreover, the levels of cardiac vascular endothelial growth factor after MI were higher in the HMGB1-TG mice than in the WT mice. The present study demonstrated that HMGB1 promotes angiogenesis and reduces the MI size by enhancing the mobilization and differentiation of bone marrow cells to EPCs as well as their migration to the border zones of the MI areas and engraftment as vascular endothelial cells in new capillaries or arterioles in the infarcted heart.

BIOLOGICAL AND BIOPHYSICAL PROPERTIES OF VASCULAR CONNEXIN CHANNELS

PubMed Central

Johnstone, Scott; Isakson, Brant; Locke, Darren

2010-01-01

Intercellular channels formed by connexin proteins play a pivotal role in the direct movement of ions and larger cytoplasmic solutes between vascular endothelial cells, between vascular smooth muscle cells, and between endothelial and smooth muscle cells. Multiple genetic and epigenetic factors modulate connexin expression levels and/or channel function, including cell type-independent and cell type-specific transcription factors, posttranslational modification and localized membrane targeting. Additionally, differences in protein-protein interactions, including those between connexins, significantly contribute to both vascular homeostasis and disease progression. The biophysical properties of the connexin channels identified in the vasculature, those formed by Cx37, Cx40, Cx43 and/or Cx45 proteins, are discussed in this review in the physiological and pathophysiological context of vessel function. PMID:19815177

Peripheral blood CD34+KDR+ endothelial progenitor cells are determinants of subclinical atherosclerosis in a middle-aged general population.

PubMed

Fadini, Gian Paolo; Coracina, Anna; Baesso, Ilenia; Agostini, Carlo; Tiengo, Antonio; Avogaro, Angelo; de Kreutzenberg, Saula Vigili

2006-09-01

Disruption of the endothelial layer is the first step in the atherogenic process. Experimental studies have shown that endothelial progenitor cells (EPCs) are involved in endothelial homeostasis and repair. Conversely, EPC depletion has been demonstrated in the setting of established atherosclerotic diseases. With this background, we evaluated whether variations in the number of EPCs are associated with subclinical atherosclerosis in healthy subjects. Carotid intima-media thickness (IMT), high-sensitive C-reactive protein (hsCRP), levels of circulating EPCs, and cardiovascular risk were compared in 137 healthy subjects. Six subpopulations of progenitor cells were determined by flow cytometry on the basis of the surface expression of CD34, CD133, and KDR antigens: CD34(+), CD133(+), CD34(+)CD133(+), CD34(+)KDR(+), CD133(+)KDR(+), and CD34(+)CD133(+)KDR(+). Among different antigenic profiles of EPCs, only CD34(+)KDR(+) cells were significantly reduced in subjects with increased IMT. Specifically, CD34(+)KDR(+) cells were inversely correlated with IMT, even after adjustment for hsCRP and 10-year Framingham risk and independently of other cardiovascular parameters. Depletion of CD34(+)KDR(+) EPCs is an independent predictor of early subclinical atherosclerosis in healthy subjects and may provide additional information beyond classic risk factors and inflammatory markers.

Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices

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

Lee, P.-F.; Yeh, Alvin T.; Bayless, Kayla J.

The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observedmore » increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress.« less

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

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

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells.

PubMed

Leckel, K; Beecken, W-D; Jonas, D; Oppermann, E; Coman, M C; Beck, K-F; Cinatl, J; Hailer, N P; Auth, M K H; Bechstein, W O; Shipkova, M; Blaheta, R A

2003-11-01

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell-endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 microm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 microm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy.

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.

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 Cell Tetrahydrobiopterin Modulates Sensitivity to Ang (Angiotensin) II-Induced Vascular Remodeling, Blood Pressure, and Abdominal Aortic Aneurysm.

PubMed

Chuaiphichai, Surawee; Rashbrook, Victoria S; Hale, Ashley B; Trelfa, Lucy; Patel, Jyoti; McNeill, Eileen; Lygate, Craig A; Channon, Keith M; Douglas, Gillian

2018-07-01

GTPCH (GTP cyclohydrolase 1, encoded by Gch1 ) is required for the synthesis of tetrahydrobiopterin; a critical regulator of endothelial NO synthase function. We have previously shown that mice with selective loss of Gch1 in endothelial cells have mild vascular dysfunction, but the consequences of endothelial cell tetrahydrobiopterin deficiency in vascular disease pathogenesis are unknown. We investigated the pathological consequence of Ang (angiotensin) II infusion in endothelial cell Gch1 deficient ( Gch1 fl/fl Tie2cre) mice. Ang II (0.4 mg/kg per day, delivered by osmotic minipump) caused a significant decrease in circulating tetrahydrobiopterin levels in Gch1 fl/fl Tie2cre mice and a significant increase in the Nω-nitro-L-arginine methyl ester inhabitable production of H 2 O 2 in the aorta. Chronic treatment with this subpressor dose of Ang II resulted in a significant increase in blood pressure only in Gch1 fl/fl Tie2cre mice. This finding was mirrored with acute administration of Ang II, where increased sensitivity to Ang II was observed at both pressor and subpressor doses. Chronic Ang II infusion in Gch1 fl/fl Tie2ce mice resulted in vascular dysfunction in resistance mesenteric arteries with an enhanced constrictor and decreased dilator response and medial hypertrophy. Altered vascular remodeling was also observed in the aorta with an increase in the incidence of abdominal aortic aneurysm formation in Gch1 fl/fl Tie2ce mice. These findings indicate a specific requirement for endothelial cell tetrahydrobiopterin in modulating the hemodynamic and structural changes induced by Ang II, through modulation of blood pressure, structural changes in resistance vessels, and aneurysm formation in the aorta. © 2018 The Authors.

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.

Chronic administration of the probiotic kefir improves the endothelial function in spontaneously hypertensive rats.

PubMed

Friques, Andreia G F; Arpini, Clarisse M; Kalil, Ieda C; Gava, Agata L; Leal, Marcos A; Porto, Marcella L; Nogueira, Breno V; Dias, Ananda T; Andrade, Tadeu U; Pereira, Thiago Melo C; Meyrelles, Silvana S; Campagnaro, Bianca P; Vasquez, Elisardo C

2015-12-30

The beverage obtained by fermentation of milk with kefir grains, a complex matrix containing acid bacteria and yeasts, has been shown to have beneficial effects in various diseases. However, its effects on hypertension and endothelial dysfunction are not yet clear. In this study, we evaluated the effects of kefir on endothelial cells and vascular responsiveness in spontaneously hypertensive rats (SHR). SHR were treated with kefir (0.3 mL/100 g body weight) for 7, 15, 30 and 60 days and compared with non-treated SHR and with normotensive Wistar-Kyoto rats. Vascular endothelial function was evaluated in aortic rings through the relaxation response to acetylcholine (ACh). The balance between reactive oxygen species (ROS) and nitric oxide (NO) synthase was evaluated through specific blockers in the ACh-induced responses and through flow cytometry in vascular tissue. Significant effects of kefir were observed only after treatment for 60 days. The high blood pressure and tachycardia exhibited by the SHR were attenuated by approximately 15 % in the SHR-kefir group. The impaired ACh-induced relaxation of the aortic rings observed in the SHR (37 ± 4 %, compared to the Wistar rats: 74 ± 5 %), was significantly attenuated in the SHR group chronically treated with kefir (52 ± 4 %). The difference in the area under the curve between before and after the NADPH oxidase blockade or NO synthase blockade of aortic rings from SHR were of approximately +90 and -60 %, respectively, when compared with Wistar rats. In the aortic rings from the SHR-kefir group, these values were reduced to +50 and -40 %, respectively. Flow cytometric analysis of aortic endothelial cells revealed increased ROS production and decreased NO bioavailability in the SHR, which were significantly attenuated by the treatment with kefir. Scanning electronic microscopy showed vascular endothelial surface injury in SHR, which was partially protected following administration of kefir for 60 days. In addition, the recruitment of endothelial progenitor cells was decreased in the non-treated SHR and partially restored by kefir treatment. Kefir treatment for 60 days was able to improve the endothelial function in SHR by partially restoring the ROS/NO imbalance and the endothelial architecture due to endothelial progenitor cells recruitment.

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.

Functional consequences of prolactin signalling in endothelial cells: a potential link with angiogenesis in pathophysiology?

PubMed Central

Reuwer, Anne Q; Nowak-Sliwinska, Patrycja; Mans, Laurie A; van der Loos, Chris M; von der Thüsen, Jan H; Twickler, Marcel Th B; Spek, C Arnold; Goffin, Vincent; Griffioen, Arjan W; Borensztajn, Keren S

2012-01-01

Prolactin is best known as the polypeptide anterior pituitary hormone, which regulates the development of the mammary gland. However, it became clear over the last decade that prolactin contributes to a broad range of pathologies, including breast cancer. Prolactin is also involved in angiogenesis via the release of pro-angiogenic factors by leukocytes and epithelial cells. However, whether prolactin also influences endothelial cells, and whether there are functional consequences of prolactin-induced signalling in the perspective of angiogenesis, remains so far elusive. In the present study, we show that prolactin induces phosphorylation of ERK1/2 and STAT5 and induces tube formation of endothelial cells on Matrigel. These effects are blocked by a specific prolactin receptor antagonist, del1-9-G129R-hPRL. Moreover, in an in vivo model of the chorioallantoic membrane of the chicken embryo, prolactin enhances vessel density and the tortuosity of the vasculature and pillar formation, which are hallmarks of intussusceptive angiogenesis. Interestingly, while prolactin has only little effect on endothelial cell proliferation, it markedly stimulates endothelial cell migration. Again, migration was reverted by del1-9-G129R-hPRL, indicating a direct effect of prolactin on its receptor. Immunohistochemistry and spectral imaging revealed that the prolactin receptor is present in the microvasculature of human breast carcinoma tissue. Altogether, these results suggest that prolactin may directly stimulate angiogenesis, which could be one of the mechanisms by which prolactin contributes to breast cancer progression, thereby providing a potential tool for intervention. PMID:22128761

Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation

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

Pan, Christopher C.; Bloodworth, Jeffrey C.; Mythreye, Karthikeyan

2012-08-03

Highlights: Black-Right-Pointing-Pointer Endoglin inhibits ERK activation in endothelial cells. Black-Right-Pointing-Pointer Endoglin is a regulator of c-Myc and cyclin D1 expression. Black-Right-Pointing-Pointer {beta}-arrestin2 interaction with endoglin is required for ERK/c-Myc repression. Black-Right-Pointing-Pointer Endoglin impedes cellular proliferation by targeting ERK-induced mitogenic signaling. -- Abstract: Endoglin is an endothelial-specific transforming growth factor beta (TGF-{beta}) co-receptor essential for angiogenesis and vascular remodeling. Endoglin regulates a wide range of cellular processes, including cell adhesion, migration, and proliferation, through TGF-{beta} signaling to canonical Smad and Smad-independent pathways. Despite its overall pro-angiogenic role in the vasculature, the underlying mechanism of endoglin action is poorly characterized. We previouslymore » identified {beta}-arrestin2 as a binding partner that causes endoglin internalization from the plasma membrane and inhibits ERK signaling towards endothelial migration. In the present study, we examined the mechanistic role of endoglin and {beta}-arrestin2 in endothelial cell proliferation. We show that endoglin impedes cell growth through sustained inhibition of ERK-induced c-Myc and cyclin D1 expression in a TGF-{beta}-independent manner. The down-regulation of c-Myc and cyclin D1, along with growth-inhibition, are reversed when the endoglin/{beta}-arrestin2 interaction is disrupted. Given that TGF-{beta}-induced Smad signaling potently represses c-Myc in most cell types, our findings here show a novel mechanism by which endoglin augments growth-inhibition by targeting ERK and key downstream mitogenic substrates.« less

O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals.

