Umbilical Vein Endothelial Cells Research Articles

Umbilical Cord Stem Cells Induce Glial Fate in Neural Progenitor Cells

s / Placenta 35 (2014) A1eA112 A93 Objectives: The Wharton’s jelly (WJ) of the umbilical cord is derived from the mesoderm of the primitive streak which invades the connecting stalk and primary chorionic villi. Mesenchymal stem cells in the mesoderm differentiate and participate in neo-vasculogenesis of the umbilical and placental circulation. Whether these cells have a continued role in placental/umbilical vascular growth and maturity is not known. Fetal stem cells have been shown to traffic and repair injured myocardial endothelium in mice (Kara et al. 2012). Their ability to interact with the uterine endothelium has not been investigated. The aim of this study was to investigate whether umbilical stem cells (WJMSC) can interact and influence umbilical vein and uterine endothelial monolayers. Methods: Human umbilical vein (HUVEC) and uterine microvascular endothelial cells (HUtMEC) were grown to 70% confluence in endothelial media and to full confluence in mixed media (endothelial: stem cell media). Isolated WJMSC, labelled with the fluorescent tracker, PKH26 was added on top. Cell-cell interactions were monitored with fluorescence time-lapse microscopy. Key events were analysed by confocal microscopy after vascular endothelial (VE) cadherin immunocytochemistry. Results: WJMSC displayed non-apoptotic blebbing, amoeboid movement and shedding of exosomes as early events. There was a time lag of 60 min before transmigration of WJMSC via the paracellular pathway to subendothelial positions. This was concomitant with disruption of VE-cadherin (statistically significant reduction of continuous junctional profiles). By 16h, a majority of WJMSC were resident underneath the endothelial monolayers; junctional profiles returned to normal values. At 22h, the cocultures showed a higher % of paracellular clefts with continuous VEcadherin labelling, indicative of increased junctional maturity. Conclusion: Our data suggests that umbilical mesenchymal stem cells can interact with and influence junctional organisation of both fetal and uterine endothelial cells. Their role, if any, in uterine vascular re-modelling during pregnancy requires investigation. P2.102-N. UMBILICAL CORD STEM CELLS INDUCE GLIAL FATE IN NEURAL PROGENITOR CELLS Byron Oppliger , Marianne J€ orger-Messerli , Camilla Marini , Ursula Reinhart , Daniel V. Surbek , Andreina Schoeberlein a Department of Obstetrics and Gynecology, University Hospital, Department of Clinical Research, University of Bern, Bern, Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences (GCB), University of Bern, Bern, Bern,

Proteomics reveals potential non-neuronal cholinergic receptor-effectors in endothelial cells.

The non-neuronal acetylcholine system (NNAS) in endothelial cells participates in modulating endothelial function, vascular tone, angiogenesis and inflammation, thus plays a critical role in cardiovascular diseases. In this study, we used a proteomic approach to study potential downstream receptor-effectors of NNAS that were involved in regulating cellular function in endothelial cells. Human umbilical vein endothelial cells were incubated in the presence of acetylcholine, oxotremorine, pilocarpine or nicotine at the concentration of 10 μmol/L for 12 h, and the expressed proteins in the cells were separated and identified with two-dimensional electrophoresis (2-DE) and LC-MS. The protein spots with the largest changes were identified by LC-MS. Biowork software was used for database search of the peptide mass fingerprints. Over 1200 polypeptides were reproducibly detected in 2-DE with a pH range of 3-10. Acetylcholine, oxotremorine, pilocarpine and nicotine treatment caused 16, 9, 8 and 9 protein spots, respectively, expressed differentially. Four protein spots were identified as destrin, FK506 binding protein 1A (FKBP1A), macrophage migration inhibitory factor (MIF) and profilin-1. Western blotting analyses showed that treatment of the cells with cholinergic agonists significantly decreased the expression of destrin, FKBP1A and MIF, and increased the expression of profilin-1. A set of proteins differentially expressed in endothelial cells in response to cholinergic agonists may have important implications for the downstream biological effects of NNAS.

A microgroove patterned multiwell cell culture plate for high-throughput studies of cell alignment.

Grooved substrates are commonly used to guide cell alignment and produce in vitro tissues that mimic certain aspects of in vivo cellular organization. These more sophisticated tissues provide valuable in vitro models for testing drugs and for dissecting out molecular mechanisms that direct tissue organization. To increase the accessibility of these tissue models we describe a simple and yet reproducible strategy to produce 1 µm-spaced grooved well plates suitable for conducting automated analysis of cellular responses. We characterize the alignment of four human cell types: retinal epithelial cells, umbilical vein endothelial cells, foreskin fibroblasts, and human pluripotent stem-cell-derived cardiac cells on grooves. We find all cells align along the grooves to differing extents at both sparse and confluent densities. To increase the sophistication of in vitro tissue organization possible, we also created hybrid substrates with controlled patterns of microgrooved and flat regions that can be identified in real-time using optical microscopy. Using our hybrid patterned surfaces we explore: (i) the ability of neighboring cells to provide a template to organize surrounding cells that are not directly exposed to grooved topographic cues, and (ii) the distance over which this template effect can operate in confluent cell sheets. We find that in fibroblast sheets, but not epithelial sheets, cells aligned on grooves can direct alignment of neighboring cells in flat regions over a limited distance of approximately 200 μm. Our hybrid surface plate provides a novel tool for studying the collective response of groups of cells exposed to differential topographical cues.