PubMed

Sawaguchi, Shogo; Varshney, Shweta; Ogawa, Mitsutaka; Sakaidani, Yuta; Yagi, Hirokazu; Takeshita, Kyosuke; Murohara, Toyoaki; Kato, Koichi; Sundaram, Subha; Stanley, Pamela; Okajima, Tetsuya

2017-04-11

The glycosyltransferase EOGT transfers O-GlcNAc to a consensus site in epidermal growth factor-like (EGF) repeats of a limited number of secreted and membrane proteins, including Notch receptors. In EOGT-deficient cells, the binding of DLL1 and DLL4, but not JAG1, canonical Notch ligands was reduced, and ligand-induced Notch signaling was impaired. Mutagenesis of O-GlcNAc sites on NOTCH1 also resulted in decreased binding of DLL4. EOGT functions were investigated in retinal angiogenesis that depends on Notch signaling. Global or endothelial cell-specific deletion of Eogt resulted in defective retinal angiogenesis, with a mild phenotype similar to that caused by reduced Notch signaling in retina. Combined deficiency of different Notch1 mutant alleles exacerbated the abnormalities in Eogt -/- retina, and Notch target gene expression was decreased in Eogt -/- endothelial cells. Thus, O-GlcNAc on EGF repeats of Notch receptors mediates ligand-induced Notch signaling required in endothelial cells for optimal vascular development.

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

PubMed Central

LECKEL, K; BEECKEN, W-D; JONAS, D; OPPERMANN, E; COMAN, M C; BECK, K-F; CINATL, J; HAILER, N P; AUTH, M K H; BECHSTEIN, W O; SHIPKOVA, M; BLAHETA, R A

2003-01-01

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell–endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 µm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 µm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy. PMID:14616783

Regulation of store-operated Ca{sup 2+} entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells

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

Kito, Hiroaki; Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto; Yamamura, Hisao

2015-04-10

Store-operated Ca{sup 2+} entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cyclemore » progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca{sup 2+} influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. - Highlights: • Orai1 is essential for SOCE activity in brain capillary endothelial cells (BCECs). • Cell cycle independent expression of Orai1 regulated SOCE and cell proliferation. • Orai2 was up-regulated only at G2/M phase and this consequently reduced SOCE. • Orai2 as well as Orai1 is a key player controlling SOCE and proliferation in BCECs.« less

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

[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.

New insights into circulating FABP4: Interaction with cytokeratin 1 on endothelial cell membranes.

PubMed

Saavedra, Paula; Girona, Josefa; Bosquet, Alba; Guaita, Sandra; Canela, Núria; Aragonès, Gemma; Heras, Mercedes; Masana, Lluís

2015-11-01

Fatty acid-binding protein 4 (FABP4) is an adipose tissue-secreted adipokine that is involved in the regulation of energetic metabolism and inflammation. Increased levels of circulating FABP4 have been detected in individuals with cardiovascular risk factors. Recent studies have demonstrated that FABP4 has a direct effect on peripheral tissues, specifically promoting vascular dysfunction; however, its mechanism of action is unknown. The objective of this work was to assess the specific interactions between exogenous FABP4 and the plasma membranes of endothelial cells. Immunofluorescence assays showed that exogenous FABP4 localized along the plasma membranes of human umbilical vein endothelial cells (HUVECs), interacting specifically with plasma membrane proteins. Anti-FABP4 immunoblotting revealed two covalent protein complexes containing FABP4 and its putative receptor; these complexes were approximately 108 kDa and 77 kDa in size. Proteomics and mass spectrometry experiments revealed that cytokeratin 1 (CK1) was the FABP4-binding protein. An anti-CK1 immunoblot confirmed the presence of CK1. FABP4-CK1 complexes were also detected in HAECs, HCASMCs, HepG2 cells and THP-1 cells. Pharmacological FABP4 inhibition by BMS309403 results in a slight decrease in the formation of these complexes, indicating that fatty acids may play a role in FABP4 functionality. In addition, we demonstrated that exogenous FABP4 crosses the plasma membrane to enter the cytoplasm and nucleus in HUVECs. These findings indicate that exogenous FABP4 interacts with plasma membrane proteins, specifically CK1. These data contribute to our current knowledge regarding the mechanism of action of circulating FABP4.

Impaired Expression of Uncoupling Protein 2 (UCP2) Causes Defective Post-ischemic Angiogenesis in Mice Deficient in AMP-activated Protein Kinase α Subunits

PubMed Central

Xu, Ming-Jiang; Song, Ping; Shirwany, Najeeb; Liang, Bin; Xing, Junjie; Viollet, Benoit; Wang, Xian; Zhu, Yi; Zou, Ming-Hui

2011-01-01

Objective The aim of the present study was to determine whether mitochondrial uncoupling protein (UCP)-2 is required for AMPK-dependent angiogenesis in ischemia in vivo. Methods and Results Angiogenesis was assayed by monitoring endothelial tube formation (a surrogate for angiogenesis) in human umbilical vein endothelial cells (HUVECs), isolated mouse aortic endothelial cells (MAECs), and pulmomary microvascular endothelial cells (PMECs), or in ischemic thigh adductor muscles from wild-type (WT) mice or mice deficient in either AMPKα1 or AMPKα2. AMPK inhibition with pharmacological inhibitor (compound C) or genetic means (transfection of AMPKα-specific siRNA) significantly lowered the tube formation in HUVECs. Consistently, compared with WT mice, tube formation in MAECs isolated from either AMPKα1−/− or AMPKα2−/− mice, which exhibited oxidative stress and reduced expression of UCP2, were significantly impaired. In addition, adenoviral overexpression of UCP2, but not adenoviruses encoding green florescence protein (GFP), normalized tube formation in MAECs from either AMPKα1−/− or AMPKα2−/− mice. Similarly, supplementation with sodium nitroprusside (SNP), a nitric oxide (NO) donor, restored tube formation. Furthermore, ischemia significantly increased angiogenesis, serine 1177 phosphorylation of endothelial NO synthase (eNOS), and UCP2 in ischemic thigh adductor muscles from WT mice, but not from either AMPKα1−/− or AMPKα2−/− mice. Conclusion We conclude that AMPK-dependent UCP2 expression in endothelial cells promotes angiogenesis in vivo. PMID:21597006

Dimethyl sulfoxide attenuates nitric oxide generation via modulation of cationic amino acid transporter-1 in human umbilical vein endothelial cells.

PubMed

Bentur, Ohad S; Chernichovski, Tamara; Ingbir, Merav; Weinstein, Talia; Schwartz, Idit F

2016-10-01

Dimethyl sulfoxide (DMSO) is a solvent that is commonly used in medicine. Conflicting data exist as to its effects on endothelial function. Endothelial cell dysfunction (ECD) is characterized by decreased endothelial nitric oxide synthase (eNOS) activity. Cationic amino acid transporter-1 (CAT-1), the specific arginine transporter for eNOS, has been shown to modulate eNOS activity. We hypothesize that DMSO inhibits eNOS activity through modulation of its selective arginine supplier CAT-1. We studied the effect of DMSO on arginine transport, NO2/NO3 generation as an index of NO production, as well as CAT-1 and Protein Kinase C alpha (PKC-α) (CAT-1 inhibitor) protein expression in human umbilical vein endothelial cell cultures (HUVECs). DMSO 2.5% and 3.5% (v/v) significantly attenuated arginine transport, a phenomenon which was prevented by co-incubation with l-arginine (1 mM). The aforementioned findings were accompanied by a decrease in NO2/NO3 generation. DMSO significantly increased the abundance of phosphorylated CAT-1 (the inactive form) and phosphorylated PKC-α protein, an effect that was attenuated by l-arginine. GO 6976 (PKC-α antagonist) prevented the decrease in arginine transport caused by DMSO. DMSO also induced profound transient morphological changes in HUVECs' structure but these were not related to its effect on arginine transport. In conclusion, DMSO inhibits NO generation by endothelial cells through modulation of CAT-1 activity. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed

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

2016-10-01

Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. Cardiolipin is a mitochondrial phospholipid required for function of the electron transport chain and ATP generation. We examined the role of cardiolipin in maintaining mitochondrial function necessary to support barrier properties of brain microvessel endothelial cells. Knockdown of the terminal enzyme of cardiolipin synthesis, cardiolipin synthase, in hCMEC/D3 cells resulted in decreased cellular cardiolipin levels compared to controls. The reduction in cardiolipin resulted in decreased mitochondrial spare respiratory capacity, increased pyruvate kinase activity, and increased 2-deoxy-[(3) H]glucose uptake and glucose transporter-1 expression and localization to membranes in hCMEC/D3 cells compared to controls. The mechanism for the increase in glucose uptake was an increase in adenosine-5'-monophosphate kinase and protein kinase B activity and decreased glycogen synthase kinase 3 beta activity. Knockdown of cardiolipin synthase did not affect permeability of fluorescent dextran across confluent hCMEC/D3 monolayers grown on Transwell(®) inserts. In contrast, knockdown of cardiolipin synthase resulted in an increase in 2-deoxy-[(3) H]glucose transport across these monolayers compared to controls. The data indicate that in hCMEC/D3 cells, spare respiratory capacity is dependent on cardiolipin. In addition, reduction in cardiolipin in these cells alters their cellular energy status and this results in increased glucose transport into and across hCMEC/D3 monolayers. Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. In human adult brain endothelial cell hCMEC/D3 monolayers cultured on Transwell(®) plates, knockdown of cardiolipin synthase results in decrease in mitochondrial cardiolipin and decreased mitochondrial spare respiratory capacity. The reduced cardiolipin results in an increased activity of adenosine monophosphate kinase (pAMPK) and protein kinase B (pAKT) and decreased activity of glycogen synthase kinase 3 beta (pGSK3β) which results in elevated glucose transporter-1 (GLUT-1) expression and association with membranes. This in turn increases 2-dexoyglucose uptake from the apical medium into the cells with a resultant 2-deoxyglucose movement into the basolateral medium. © 2016 International Society for Neurochemistry.

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

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

Stabiliztin of VEGFR2 Signaling by Cerebral Cavernous Malformation 3 is Critical for Vascular Development

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

Y He; H Zhang; L Yu

2011-12-31

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: CCM1, CCM2, and CCM3. CCM3, also known as PDCD10 (programmed cell death 10), was initially identified as a messenger RNA whose abundance was induced by apoptotic stimuli in vitro. However, the in vivo function of CCM3 has not been determined. Here, we describe mice with a deletion of the CCM3 gene either ubiquitously or specifically in the vascular endothelium, smooth muscle cells, or neurons. Mice with global or endothelial cell-specific deletion of CCM3 exhibited defects in embryonic angiogenesis and died at an earlymore » embryonic stage. CCM3 deletion reduced vascular endothelial growth factor receptor 2 (VEGFR2) signaling in embryos and endothelial cells. In response to VEGF stimulation, CCM3 was recruited to and stabilized VEGFR2, and the carboxyl-terminal domain of CCM3 was required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients lacking the carboxyl-terminal domain were labile and were unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 promotes VEGFR2 signaling during vascular development.« less

Reversing resistance to vascular-disrupting agents by blocking late mobilization of circulating endothelial progenitor cells.

PubMed

Taylor, Melissa; Billiot, Fanny; Marty, Virginie; Rouffiac, Valérie; Cohen, Patrick; Tournay, Elodie; Opolon, Paule; Louache, Fawzia; Vassal, Gilles; Laplace-Builhé, Corinne; Vielh, Philippe; Soria, Jean-Charles; Farace, Françoise

2012-05-01

The prevailing concept is that immediate mobilization of bone marrow-derived circulating endothelial progenitor cells (CEP) is a key mechanism mediating tumor resistance to vascular-disrupting agents (VDA). Here, we show that administration of VDA to tumor-bearing mice induces 2 distinct peaks in CEPs: an early, unspecific CEP efflux followed by a late yet more dramatic tumor-specific CEP burst that infiltrates tumors and is recruited to vessels. Combination with antiangiogenic drugs could not disrupt the early peak but completely abrogated the late VDA-induced CEP burst, blunted bone marrow-derived cell recruitment to tumors, and resulted in striking antitumor efficacy, indicating that the late CEP burst might be crucial to tumor recovery after VDA therapy. CEP and circulating endothelial cell kinetics in VDA-treated patients with cancer were remarkably consistent with our preclinical data. These findings expand the current understanding of vasculogenic "rebounds" that may be targeted to improve VDA-based strategies. Our findings suggest that resistance to VDA therapy may be strongly mediated by late, rather than early, tumor-specific recruitment of CEPs, the suppression of which resulted in increased VDA-mediated antitumor efficacy. VDA-based therapy might thus be significantly enhanced by combination strategies targeting late CEP mobilization. © 2012 AACR

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.

Exercise training improves obesity‐related lymphatic dysfunction

PubMed Central

Hespe, Geoffrey E.; Kataru, Raghu P.; Savetsky, Ira L.; García Nores, Gabriela D.; Torrisi, Jeremy S.; Nitti, Matthew D.; Gardenier, Jason C.; Zhou, Jie; Yu, Jessie Z.; Jones, Lee W.

2016-01-01

Key points Obesity results in perilymphatic inflammation and lymphatic dysfunction.Lymphatic dysfunction in obesity is characterized by decreased lymphatic vessel density, decreased collecting lymphatic vessel pumping frequency, decreased lymphatic trafficking of immune cells, increased lymphatic vessel leakiness and changes in the gene expression patterns of lymphatic endothelial cells.Aerobic exercise, independent of weight loss, decreases perilymphatic inflammatory cell accumulation, improves lymphatic function and reverses pathological changes in gene expression in lymphatic endothelial cells. Abstract Although previous studies have shown that obesity markedly decreases lymphatic function, the cellular mechanisms that regulate this response remain unknown. In addition, it is unclear whether the pathological effects of obesity on the lymphatic system are reversible with behavioural modifications. The purpose of this study, therefore, was to analyse lymphatic vascular changes in obese mice and to determine whether these pathological effects are reversible with aerobic exercise. We randomized obese mice to either aerobic exercise (treadmill running for 30 min per day, 5 days a week, for 6 weeks) or a sedentary group that was not exercised and analysed lymphatic function using a variety of outcomes. We found that sedentary obese mice had markedly decreased collecting lymphatic vessel pumping capacity, decreased lymphatic vessel density, decreased lymphatic migration of immune cells, increased lymphatic vessel leakiness and decreased expression of lymphatic specific markers compared with lean mice (all P < 0.01). Aerobic exercise did not cause weight loss but markedly improved lymphatic function compared with sedentary obese mice. Exercise had a significant anti‐inflammatory effect, resulting in decreased perilymphatic accumulation of inflammatory cells and inducible nitric oxide synthase expression. In addition, exercise normalized isolated lymphatic endothelial cell gene expression of lymphatic specific genes, including VEGFR‐3 and Prox1. Taken together, our findings suggest that obesity impairs lymphatic function via multiple mechanisms and that these pathological changes can be reversed, in part, with aerobic exercise, independent of weight loss. In addition, our study shows that obesity‐induced lymphatic endothelial cell gene expression changes are reversible with behavioural modifications. PMID:26931178

Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate.

PubMed

Siegel, Georg; Fleck, Erika; Elser, Stefanie; Hermanutz-Klein, Ursula; Waidmann, Marc; Northoff, Hinnak; Seifried, Erhard; Schäfer, Richard

2018-05-01

Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10 -8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 10 3 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP. © 2018 AABB.

Activation of RelA homodimers by tumour necrosis factor α: a possible transcriptional activator in human vascular endothelial cells

PubMed Central

2005-01-01

In vascular endothelial cells, cytokines induce genes that are expressed in inflammatory lesions partly through the activation of transcription factor NF-κB (nuclear factor-κB). Among the members of the NF-κB/rel protein family, homodimers of the RelA subunit of NF-κB can also function as strong transactivators when expressed in cells. However, the functional role of endogenous RelA homodimers has not been clearly elucidated. We investigated whether RelA homodimers are induced in cytokine-treated vascular endothelial cells. Gel mobility-shift and supershift assays revealed that a cytokine TNFα (tumour necrosis factor α) activated both NF-κB1/RelA heterodimers and RelA homodimers that bound to a canonical κB sequence, IgκB (immunoglobulin κB), in SV40 (simian virus 40) immortalized HMEC-1 (human dermal microvascular endothelial cell line 1). In HMEC-1 and HUVEC (human umbilical-vein endothelial cells), TNFα also induced RelA homodimers that bound to the sequence 65-2κB, which specifically binds to RelA homodimers but not to NF-κB1/RelA heterodimers in vitro. Deoxycholic acid, a detergent that can dissociate the NF-κB–IκB complex (where IκB stands for inhibitory κB), induced the binding of the RelA homodimers to 65-2κB from the cytosolic fraction of resting HMEC-1. Furthermore, TNFα induced the transcriptional activity of a reporter gene that was driven by 65-2κB in HMEC-1. These results suggest that in addition to NF-κB1/RelA heterodimers, TNFα also induces RelA homodimers that are functionally active. Thus RelA homodimers may actively participate in cytokine regulation of gene expression in human vascular endothelial cells. PMID:15876188

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.

At the interface of antioxidant signalling and cellular function: Key polyphenol effects

PubMed Central

Kerimi, Asimina

2016-01-01

The hypothesis that dietary (poly)phenols promote well‐being by improving chronic disease‐risk biomarkers, such as endothelial dysfunction, chronic inflammation and plasma uric acid, is the subject of intense current research, involving human interventions studies, animal models and in vitro mechanistic work. The original claim that benefits were due to the direct antioxidant properties of (poly)phenols has been mostly superseded by detailed mechanistic studies on specific molecular targets. Nevertheless, many proposed mechanisms in vivo and in vitro are due to modulation of oxidative processes, often involving binding to specific proteins and effects on cell signalling. We review the molecular mechanisms for 3 actions of (poly)phenols on oxidative processes where there is evidence in vivo from human intervention or animal studies. (1) Effects of (poly) phenols on pathways of chronic inflammation leading to prevention of some of the damaging effects associated with the metabolic syndrome. (2) Interaction of (poly)phenols with endothelial cells and smooth muscle cells, leading to effects on blood pressure and endothelial dysfunction, and consequent reduction in cardiovascular disease risk. (3) The inhibition of xanthine oxidoreductase leading to modulation of intracellular superoxide and plasma uric acid, a risk factor for developing type 2 diabetes. PMID:26887821

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.

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.

Upregulation of human heme oxygenase gene expression by Ets-family proteins.

PubMed

Deramaudt, B M; Remy, P; Abraham, N G

1999-03-01

Overexpression of human heme oxygenase-1 has been shown to have the potential to promote EC proliferation and angiogenesis. Since Ets-family proteins have been shown to play an important role in angiogenesis, we investigated the presence of ETS binding sites (EBS), GGAA/T, and ETS protein contributing to human HO-1 gene expression. Several chloramphenicol acetyltransferase constructs were examined in order to analyze the effect of ETS family proteins on the transduction of HO-1 in Xenopus oocytes and in microvessel endothelial cells. Heme oxygenase promoter activity was up-regulated by FLI-1ERGETS-1 protein(s). Chloramphenicol acetyltransferase (CAT) assays demonstrated that the promoter region (-1500 to +19) contains positive and negative control elements and that all three members of the ETS protein family were responsible for the up-regulation of HHO-1. Electrophoretic mobility shift assays (EMSA), performed with nuclear extracts from endothelial cells overexpressing HHO-1 gene, and specific HHO-1 oligonucleotides probes containing putative EBS resulted in a specific and marked bandshift. Synergistic binding was observed in EMSA between AP-1 on the one hand, FLI-1, ERG, and ETS-1 protein on the other. Moreover, 5'-deletion analysis demonstrated the existence of a negative control element of HHO-1 expression located between positions -1500 and -120 on the HHO-1 promoter. The presence of regulatory sequences for transcription factors such as ETS-1, FLI-1, or ERG, whose activity is associated with cell proliferation, endothelial cell differentiation, and matrix metalloproteinase transduction, may be an indication of the important role that HO-1 may play in coronary collateral circulation, tumor growth, angiogenesis, and hemoglobin-induced endothelial cell injuries.

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.

Adrenomedullin and adrenomedullin binding protein-1 attenuate vascular endothelial cell apoptosis in sepsis.

PubMed

Zhou, Mian; Simms, H Hank; Wang, Ping

2004-08-01

To determine whether vascular endothelial cell apoptosis occurs in the late stage of sepsis and, if so, whether administration of a potent vasodilatory peptide adrenomedullin and its newly reported specific binding protein (AM/AMBP-1) prevents sepsis-induced endothelial cell apoptosis. Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Our recent studies have shown that administration of AM/AMBP-1 delays or even prevents the transition from the hyperdynamic phase to the hypodynamic phase of sepsis, attenuates tissue injury, and decreases sepsis-induced mortality. However, the mechanisms responsible for the beneficial effects of AM/AMBP-1 in sepsis remain unknown. Polymicrobial sepsis was induced by cecal ligation and puncture in adult male rats. Human AMBP-1 (40 microg/kg body weight) was infused intravenously at the beginning of sepsis for 20 minutes and synthetic AM (12 microg/kg body weight) was continuously administered for the entire study period using an Alzert micro-osmotic pump, beginning 3 hours prior to the induction of sepsis. The thoracic aorta and pulmonary tissues were harvested at 20 hours after cecal ligation and puncture (ie, the late stage of sepsis). Apoptosis was determined using TUNEL assay, M30 Cytodeath immunostaining, and electromicroscopy. In addition, anti-apoptotic Bcl-2 and pro-apoptotic Bax gene expression and protein levels were assessed by RT-PCR and Western blot analysis, respectively. Vascular endothelial cells underwent apoptosis formation at 20 hours after cecal ligation and puncture as determined by three different methods. Moreover, partial detached endothelial cell in the aorta was observed. Bcl-2 mRNA and protein levels decreased significantly at 20 hours after the onset of sepsis while Bax was not altered. Administration of AM/AMBP-1 early after sepsis, however, significantly reduced the number of apoptotic endothelial cells. This was associated with significantly increased Bcl-2 protein levels and decreased Bax gene expression in the aortic and pulmonary tissues. The above results suggest that vascular endothelial cell apoptosis occurs in late sepsis and the anti-apoptotic effects of AM/AMBP-1 appear to be in part responsible for their beneficial effects observed under such conditions.

Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy.

PubMed

Denmeade, Samuel R; Mhaka, Annastasiah M; Rosen, D Marc; Brennen, W Nathaniel; Dalrymple, Susan; Dach, Ingrid; Olesen, Claus; Gurel, Bora; Demarzo, Angelo M; Wilding, George; Carducci, Michael A; Dionne, Craig A; Møller, Jesper V; Nissen, Poul; Christensen, S Brøgger; Isaacs, John T

2012-06-27

Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer.

Ca2+-dependent nitric oxide release in the injured endothelium of excised rat aorta: a promising mechanism applying in vascular prosthetic devices in aging patients

PubMed Central

2013-01-01

Background Nitric oxide is key to endothelial regeneration, but it is still unknown whether endothelial cell (EC) loss results in an increase in NO levels at the wound edge. We have already shown that endothelial damage induces a long-lasting Ca2+ entry into surviving cells though connexin hemichannels (CxHcs) uncoupled from their counterparts on ruptured cells. The physiological outcome of injury-induced Ca2+ inflow is, however, unknown. Methods In this study, we sought to determine whether and how endothelial scraping induces NO production (NOP) in the endothelium of excised rat aorta by exploiting the NO-sensitive fluorochrome, DAF-FM diacetate and the Ca2+-sensitive fluorescent dye, Fura-2/AM. Results We demonstrated that injury-induced NOP at the lesion site is prevented in presence of the endothelial NO synthase inhibitor, L-NAME, and in absence of extracellular Ca2+. Unlike ATP-dependent NO liberation, the NO response to injury is insensitive to BTP-2, which selectively blocks store-operated Ca2+ inflow. However, injury-induced NOP is significantly reduced by classic gap junction blockers, and by connexin mimetic peptides specifically targeting Cx37Hcs, Cx40HCs, and Cx43Hcs. Moreover, disruption of caveolar integrity prevents injury-elicited NO signaling, but not the accompanying Ca2+ response. Conclusions The data presented provide the first evidence that endothelial scraping stimulates NO synthesis at the wound edge, which might both exert an immediate anti-thrombotic and anti-inflammatory action and promote the subsequent re-endothelialization. PMID:24266895

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.

Inhibition of GTPase Rac1 in endothelium by 6-mercaptopurine results in immunosuppression in nonimmune cells: new target for an old drug.

PubMed

Marinković, Goran; Kroon, Jeffrey; Hoogenboezem, Mark; Hoeben, Kees A; Ruiter, Matthijs S; Kurakula, Kondababu; Otermin Rubio, Iker; Vos, Mariska; de Vries, Carlie J M; van Buul, Jaap D; de Waard, Vivian

2014-05-01

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are well established immunosuppressive drugs. Common understanding of their immunosuppressive properties is largely limited to immune cells. However, in this study, the mechanism underlying the protective role of 6-MP in endothelial cell activation is investigated. Because 6-MP and its derivative 6-thioguanosine-5'-triphosphate (6-T-GTP) were shown to block activation of GTPase Rac1 in T lymphocytes, we focused on Rac1-mediated processes in endothelial cells. Indeed, 6-MP and 6-T-GTP decreased Rac1 activation in endothelial cells. As a result, the compounds inhibited TNF-α-induced downstream signaling via JNK and reduced activation of transcription factors c-Jun, activating transcription factor-2 and, in addition, NF κ-light-chain-enhancer of activated B cells (NF-κB), which led to decreased transcription of proinflammatory cytokines. Moreover, 6-MP and 6-T-GTP selectively decreased TNF-α-induced VCAM-1 but not ICAM-1 protein levels. Rac1-mediated generation of cell membrane protrusions, which form docking structures to capture leukocytes, also was reduced by 6-MP/6-T-GTP. Consequently, leukocyte transmigration was inhibited after 6-MP/6-T-GTP treatment. These data underscore the anti-inflammatory effect of 6-MP and 6-T-GTP on endothelial cells by blocking Rac1 activation. Our data provide mechanistic insight that supports development of novel Rac1-specific therapeutic approaches against chronic inflammatory diseases.

Neurotrophins promote revascularization by local recruitment of TrkB+ endothelial cells and systemic mobilization of hematopoietic progenitors

PubMed Central

Kermani, Pouneh; Rafii, Dahlia; Jin, David K.; Whitlock, Paul; Schaffer, Wendy; Chiang, Anne; Vincent, Loic; Friedrich, Matthias; Shido, Koji; Hackett, Neil R.; Crystal, Ronald G.; Rafii, Shahin; Hempstead, Barbara L.

2005-01-01

The neurotrophin brain-derived neurotrophic factor (BDNF) is required for the maintenance of cardiac vessel wall stability during embryonic development through direct angiogenic actions on endothelial cells expressing the tropomysin receptor kinase B (TrkB). However, the role of BDNF and a related neurotrophin ligand, neurotrophin-4 (NT-4), in the regulation of revascularization of the adult tissues is unknown. To study the potential angiogenic capacity of BDNF in mediating the neovascularization of ischemic and non-ischemic adult mouse tissues, we utilized a hindlimb ischemia and a subcutaneous Matrigel model. Recruitment of endothelial cells and promotion of channel formation within the Matrigel plug by BDNF and NT-4 was comparable to that induced by VEGF-A. The introduction of BDNF into non-ischemic ears or ischemic limbs induced neoangiogenesis, with a 2-fold increase in the capillary density. Remarkably, treatment with BDNF progressively increased blood flow in the ischemic limb over 21 days, similar to treatment with VEGF-A. The mechanism by which BDNF enhances capillary formation is mediated in part through local activation of the TrkB receptor and also by recruitment of Sca-1+CD11b+ pro-angiogenic hematopoietic cells. BDNF induces a potent direct chemokinetic action on subsets of marrow-derived Sca-1+ hematopoietic cells co-expressing TrkB. These studies suggest that local regional delivery of BDNF may provide a novel mechanism for inducing neoangiogenesis through both direct actions on local TrkB-expressing endothelial cells in skeletal muscle and recruitment of specific subsets of TrkB+ bone marrow–derived hematopoietic cells to provide peri-endothelial support for the newly formed vessels. PMID:15765148

Inhibitory effect of trans-caryophyllene (TC) on leukocyte-endothelial attachment.

PubMed

Zhang, Zhen; Yang, Chunfeng; Dai, Xinlun; Ao, Yu; Li, Yumei

2017-08-15

trans-Caryophyllene (TC) is a major component found in the essential oils of many spices and foods/medicinal plants. It is a natural sesquiterpene and has been the subject of numerous studies. However, the effects of TC on vascular inflammation remain unknown. In this study, we reported that TC treatment in human umbilical vein endothelial cells (HUVECs) prevented attachment of monocytic leukemia cell line THP-1 cells to endothelial cells. In addition, in vivo results indicate that TC inhibited macrophage infiltration to the aortic surface and reduced total serum levels of cholesterol and triglycerides. Importantly, administration of TC could inhibit the induction of vascular cell adhesion molecule-1 (VCAM-1) both in vitro and in vivo. Notably, our data indicate that the inhibitory effects of TC on the expression of VCAM-1 are mediated by the JAK2/STAT1/IRF-1 pathway. TC is a specific agonist of the type 2 cannabinoid receptor (CB2R). Importantly, we further verified that the inhibitory effects of TC on the expression of IRF-1 and VCAM-1 are dependent on activation of CB2R. Inhibition of CB2R by either specific inhibitors or RNA interference abolished the inhibitory effects of TC on the expression of IRF-1 and VCAM-1. Our results suggest that TC might have a capacity to suppress the development of atherosclerosis. Copyright © 2017 Elsevier Inc. All rights reserved.

A 3D Human Renal Cell Carcinoma-on-a-Chip for the Study of Tumor Angiogenesis.

PubMed

Miller, Chris P; Tsuchida, Connor; Zheng, Ying; Himmelfarb, Jonathan; Akilesh, Shreeram

2018-06-01

Tractable human tissue-engineered 3D models of cancer that enable fine control of tumor growth, metabolism, and reciprocal interactions between different cell types in the tumor microenvironment promise to accelerate cancer research and pharmacologic testing. Progress to date mostly reflects the use of immortalized cancer cell lines, and progression to primary patient-derived tumor cells is needed to realize the full potential of these platforms. For the first time, we report endothelial sprouting induced by primary patient tumor cells in a 3D microfluidic system. Specifically, we have combined primary human clear cell renal cell carcinoma (ccRCC) cells from six independent donors with human endothelial cells in a vascularized, flow-directed, 3D culture system ("ccRCC-on-a-chip"). The upregulation of key angiogenic factors in primary human ccRCC cells, which exhibited unique patterns of donor variation, was further enhanced when they were cultured in 3D clusters. When embedded in the matrix surrounding engineered human vessels, these ccRCC tumor clusters drove potent endothelial cell sprouting under continuous flow, thus recapitulating the critical angiogenic signaling axis between human ccRCC cells and endothelial cells. Importantly, this phenotype was driven by a primary tumor cell-derived biochemical gradient of angiogenic growth factor accumulation that was subject to pharmacological blockade. Our novel 3D system represents a vascularized tumor model that is easy to image and quantify and is fully tunable in terms of input cells, perfusate, and matrices. We envision that this ccRCC-on-a-chip will be valuable for mechanistic studies, for studying tumor-vascular cell interactions, and for developing novel and personalized antitumor therapies. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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.

Tumor-targeting CTL expressing a single-chain Fv specific for VEGFR2.

PubMed

Kanagawa, Naoko; Yanagawa, Tatsuya; Mukai, Yohei; Yoshioka, Yasuo; Okada, Naoki; Nakagawa, Shinsaku

2010-03-26

Cytotoxic T lymphocytes (CTL) are critical effector cells in tumor immunity. Adoptive transfer therapy with in vitro-expanded tumor-specific CTL is a promising approach for preventing cancer metastasis and recurrence. Transferred CTL are not effective in clinical trials, however, due to inadequate tumor-infiltration. Therefore, the development of functionally modified CTL, such as tumor-targeting CTL, is widely desired. Here, we designed the tumor-targeting CTL expressing a single-chain antibody fragment (scFv-CTL) specific for vascular endothelial growth factor receptor 2 (VEGFR2/flk1) by transducing the CTL with a retroviral vector. The scFv-CTL bound to VEGFR2/flk1-expressing cells and retained their cytotoxic activity against tumor cells. In addition, adoptive transfer of scFv-CTL into tumor-bearing mice effectively suppressed tumor growth due to the augmented accumulation of the transferred CTL in the tumor tissue. These findings indicate that the creation of CTL capable of targeting tumor vascular endothelial cells by scFv-expression technique is considerably promising for improvement of efficacy in adoptive immunotherapy. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Tissue factor is an angiogenic-specific receptor for factor VII-targeted immunotherapy and photodynamic therapy.

PubMed

Hu, Zhiwei; Cheng, Jijun; Xu, Jie; Ruf, Wolfram; Lockwood, Charles J

2017-02-01

Identification of target molecules specific for angiogenic vascular endothelial cells (VEC), the inner layer of pathological neovasculature, is critical for discovery and development of neovascular-targeting therapy for angiogenesis-dependent human diseases, notably cancer, macular degeneration and endometriosis, in which vascular endothelial growth factor (VEGF) plays a central pathophysiological role. Using VEGF-stimulated vascular endothelial cells (VECs) isolated from microvessels, venous and arterial blood vessels as in vitro angiogenic models and unstimulated VECs as a quiescent VEC model, we examined the expression of tissue factor (TF), a membrane-bound receptor on the angiogenic VEC models compared with quiescent VEC controls. We found that TF is specifically expressed on angiogenic VECs in a time-dependent manner in microvessels, venous and arterial vessels. TF-targeted therapeutic agents, including factor VII (fVII)-IgG1 Fc and fVII-conjugated photosensitizer, can selectively bind angiogenic VECs, but not the quiescent VECs. Moreover, fVII-targeted photodynamic therapy can selectively and completely eradicate angiogenic VECs. We conclude that TF is an angiogenic-specific receptor and the target molecule for fVII-targeted therapeutics. This study supports clinical trials of TF-targeted therapeutics for the treatment of angiogenesis-dependent diseases such as cancer, macular degeneration and endometriosis.

Matrix-specific protein kinase A signaling regulates p21 activated kinase activation by flow in endothelial cells

PubMed Central

Funk, Steven Daniel; Yurdagul, Arif; Green, Jonette M.; Jhaveri, Krishna A.; Schwartz, Martin Alexander; Orr, A. Wayne

2010-01-01

Rationale Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and NF-κB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-κB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-κB. Objective To elucidate the mechanisms regulating matrix-specific PAK activation. Methods and Results We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead basement membrane proteins enhance flow-induced protein kinase A (PKA) activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-κB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the PGI2 analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKI injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI. Conclusions Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK. PMID:20224042

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

Crucial Role of the Aryl Hydrocarbon Receptor (AhR) in Indoxyl Sulfate-Induced Vascular Inflammation.

PubMed

Ito, Shunsuke; Osaka, Mizuko; Edamatsu, Takeo; Itoh, Yoshiharu; Yoshida, Masayuki

2016-08-01

The aryl hydrocarbon receptor (AhR), a ligand-inducible transcription factor mediating toxic effects of dioxins and uremic toxins, has recently emerged as a pathophysiological regulator of immune-inflammatory conditions. Indoxyl sulfate, a uremic toxin, is associated with cardiovascular disease in patients with chronic kidney disease and has been shown to be a ligand for AhR. The aim of this study was to investigate the potential role of AhR in indoxyl sulfate-induced leukocyte-endothelial interactions. Endothelial cell-specific AhR knockout (eAhR KO) mice were produced by crossing AhR floxed mice with Tie2 Cre mice. Indoxyl sulfate was administered for 2 weeks, followed by injection of TNF-α. Leukocyte recruitment to the femoral artery was assessed by intravital microscopy. Vascular endothelial cells were transfected with siRNA specific to AhR (siAhR) and treated with indoxyl sulfate, followed by stimulation with TNF-α. Indoxyl sulfate dramatically enhanced TNF-α-induced leukocyte recruitment to the vascular wall in control animals but not in eAhR KO mice. In endothelial cells, siAhR significantly reduced indoxyl sulfate-enhanced leukocyte adhesion as well as E-selectin expression, whereas the activation of JNK and nuclear factor-κB was not affected. A luciferase assay revealed that the region between －153 and －146 bps in the E-selectin promoter was responsible for indoxyl sulfate activity via AhR. Mutational analysis of this region revealed that activator protein-1 (AP-1) is responsible for indoxyl sulfate-triggered E-selectin expression via AhR. AhR mediates indoxyl sulfate-enhanced leukocyte-endothelial interactions through AP-1 transcriptional activity, which may constitute a new mechanism of vascular inflammation in patients with renal disease.

Effect of specific surface microstructures on substrate endothelialisation and thrombogenicity: Importance for stent design.

PubMed

Lutter, Christoph; Nothhaft, Matthias; Rzany, Alexander; Garlichs, Christoph D; Cicha, Iwona

2015-01-01

In coronary artery disease, highly stenosed arteries are frequently treated by stent implantation, which thereafter necessitates a dual-antiplatelet therapy (DAPT) in order to prevent stent-thrombosis. We hypothesized that specific patterns of microstructures on stents can accelerate endothelialisation thereby reducing their thrombogenicity and the DAPT duration. Differently designed, 2-5 μm high elevations or hollows were lithographically etched on silicon plates, subsequently coated with silicon carbide. Smooth silicon plates and bare metal substrates were used as controls. To assess attachment and growth of human umbilical vein endothelial cells under static or flow conditions, actin cytoskeleton was visualised with green phalloidin. Endothelial migration was assessed in a modified barrier assay. To investigate surface thrombogenicity, platelets were incubated on the structured surfaces in static and flow conditions, and visualised with fluorescein-conjugated P-selectin antibody. Images were taken with incident-light fluorescent microscope for non-transparent objects. Compared to smooth surface, flat cubic elevations (5 μm edge length) improved endothelial cell attachment and growth under static and dynamic conditions, whereas smaller, spiky structures (2 μm edge length) had a negative influence on endothelialisation. Endothelial cell migration was fastest on flat cubic elevations, hollows, and smooth surfaces, whereas spiky structures and bare metal had a negative effect on endothelial migration. Thrombogenicity assays under static and flow conditions showed that platelet adhesion was reduced on the flat elevations and the smooth surface, as compared to the spiky structures, the hollow design and the bare metal substrates. Surface microstructures strongly influence endothelialisation of substrates. Designing stents with surface topography which accelerates endothelialisation and reduces thrombogenicity may be of clinical benefit by improving the safety profile of coronary interventions.

Patterning of Endothelial Cells and Mesenchymal Stem Cells by Laser-Assisted Bioprinting to Study Cell Migration.

PubMed

Bourget, Jean-Michel; Kérourédan, Olivia; Medina, Manuela; Rémy, Murielle; Thébaud, Noélie Brunehilde; Bareille, Reine; Chassande, Olivier; Amédée, Joëlle; Catros, Sylvain; Devillard, Raphaël

2016-01-01

Tissue engineering of large organs is currently limited by the lack of potent vascularization in vitro . Tissue-engineered bone grafts can be prevascularized in vitro using endothelial cells (ECs). The microvascular network architecture could be controlled by printing ECs following a specific pattern. Using laser-assisted bioprinting, we investigated the effect of distance between printed cell islets and the influence of coprinted mesenchymal cells on migration. When printed alone, ECs spread out evenly on the collagen hydrogel, regardless of the distance between cell islets. However, when printed in coculture with mesenchymal cells by laser-assisted bioprinting, they remained in the printed area. Therefore, the presence of mesenchymal cell is mandatory in order to create a pattern that will be conserved over time. This work describes an interesting approach to study cell migration that could be reproduced to study the effect of trophic factors.

Patterning of Endothelial Cells and Mesenchymal Stem Cells by Laser-Assisted Bioprinting to Study Cell Migration

PubMed Central

Medina, Manuela; Rémy, Murielle; Thébaud, Noélie Brunehilde; Bareille, Reine; Chassande, Olivier; Amédée, Joëlle; Catros, Sylvain

2016-01-01

Tissue engineering of large organs is currently limited by the lack of potent vascularization in vitro. Tissue-engineered bone grafts can be prevascularized in vitro using endothelial cells (ECs). The microvascular network architecture could be controlled by printing ECs following a specific pattern. Using laser-assisted bioprinting, we investigated the effect of distance between printed cell islets and the influence of coprinted mesenchymal cells on migration. When printed alone, ECs spread out evenly on the collagen hydrogel, regardless of the distance between cell islets. However, when printed in coculture with mesenchymal cells by laser-assisted bioprinting, they remained in the printed area. Therefore, the presence of mesenchymal cell is mandatory in order to create a pattern that will be conserved over time. This work describes an interesting approach to study cell migration that could be reproduced to study the effect of trophic factors. PMID:27833916

Effector T-cell infiltration positively impacts survival of glioblastoma patients and is impaired by tumor-derived TGF-β.

PubMed

Lohr, Jennifer; Ratliff, Thomas; Huppertz, Andrea; Ge, Yingzi; Dictus, Christine; Ahmadi, Rezvan; Grau, Stefan; Hiraoka, Nobuyoshi; Eckstein, Volker; Ecker, Rupert C; Korff, Thomas; von Deimling, Andreas; Unterberg, Andreas; Beckhove, Philipp; Herold-Mende, Christel

2011-07-01

In glioma-in contrast to various other cancers-the impact of T-lymphocytes on clinical outcome is not clear. We investigated the clinical relevance and regulation of T-cell infiltration in glioma. T-cell subpopulations from entire sections of 93 WHO°II-IV gliomas were computationally identified using markers CD3, CD8, and Foxp3; survival analysis was then done on primary glioblastomas (pGBM). Endothelial cells expressing cellular adhesion molecules (CAM) were similarly computationally quantified from the same glioma tissues. Influence of prominent cytokines (as measured by ELISA from 53 WHO°II-IV glioma lysates) on CAM-expression in GBM-isolated endothelial cells was determined using flow cytometry. The functional relevance of the cytokine-mediated CAM regulation was tested in a transmigration assay using GBM-derived endothelial cells and autologous T-cells. Infiltration of all T-cell subsets increased in high-grade tumors. Most strikingly, within pGBM, elevated numbers of intratumoral effector T cells (T(eff), cytotoxic and helper) significantly correlated with a better survival; regulatory T cells were infrequently present and not associated with GBM patient outcome. Interestingly, increased infiltration of T(eff) cells was related to the expression of ICAM-1 on the vessel surface. Transmigration of autologous T cells in vitro was markedly reduced in the presence of CAM-blocking antibodies. We found that TGF-β molecules impeded transmigration and downregulated CAM-expression on GBM-isolated endothelial cells; blocking TGF-β receptor signaling increased transmigration. This study provides comprehensive and novel insights into occurrence and regulation of T-cell infiltration in glioma. Specifically, targeting TGF-β1 and TGF-β2 might improve intratumoral T-cell infiltration and thus enhance effectiveness of immunotherapeutic approaches.

Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction.

PubMed

Rouhani, Sherin J; Eccles, Jacob D; Riccardi, Priscila; Peske, J David; Tewalt, Eric F; Cohen, Jarish N; Liblau, Roland; Mäkinen, Taija; Engelhard, Victor H

2015-04-10

Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3.

Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction

PubMed Central

Rouhani, Sherin J.; Eccles, Jacob D.; Riccardi, Priscila; Peske, J. David; Tewalt, Eric F.; Cohen, Jarish N.; Liblau, Roland; Mäkinen, Taija; Engelhard, Victor H.

2015-01-01

Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3. PMID:25857745

Vascular tissue engineering by computer-aided laser micromachining.

PubMed

Doraiswamy, Anand; Narayan, Roger J

2010-04-28

Many conventional technologies for fabricating tissue engineering scaffolds are not suitable for fabricating scaffolds with patient-specific attributes. For example, many conventional technologies for fabricating tissue engineering scaffolds do not provide control over overall scaffold geometry or over cell position within the scaffold. In this study, the use of computer-aided laser micromachining to create scaffolds for vascular tissue networks was investigated. Computer-aided laser micromachining was used to construct patterned surfaces in agarose or in silicon, which were used for differential adherence and growth of cells into vascular tissue networks. Concentric three-ring structures were fabricated on agarose hydrogel substrates, in which the inner ring contained human aortic endothelial cells, the middle ring contained HA587 human elastin and the outer ring contained human aortic vascular smooth muscle cells. Basement membrane matrix containing vascular endothelial growth factor and heparin was to promote proliferation of human aortic endothelial cells within the vascular tissue networks. Computer-aided laser micromachining provides a unique approach to fabricate small-diameter blood vessels for bypass surgery as well as other artificial tissues with complex geometries.

Knockdown of long noncoding RNA XIST alleviates oxidative low-density lipoprotein-mediated endothelial cells injury through modulation of miR-320/NOD2 axis.

PubMed

Xu, Xiaohui; Ma, Congmin; Liu, Chao; Duan, Zhihui; Zhang, Li

2018-06-14

Atherosclerosis remains to be one of the most common vascular disorders resulting in morbidity and mortality in the world. Recent studies suggested that endothelial cells (ECs) injury caused by oxidative low-density lipoprotein (ox-LDL) is an early marker for atherosclerosis. Nevertheless, the mechanisms of ox-LDL-induced ECs injury are complicated and largely unknown. Here, we found lncRNA XIST (X-inactive specific transcript) was upregulated in human umbilical vein endothelial cells (HUVECs) stimulated by ox-LDL. Knockdown of XIST boosted the cell viability and suppressed cell apoptosis under ox-LDL stimuli. Further experiments identified XIST regulated the expression of Nucleotide-Binding Oligomerization Domain 2 (NOD2) by sponging miR-320. XIST silencing exerted a protective effect on ox-LDL-induced HUVECs injury via miR-320/NOD2 regulatory network. Our data provide insight into the role of the lncRNA XIST in ox-LDL mediated ECs injury, which can aid in developing new therapeutic strategies for the treatment of atherosclerosis. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

Regulation and function of endothelial glycocalyx layer in vascular diseases.

PubMed

Sieve, Irina; Münster-Kühnel, Anja K; Hilfiker-Kleiner, Denise

2018-01-01

In the vascular system, the endothelial surface layer (ESL) as the inner surface of blood vessels affects mechanotransduction, vascular permeability, rheology, thrombogenesis, and leukocyte adhesion. It creates barriers between endothelial cells and blood and neighbouring cells. The glycocalyx, composed of glycoconjugates and proteoglycans, is an integral component of the ESL and a key element in inter- and intracellular communication and tissue homeostasis. In pathophysiological conditions (atherosclerosis, infection, ischemia/reperfusion injury, diabetes, trauma and acute lung injury) glycocalyx-degrading factors, i.e. reactive oxygen and nitrogen species, matrix metalloproteinases, heparanase and sialidases, damage the ESL, thereby impairing endothelial functions. This leads to increased capillary permeability, leucocyte-endothelium interactions, thrombosis and vascular inflammation, the latter further driving glycocalyx destruction. The present review highlights current knowledge on the vasculoprotective role of the ESL, with specific emphasis on its remodelling in inflammatory vascular diseases and discusses its potential as a novel therapeutic target to treat vascular pathologies. Copyright © 2017 Elsevier Inc. All rights reserved.

A Two-Tier Golgi-Based Control of Organelle Size Underpins the Functional Plasticity of Endothelial Cells

PubMed Central

Ferraro, Francesco; Kriston-Vizi, Janos; Metcalf, Daniel J.; Martin-Martin, Belen; Freeman, Jamie; Burden, Jemima J.; Westmoreland, David; Dyer, Clare E.; Knight, Alex E.; Ketteler, Robin; Cutler, Daniel F.

2014-01-01

Summary Weibel-Palade bodies (WPBs), endothelial-specific secretory granules that are central to primary hemostasis and inflammation, occur in dimensions ranging between 0.5 and 5 μm. How their size is determined and whether it has a functional relevance are at present unknown. Here, we provide evidence for a dual role of the Golgi apparatus in controlling the size of these secretory carriers. At the ministack level, cisternae constrain the size of nanostructures (“quanta”) of von Willebrand factor (vWF), the main WPB cargo. The ribbon architecture of the Golgi then allows copackaging of a variable number of vWF quanta within the continuous lumen of the trans-Golgi network, thereby generating organelles of different sizes. Reducing the WPB size abates endothelial cell hemostatic function by drastically diminishing platelet recruitment, but, strikingly, the inflammatory response (the endothelial capacity to engage leukocytes) is unaltered. Size can thus confer functional plasticity to an organelle by differentially affecting its activities. PMID:24794632

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).

Low-level laser therapy prevents endothelial cells from TNF-α/cycloheximide-induced apoptosis.

PubMed

Chu, Yu-Hsiu; Chen, Shu-Ya; Hsieh, Yueh-Ling; Teng, Yi-Hsien; Cheng, Yu-Jung

2018-02-01

Low-level laser therapy (LLLT), widely used in physiotherapy, has been known to enhance wound healing and stimulate cell proliferation, including fibroblast and endothelial cells. Applying LLLT can increase cell proliferation in many kinds of cells including fibroblasts and endothelial cells. However, the protective mechanisms of LLLT on endothelial apoptosis remain unclear. We hypothesized LLLT can protect endothelial cells from inflammation-induced apoptosis. Human endothelial cell line, EA.hy926 cells, and TNF-α/cycloheximide (TNF/CHX) were used to explore the protective effects of LLLT (660 nm) on inflammation-induced endothelial apoptosis. Cell viability, apoptosis, caspase-3/7/8/9 activity, MAPKs signaling, NF-κB activity, and inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression were measured. Our results showed that LLLT increased EA.hy926 cell proliferation, attenuated the TNF/CHX-induced apoptosis, and reduced the TNF/CHX-mediated caspase-3/7/8/9 activation. In addition, LLLT increased ERK MAPK phosphorylation and suppressed the TNF/CHX-increased p38 MAPK, JNK, IKK phosphorylation, NF-κB translocation, and iNOS expression. The caspases-3 cleavage and cell death were not increased in cells treating with ERK inhibitor U0126, which implicated that ERK is not to be responsible for the protective effects of LLLT. After treating with p38 mitogen-activated protein kinase (MAPK) activator, the protection of LLLT in cell apoptosis was no longer existed, showing that LLLT protected the endothelial cells by suppressing p38 MAPK signaling. Our results provide a new insight into the possible molecular mechanisms in which LLLT protects against inflammatory-induced endothelial dysfunction.

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.

Regulation of store-operated Ca2+ entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells.

PubMed

Kito, Hiroaki; Yamamura, Hisao; Suzuki, Yoshiaki; Yamamura, Hideto; Ohya, Susumu; Asai, Kiyofumi; Imaizumi, Yuji

2015-04-10

Store-operated Ca(2+) entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca(2+) influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction

NASA Astrophysics Data System (ADS)

Bethel, Kelly; Luttgen, Madelyn S.; Damani, Samir; Kolatkar, Anand; Lamy, Rachelle; Sabouri-Ghomi, Mohsen; Topol, Sarah; Topol, Eric J.; Kuhn, Peter

2014-02-01

Elevated levels of circulating endothelial cells (CECs) occur in response to various pathological conditions including myocardial infarction (MI). Here, we adapted a fluid phase biopsy technology platform that successfully detects circulating tumor cells in the blood of cancer patients (HD-CTC assay), to create a high-definition circulating endothelial cell (HD-CEC) assay for the detection and characterization of CECs. Peripheral blood samples were collected from 79 MI patients, 25 healthy controls and six patients undergoing vascular surgery (VS). CECs were defined by positive staining for DAPI, CD146 and von Willebrand Factor and negative staining for CD45. In addition, CECs exhibited distinct morphological features that enable differentiation from surrounding white blood cells. CECs were found both as individual cells and as aggregates. CEC numbers were higher in MI patients compared with healthy controls. VS patients had lower CEC counts when compared with MI patients but were not different from healthy controls. Both HD-CEC and CellSearch® assays could discriminate MI patients from healthy controls with comparable accuracy but the HD-CEC assay exhibited higher specificity while maintaining high sensitivity. Our HD-CEC assay may be used as a robust diagnostic biomarker in MI patients.

Brain endothelial adhesion molecule expression in experimental colitis.

PubMed

Sans, M; Kawachi, S; Soriano, A; Palacín, A; Morise, Z; Granger, D N; Piqué, J M; Grisham, M B; Panés, J

2001-04-01

1) To determine if endothelial expression of adhesion molecules involved in leukocyte recruitment is increased in the brain and other organs in four different models of experimental colitis, and 2) to investigate whether leukocyte infiltration occurs in the brain of colitic animals. Endothelial vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression was quantified, using the dual radiolabeled antibody technique in rats with trinitrobenzenesulfonic acid (TNBS)-induced colitis, in mice with dextran sulfate sodium (DSS)-induced colitis, in SCID mice reconstituted with CD45RBhigh T-cells, and in IL-10-/- mice. Leukocyte infiltration in the brain of TNBS-induced colitic rats was assessed by myeloperoxidase activity and immunohistochemical staining with anti-CD45 monoclonal antibody. Marked upregulation of brain endothelial VCAM-1 (2- to 5.5-fold) was consistently found in colitic animals in the four models studied. Brain VCAM-1 strongly correlated with colon VCAM-1 and colon weight. By contrast, upregulation of brain ICAM-1 in colitic animals was only observed in the CD45RBhigh transfer (3-fold) and the TNBS-induced (1.5-fold models). Heart and muscle VCAM-1 and ICAM-1 were not upregulated in colitic animals in the majority of models studied. There was no leukocyte infiltration into the brain of TNBS-induced colitic rats. Our study demonstrates a marked and specific upregulation of endothelial VCAM-1 in the brain of colitic animals. This activation of cerebral endothelial cells was not associated with an infiltration of leukocytes into brain tissue.

Mechanism for the Increased Permeability in Endothelial Monolayers Induced by Elastase

PubMed Central

Ishii, Y.; Kitamura, S.

1994-01-01

The aim of this study was to investigate the mechanism for the increase in endothelial permeability induced by human neutrophil elastase (HNE). Pretreatment of bovine pulmonary artery endothelial cells (BPAEC) with HNE(0-30 μg/ml) for 1 h produced a concentration dependent increase in 125I-albumin clearance. The effect was reversible and was not due to cytolysis. Pretreatment of BPAEC with sodium tungstate, which depletes xanthine oxidase, or with oxypurinol, did not prevent HNE induced increased permeability. Heparin, which neutralizes the cationic charge of HNE, also had no protective effect. Pretreatment with heat inactivated HNE, which still had positive charge sites, did not result in increased endothelial permeability. Also, ONO-5046, a novel specific inhibitor of HNE, did prevent increased permeability. These results suggest that elastase increases endothelial permeability mainly through its proteolytic effects. PMID:18472917

Targeting of MPEG-protected polyamino acid carrier to human E-selectin in vitro.

PubMed

Kang, H W; Weissleder, R; Bogdanov, A

2002-01-01

Targeted diagnostic agents are expected to have a significant impact in molecular imaging of cell-surface associated markers of proliferation, inflammation and angiogenesis. In this communication, we describe a new class of targeted polyamino acid-based protected graft copolymers (PGC) of poly-(L-lysine) and methyl poly-(ethylene glycol) (PGC) covalently conjugated with a monoclonal antibody fragment, F(ab')(2). We utilized targeted PGC conjugates as carriers of near-infrared indocyanine fluorophores (Cy5.5) for optical imaging of endothelial cell populations expressing IL-1 beta inducible proinflammatory marker E-selectin. We compared two conjugation chemistries, involving either introduction of sulfhydryl group to F(ab')(2), or via direct attachment of the antibody fragment directly to the chemically activated PGC. Both PGC-based targeted agents demonstrated high binding specificity (20-30 fold over non-specific uptake) and were utilized for imaging E-selectin expression on human endothelial cells activated with IL-1 beta.

PECAM1 regulates flow-mediated Gab1 tyrosine phosphorylation and signaling

PubMed Central

Xu, Suowen; Ha, Chang Hoon; Wang, Weiye; Xu, Xiangbin; Yin, Meimei; Jin, Felix Q.; Mastrangelo, Michael; Koroleva, Marina; Fujiwara, Keigi; Jin, Zheng Gen

2016-01-01

Endothelial dysfunction, characterized by impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued decrease of NO production, is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Laminar blood flow-mediated specific signaling cascades modulate vascular endothelial cells (ECs) structure and functions. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation in ECs, which in part confers laminar flow atheroprotective action. However, the molecular mechanisms whereby flow regulates Gab1 tyrosine phosphorylation and its downstream signaling events remain unclear. Here we show that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in flow signaling and HGF signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K decreased flow-, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs. PMID:26706435

Breast cancer drugs dampen vascular functions by interfering with nitric oxide signaling in endothelium

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

Gajalakshmi, Palanivel; Priya, Mani Krishna; Pradeep, Thangaraj

Widely used chemotherapeutic breast cancer drugs such as Tamoxifen citrate (TC), Capecitabine (CP) and Epirubicin (EP) are known to cause various cardiovascular side-effects among long term cancer survivors. Vascular modulation warrants nitric oxide (NO) signal transduction, which targets the vascular endothelium. We hypothesize that TC, CP and EP interference with the nitric oxide downstream signaling specifically, could lead to cardiovascular dysfunctions. The results demonstrate that while all three drugs attenuate NO and cyclic guanosine mono-phosphate (cGMP) production in endothelial cells, they caused elevated levels of NO in the plasma and RBC. However, PBMC and platelets did not show any significantmore » changes under treatment. This implies that the drug effects are specific to the endothelium. Altered eNOS and phosphorylated eNOS (Ser-1177) localization patterns in endothelial cells were observed following drug treatments. Similarly, the expression of phosphorylated eNOS (Ser-1177) protein was decreased under the treatment of drugs. Altered actin polymerization was also observed following drug treatment, while addition of SpNO and 8Br-cGMP reversed this effect. Incubation with the drugs decreased endothelial cell migration whereas addition of YC-1, SC and 8Br-cGMP recovered the effect. Additionally molecular docking studies showed that all three drugs exhibited a strong binding affinity with the catalytic domain of human sGC. In conclusion, results indicate that TC, CP and EP cause endothelial dysfunctions via the NO–sGC–cGMP pathway and these effects could be recovered using pharmaceutical agonists of NO signaling pathway. Further, the study proposes a combination therapy of chemotherapeutic drugs and cGMP analogs, which would confer protection against chemotherapy mediated vascular dysfunctions in cancer patients. - Highlights: • NO production is reduced in endothelial cells under breast cancer drug treatment. • Cellular cGMP level is decreased under the treatments of breast cancer drugs. • Breast cancer drugs induce vasoconstriction by interfering with NO pathway. • NO donors, cGMP analogs rescue breast cancer drug induced endothelial dysfunctions.« less

Endothelial cell culture in microfluidic devices for investigating microvascular processes.

PubMed

Mannino, Robert G; Qiu, Yongzhi; Lam, Wilbur A

2018-07-01

Numerous conditions and disease states such as sickle cell disease, malaria, thrombotic microangiopathy, and stroke significantly impact the microvasculature function and its role in disease progression. Understanding the role of cellular interactions and microvascular hemodynamic forces in the context of disease is crucial to understanding disease pathophysiology. In vivo models of microvascular disease using animal models often coupled with intravital microscopy have long been utilized to investigate microvascular phenomena. However, these methods suffer from some major drawbacks, including the inability to tightly and quantitatively control experimental conditions, the difficulty of imaging multiple microvascular beds within a living organism, and the inability to isolate specific microvascular geometries such as bifurcations. Thus, there exists a need for in vitro microvascular models that can mitigate the drawbacks associated with in vivo systems. To that end, microfluidics has been widely used to develop such models, as it allows for tight control of system inputs, facile imaging, and the ability to develop robust and repeatable systems with well-defined geometries. Incorporating endothelial cells to branching microfluidic models allows for the development of "endothelialized" systems that accurately recapitulate physiological microvessels. In this review, we summarize the field of endothelialized microfluidics, specifically focusing on fabrication methods, limitations, and applications of these systems. We then speculate on future directions and applications of these cutting edge technologies. We believe that this review of the field is of importance to vascular biologists and bioengineers who aim to utilize microfluidic technologies to solve vascular problems.

Molecular and functional characterization of riboflavin specific transport system in rat brain capillary endothelial cells.

PubMed

Patel, Mitesh; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Mitra, Ashim K

2012-08-15

Riboflavin is an important water soluble vitamin (B2) required for metabolic reactions, normal cellular growth, differentiation and function. Mammalian brain cells cannot synthesize riboflavin and must import from systemic circulation. However, the uptake mechanism, cellular translocation and intracellular trafficking of riboflavin in brain capillary endothelial cells are poorly understood. The primary objective of this study is to investigate the existence of a riboflavin-specific transport system and delineate the uptake and intracellular regulation of riboflavin in immortalized rat brain capillary endothelial cells (RBE4). The uptake of [3H]-riboflavin is sodium, temperature and energy dependent but pH independent. [3H]-Riboflavin uptake is saturable with K(m) and V(max) values of 19 ± 3 μM and 0.235 ± 0.012 pmol/min/mg protein, respectively. The uptake process is inhibited by unlabelled structural analogs (lumiflavin, lumichrome) but not by structurally unrelated vitamins. Ca(++)/calmodulin and protein kinase A (PKA) pathways are found to play an important role in the intracellular regulation of [3H]-riboflavin. Apical and baso-lateral uptake of [3H]-riboflavin clearly indicates that a riboflavin specific transport system is predominantly localized on the apical side of RBE4 cells. A 628 bp band corresponding to a riboflavin transporter is revealed in RT-PCR analysis. These findings, for the first time report the existence of a specialized and high affinity transport system for riboflavin in RBE4 cells. The blood-brain barrier (BBB) is a major obstacle limiting drug transport inside the brain as it regulates drug permeation from systemic circulation. This transporter can be utilized for targeted delivery in enhancing brain permeation of highly potent drugs on systemic administration. Copyright © 2012 Elsevier B.V. All rights reserved.

Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor.

PubMed Central

Colman, R W; Pixley, R A; Najamunnisa, S; Yan, W; Wang, J; Mazar, A; McCrae, K R

1997-01-01

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties. PMID:9294114

Moderate Hypoxia Exhibits Increased Endothelial Progenitor Vessel-forming Ability However Gestational Diabetes Caused to Impede Compensatory Defense Reaction.

PubMed

Dincer, U Deniz

2016-05-30

Endothelium represents a defense barrier and responds and integrates neuro humoral stimulus which describes as a compensatory mechanism. Endothelium formed with endothelial cells (ECs) and their progenitors. Endothelial progenitor cells (EPCs) represent minor subpopulation of mononuclear cells in the blood. During acute hypoxia, larger amount of EPCs mobilize into the peripheral blood and they directly contribute revascularization process. One of the subtypes of EPC is termed endothelial colony forming cells (ECFCs) which they possess de novo vessel-forming ability. The present study aims to investigate the role of hypoxia in EPCs functional and vessel-forming ability. Furthermore, it was investigated whether fetal exposure to a diabetic intrauterine environment influence EPCs adaptation ability. Human umbilical cord blood (HUCB) derived ECFCs were selected in all experimental procedures obtained from normal and gestational diabetes mellitus (GDM) subjects via in vitro cell culture methods. Early passage (<5) HUCB ECFCs obtain from GDM (n; 5) and control (n; 5) subjects were cultured with plates pre-coated with collagen in vitro 72 h hypoxic as well as normoxic condition. Endothelial, angiogenic and hypoxia associated gene specific primers designed to perform Real-time PCR. Senescenes assay conducted onto HUCB ECFCs to investigate their functional clonogenic ability. To quantify their vessel forming ability matrigel assay was applied. These data demonstrates that moderate hypoxia results increased vessel-forming ability and VEGFA expression in HUCB ECFCs obtained from control subjects. However, GDM caused to impede compensatory defense reaction against hypoxia which observed in control subjects. Thus, it illuminates beneficial information related future therapeutic modalities.

G-protein-coupled receptor-2-interacting protein-1 is required for endothelial cell directional migration and tumor angiogenesis via cortactin-dependent lamellipodia formation.

PubMed

Majumder, Syamantak; Sowden, Mark P; Gerber, Scott A; Thomas, Tamlyn; Christie, Christine K; Mohan, Amy; Yin, Guoyong; Lord, Edith M; Berk, Bradford C; Pang, Jinjiang

2014-02-01

Recent evidence suggests G-protein-coupled receptor-2-interacting protein-1 (GIT1) overexpression in several human metastatic tumors, including breast, lung, and prostate. Tumor metastasis is associated with an increase in angiogenesis. We have showed previously that GIT1 is required for postnatal angiogenesis during lung development. However, the functional role of GIT1 in pathological angiogenesis during tumor growth is unknown. In the present study, we show inhibition of angiogenesis in matrigel implants as well as reduced tumor angiogenesis and melanoma tumor growth in GIT1-knockout mice. We demonstrate that this is a result of impaired directional migration of GIT1-depleted endothelial cells toward a vascular endothelial growth factor gradient. Cortactin-mediated lamellipodia formation in the leading edge is critical for directional migration. We observed a significant reduction in cortactin localization and lamellipodia formation in the leading edge of GIT1-depleted endothelial cells. We specifically identified that the Spa homology domain (aa 250-420) of GIT1 is required for GIT1-cortactin complex localization to the leading edge. The mechanisms involved extracellular signal-regulated kinases 1 and 2-mediated Cortactin-S405 phosphorylation and activation of Rac1/Cdc42. Finally, using gain of function studies, we show that a constitutively active mutant of cortactin restored directional migration of GIT1-depleted cells. Our data demonstrated that a GIT1-cortactin association through GIT1-Spa homology domain is required for cortactin localization to the leading edge and is essential for endothelial cell directional migration and tumor angiogenesis.

Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis

PubMed Central

Daehn, Ilse; Casalena, Gabriella; Zhang, Taoran; Shi, Shaolin; Fenninger, Franz; Barasch, Nicholas; Yu, Liping; D’Agati, Vivette; Schlondorff, Detlef; Kriz, Wilhelm; Haraldsson, Borje; Bottinger, Erwin P.

2014-01-01

Focal segmental glomerular sclerosis (FSGS) is a primary kidney disease that is commonly associated with proteinuria and progressive loss of glomerular function, leading to development of chronic kidney disease (CKD). FSGS is characterized by podocyte injury and depletion and collapse of glomerular capillary segments. Progression of FSGS is associated with TGF-β activation in podocytes; however, it is not clear how TGF-β signaling promotes disease. Here, we determined that podocyte-specific activation of TGF-β signaling in transgenic mice and BALB/c mice with Adriamycin-induced glomerulosclerosis is associated with endothelin-1 (EDN1) release by podocytes, which mediates mitochondrial oxidative stress and dysfunction in adjacent endothelial cells via paracrine EDN1 receptor type A (EDNRA) activation. Endothelial dysfunction promoted podocyte apoptosis, and inhibition of EDNRA or scavenging of mitochondrial-targeted ROS prevented podocyte loss, albuminuria, glomerulosclerosis, and renal failure. We confirmed reciprocal crosstalk between podocytes and endothelial cells in a coculture system. Biopsies from patients with FSGS exhibited increased mitochondrial DNA damage, consistent with EDNRA-mediated glomerular endothelial mitochondrial oxidative stress. Our studies indicate that segmental glomerulosclerosis develops as a result of podocyte-endothelial crosstalk mediated by EDN1/EDNRA-dependent mitochondrial dysfunction and suggest that targeting the reciprocal interaction between podocytes and endothelia may provide opportunities for therapeutic intervention in FSGS. PMID:24590287

Intravenously injected human multilineage-differentiating stress-enduring cells selectively engraft into mouse aortic aneurysms and attenuate dilatation by differentiating into multiple cell types.

PubMed

Hosoyama, Katsuhiro; Wakao, Shohei; Kushida, Yoshihiro; Ogura, Fumitaka; Maeda, Kay; Adachi, Osamu; Kawamoto, Shunsuke; Dezawa, Mari; Saiki, Yoshikatsu

2018-06-01

Aortic aneurysms result from the degradation of multiple components represented by endothelial cells, vascular smooth muscle cells, and elastic fibers. Cells that can replenish these components are desirable for cell-based therapy. Intravenously injected multilineage-differentiating stress-enduring (Muse) cells, endogenous nontumorigenic pluripotent-like stem cells, reportedly integrate into the damaged site and repair the tissue through spontaneous differentiation into tissue-compatible cells. We evaluated the therapeutic efficacy of Muse cells in a murine aortic aneurysm model. Human bone marrow Muse cells, isolated as stage-specific embryonic antigen-3 + from bone marrow mesenchymal stem cells, or non-Muse cells (stage-specific embryonic antigen-3 - cells in mesenchymal stem cells), bone marrow mesenchymal stem cells, or vehicle was intravenously injected at day 0, day 7, and 2 weeks (20,000 cells/injection) after inducing aortic aneurysms by periaortic incubation of CaCl 2 and elastase in severe combined immunodeficient mice. At 8 weeks, infusion of human Muse cells attenuated aneurysm dilation, and the aneurysmal size in the Muse group corresponded to approximately 62.5%, 55.6%, and 45.6% in the non-Muse, mesenchymal stem cell, and vehicle groups, respectively. Multiphoton laser confocal microscopy revealed that infused Muse cells migrated into aneurysmal tissue from the adventitial side and penetrated toward the luminal side. Histologic analysis demonstrated robust preservation of elastic fibers and spontaneous differentiation into endothelial cells and vascular smooth muscle cells. After intravenous injection, Muse cells homed and expanded to the aneurysm from the adventitial side. Subsequently, Muse cells differentiated spontaneously into vascular smooth muscle cells and endothelial cells, and elastic fibers were preserved. These Muse cell features together led to substantial attenuation of aneurysmal dilation. Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood–brain barrier model for drug permeability studies

PubMed Central

2013-01-01

Background Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Methods Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. Results The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Conclusions Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products. PMID:24262108

Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.

PubMed

Eigenmann, Daniela E; Xue, Gongda; Kim, Kwang S; Moses, Ashlee V; Hamburger, Matthias; Oufir, Mouhssin

2013-11-22

Reliable human in vitro blood-brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time.Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products.

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.

CXCL4L1 inhibits angiogenesis and induces undirected endothelial cell migration without affecting endothelial cell proliferation and monocyte recruitment.

PubMed

Sarabi, A; Kramp, B K; Drechsler, M; Hackeng, T M; Soehnlein, O; Weber, C; Koenen, R R; Von Hundelshausen, P

2011-01-01

The non-allelic variant of CXCL4/PF4, CXCL4L1/PF4alt, differs from CXCL4 in three amino acids of the C-terminal α-helix and has been characterized as a potent anti-angiogenic regulator. Although CXCL4 structurally belongs to the chemokine family, it does not behave like a 'classical' chemokine, lacking significant chemotactic properties. Specific hallmarks are its angiostatic, anti-proliferative activities, and proinflammatory functions, which can be conferred by heteromer-formation with CCL5/RANTES enhancing monocyte recruitment. Here we show that tube formation of endothelial cells was inhibited by CXCL4L1 and CXCL4, while only CXCL4L1 triggered chemokinesis of endothelial cells. The chemotactic response towards VEGF and bFGF was attenuated by both variants and CXCL4L1-induced chemokinesis was blocked by bFGF or VEGF. Endothelial cell proliferation was inhibited by CXCL4 (IC(50) 6.9 μg mL(-1)) but not by CXCL4L1, while both chemokines bound directly to VEGF and bFGF. Moreover, CXCL4 enhanced CCL5-induced monocyte arrest in flow adhesion experiments and monocyte recruitment into the mouse peritoneal cavity in vivo, whereas CXCL4L1 had no effect. CXCL4L1 revealed lower affinity to CCL5 than CXCL4, as quantified by isothermal fluorescence titration. As evidenced by the reduction of the activated partial thromboplastin time, CXCL4L1 showed a tendency towards less heparin-neutralizing activity than CXCL4 (IC(50) 2.45 vs 0.98 μg mL(-1)).  CXCL4L1 may act angiostatically by causing random endothelial cell locomotion, disturbing directed migration towards angiogenic chemokines, serving as a homeostatic chemokine with a moderate structural distinction yet different functional profile from CXCL4. © 2010 International Society on Thrombosis and Haemostasis.

Development and characterization of monoclonal antibodies specific for chicken IL-8

USDA-ARS?s Scientific Manuscript database

Interleukin-8/CXCL8 (IL-8) is a CXC-family chemokine produced by fibroblasts and other cell types, including epithelial cells, endothelial cells, neutrophils, and macrophages. Given that IL-8 attracts lymphocytes to the sites of tissue damage, IL-8 plays a role in the inflammatory response and woun...

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.

Synthetic PAMAM-RGD conjugates target and bind to odontoblast-like MDPC 23 cells and the predentin in tooth organ cultures.

PubMed

Hill, Elliott; Shukla, Rameshwer; Park, Steve S; Baker, James R

2007-01-01

Screening techniques now allow for the identification of small peptides that bind specifically to molecules like cells. However, despite the enthusiasm for this approach, single peptides often lack the binding affinity to target in vivo and regulate cell function. We took peptides containing the Arg-Gly Asp(RGD) motif that bind to the alpha Vbeta 3 integrin and have shown potential as therapeutics. To improve their binding affinity, we synthesized polyamidoamine (PAMAM) dendrimer-RGD conjugates that that contain 12-13 copies of the peptide. When cultured with human dermal microvessel endothelial cells (HDMEC), human vascular endothelial cells (HUVEC), or odontoblast-like MDPC-23 cells, the PAMAM dendrimer conjugate targets this receptor in a manner that is both time- and dose-dependent. Finally, this conjugate selectively targets RGD binding sites in the predentin of human tooth organ cultures. Taken together, these studies provide proof of principle that synthetic PAMAM-RGD conjugates could prove useful as carriers for the tissue-specific delivery of integrin-targeted therapeutics or imaging agents and could be used to engineer tissue regeneration.

Plasmin-Cleaved β-2-Glycoprotein 1 Is an Inhibitor of Angiogenesis

PubMed Central

Sakai, Taro; Balasubramanian, Krishnakumar; Maiti, Sourindra; Halder, Jyotsna B.; Schroit, Alan J.

2007-01-01

β-2-Glycoprotein 1, an abundant plasma glycoprotein, binds anionic cell surfaces and functions as a regulator of thrombosis. Here, we show that cleavage of the kringle domain at Lys317/Thr318 switches its function to a regulator of angiogenesis. In vitro, the cleaved protein specifically inhibited the proliferation and migration of endothelial cells. The protein was without effect on preformed endothelial cell tubes. In vivo, the cleaved protein inhibited neovascularization into subcutaneously implanted Matrigel and Gelfoam sponge implants and the growth of orthotopically injected tumors. Collectively, these data indicate that plasmin-cleaved β-2-glycoprotein 1 is a potent antiangiogenic and antitumor molecule of potential therapeutic significance. PMID:17872974

Protein kinase C-α and arginase I mediate pneumolysin-induced pulmonary endothelial hyperpermeability.

PubMed

Lucas, Rudolf; Yang, Guang; Gorshkov, Boris A; Zemskov, Evgeny A; Sridhar, Supriya; Umapathy, Nagavedi S; Jezierska-Drutel, Agnieszka; Alieva, Irina B; Leustik, Martin; Hossain, Hamid; Fischer, Bernhard; Catravas, John D; Verin, Alexander D; Pittet, Jean-François; Caldwell, Ruth B; Mitchell, Timothy J; Cederbaum, Stephen D; Fulton, David J; Matthay, Michael A; Caldwell, Robert W; Romero, Maritza J; Chakraborty, Trinad

2012-10-01

Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-α activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-α inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-α activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI)(+/-)/arginase II (AII)(-/-) C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI(+/+)/AII(-/-) counterparts, indicating an important role for arginase I in PLY-induced endothelial hyperpermeability. These results identify PKC-α and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.

Protein Kinase C-α and Arginase I Mediate Pneumolysin-Induced Pulmonary Endothelial Hyperpermeability

PubMed Central

Yang, Guang; Gorshkov, Boris A.; Zemskov, Evgeny A.; Sridhar, Supriya; Umapathy, Nagavedi S.; Jezierska-Drutel, Agnieszka; Alieva, Irina B.; Leustik, Martin; Hossain, Hamid; Fischer, Bernhard; Catravas, John D.; Verin, Alexander D.; Pittet, Jean-François; Caldwell, Ruth B.; Mitchell, Timothy J.; Cederbaum, Stephen D.; Fulton, David J.; Matthay, Michael A.; Caldwell, Robert W.; Romero, Maritza J.; Chakraborty, Trinad

2012-01-01

Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-α activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-α inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-α activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI)+/−/arginase II (AII)−/− C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI+/+/AII−/− counterparts, indicating an important role for arginase I in PLY-induced endothelial hyperpermeability. These results identify PKC-α and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction. PMID:22582175

FOXO3 Modulates Endothelial Gene Expression and Function by Classical and Alternative Mechanisms*

PubMed Central

Czymai, Tobias; Viemann, Dorothee; Sticht, Carsten; Molema, Grietje; Goebeler, Matthias; Schmidt, Marc

2010-01-01

FOXO transcription factors represent targets of the phosphatidylinositol 3-kinase/protein kinase B survival pathway controlling important biological processes, such as cell cycle progression, apoptosis, vascular remodeling, stress responses, and metabolism. Recent studies suggested the existence of alternative mechanisms of FOXO-dependent gene expression beyond classical binding to a FOXO-responsive DNA-binding element (FRE). Here we analyzed the relative contribution of those mechanisms to vascular function by comparing the transcriptional and cellular responses to conditional activation of FOXO3 and a corresponding FRE-binding mutant in human primary endothelial cells. We demonstrate that FOXO3 controls expression of vascular remodeling genes in an FRE-dependent manner. In contrast, FOXO3-induced cell cycle arrest and apoptosis occurs independently of FRE binding, albeit FRE-dependent gene expression augments the proapoptotic response. These findings are supported by bioinformatical analysis, which revealed a statistical overrepresentation of cell cycle regulators and apoptosis-related genes in the group of co-regulated genes. Molecular analysis of FOXO3-induced endothelial apoptosis excluded modulators of the extrinsic death receptor pathway and demonstrated important roles for the BCL-2 family members BIM and NOXA in this process. Although NOXA essentially contributed to FRE-dependent apoptosis, BIM was effectively induced in the absence of FRE-binding, and small interfering RNA-mediated BIM depletion could rescue apoptosis induced by both FOXO3 mutants. These data suggest BIM as a critical cell type-specific mediator of FOXO3-induced endothelial apoptosis, whereas NOXA functions as an amplifying factor. Our study provides the first comprehensive analysis of alternatively regulated FOXO3 targets in relevant primary cells and underscores the importance of such genes for endothelial function and integrity. PMID:20123982

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

Simvastatin and Benznidazole-Mediated Prevention of Trypanosoma cruzi-Induced Endothelial Activation: Role of 15-epi-lipoxin A4 in the Action of Simvastatin

PubMed Central

Campos-Estrada, Carolina; Liempi, Ana; González-Herrera, Fabiola; Lapier, Michel; Kemmerling, Ulrike; Pesce, Barbara; Ferreira, Jorge; López-Muñoz, Rodrigo; Maya, Juan D.

2015-01-01

Trypanosoma cruzi is the causal agent of Chagas Disease that is endemic in Latin American, afflicting more than ten million people approximately. This disease has two phases, acute and chronic. The acute phase is often asymptomatic, but with time it progresses to the chronic phase, affecting the heart and gastrointestinal tract and can be lethal. Chronic Chagas cardiomyopathy involves an inflammatory vasculopathy. Endothelial activation during Chagas disease entails the expression of cell adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) through a mechanism involving NF-κB activation. Currently, specific trypanocidal therapy remains on benznidazole, although new triazole derivatives are promising. A novel strategy is proposed that aims at some pathophysiological processes to facilitate current antiparasitic therapy, decreasing treatment length or doses and slowing disease progress. Simvastatin has anti-inflammatory actions, including improvement of endothelial function, by inducing a novel pro-resolving lipid, the 5-lypoxygenase derivative 15-epi-lipoxin A4 (15-epi-LXA4), which belongs to aspirin-triggered lipoxins. Herein, we propose modifying endothelial activation with simvastatin or benznidazole and evaluate the pathways involved, including induction of 15-epi-LXA4. The effect of 5 μM simvastatin or 20 μM benznidazole upon endothelial activation was assessed in EA.hy926 or HUVEC cells, by E-selectin, ICAM-1 and VCAM-1 expression. 15-epi-LXA4 production and the relationship of both drugs with the NFκB pathway, as measured by IKK-IKB phosphorylation and nuclear migration of p65 protein was also assayed. Both drugs were administered to cell cultures 16 hours before the infection with T. cruzi parasites. Indeed, 5 μM simvastatin as well as 20 μM benznidazole prevented the increase in E-selectin, ICAM-1 and VCAM-1 expression in T. cruzi-infected endothelial cells by decreasing the NF-κB pathway. In conclusion, Simvastatin and benznidazole prevent endothelial activation induced by T. cruzi infection, and the effect of simvastatin is mediated by the inhibition of the NFκB pathway by inducing 15-epi-LXA4 production. PMID:25978361