The Peptide-binding Cavity Is Essential for Als3-mediated Adhesion of Candida albicans to Human Cells

The adhesive phenotype of Candida albicans contributes to its ability to colonize the host and cause disease. Als proteins are one of the most widely studied C. albicans virulence attributes; deletion of ALS3 produces the greatest reduction in adhesive function. Although adhesive activity is thought to reside within the N-terminal domain of Als proteins (NT-Als), the molecular mechanism of adhesion remains unclear. We designed mutations in NT-Als3 that test the contribution of the peptide-binding cavity (PBC) to C. albicans adhesion and assessed the adhesive properties of other NT-Als3 features in the absence of a functional PBC. Structural analysis of purified loss-of-PBC-function mutant proteins showed that the mutations did not alter the overall structure or surface properties of NT-Als3. The mutations were incorporated into full-length ALS3 and integrated into the ALS3 locus of a deletion mutant, under control of the native ALS3 promoter. The PBC mutant phenotype was evaluated in assays using monolayers of human pharyngeal epithelial and umbilical vein endothelial cells, and freshly collected human buccal epithelial cells in suspension. Loss of PBC function resulted in an adhesion phenotype that was indistinguishable from the Δals3/Δals3 strain. The adhesive contribution of the Als3 amyloid-forming-region (AFR) was also tested using these methods. C. albicans strains producing cell surface Als3 in which the amyloidogenic potential was destroyed showed little contribution of the AFR to adhesion, instead suggesting an aggregative function for the AFR. Collectively, these results demonstrate the essential and principal role of the PBC in Als3 adhesion.

74 Pro-Angiogenic Effects of the Prolyl-hydroxylase Inhibitor FG-2216/BIQ: Therapeutic Angiogenesis for the Treatment of Chronic Total Occlusions

Percutaneous coronary intervention (PCI) is now the dominant revascularisation therapy for coronary artery disease, with nearly 3 million PCIs undertaken per annum world-wide. One of the last remaining challenges to...

Influence of HMG-CoA Reductase Inhibitors on Leptin-Induced Endothelial Cell Proliferation, Migration, and Capillary-Like Tube Formation

Objective: This study investigated the impact of the hepatic hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on the leptin-induced human umbilical vein endothelial (HUVE) cell proliferation, migration, and capillary-like tube formation. Methods: The HUVE cells were isolated and cultured, and stimulated with leptin, statins (cerivastatin, fluvastatin, simvastatin), mevalonate, farnesyl pyrophosphate, geranylgeranyl pyrophosphate, or methyl-β-cyclodextrin. The endothelial cell proliferation was assessed using the Neubauer counting chamber. The migration of HUVE cells was examined with the planar migration assay. In vitro capillary sprouting was quantified by measuring the sprout length, number, and cumulative sprout length. Results: The HMG-CoA reductase inhibitors significantly reduced leptin-induced proliferation and migration, which was reversed by mevalonate. Further, the inhibitory effect of the statins on leptin-induced migration was shown to be modulated by the prenylation of farnesyl pyrophosphate and geranylgeranyl pyrophosphate. Although stimulation with a leptin showed no significant effect, a marked increase in capillary-like tube formation was observed with a joint stimulation with HMG-CoA reductase inhibitors. Although statins caused inhibition of proliferation and migration, the same dose of the agents amplified the selective growth of capillary-like tube formation. Membranous cholesterol depletion by methyl-β-cyclodextrin showed a weaker effect compared with statins. Conclusion: Through modulation of prenylation, leptin-induced pro-atherosclerotic events including proliferation and migration were inhibited by HMG-CoA reductase inhibitors.

Arecoline is cytotoxic for human endothelial cells

Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4h with arecoline at 333μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis.

20-Hydroxyeicosatetraenoic acid impairs endothelial insulin signaling by inducing phosphorylation of the insulin receptor substrate-1 at Ser616.

20-hydroxyeicosatetraenoic acid (20-HETE) induces endothelial dysfunction and is correlated with diabetes. This study was designed to investigate the effects of 20-HETE on endothelial insulin signaling.Human umbilical vein endothelial cells (HUVECs) or C57BL/6J mice were treated with 20-HETE in the presence or absence of insulin, and p-ERK1/2, p-JNK, IRS-1/PI3K/AKT/eNOS pathway, were examined in endothelial cells and aortas by immunoblotting. eNOS activity and nitric oxide production were measured. 20-HETE increased ERK1/2 phosphorylation and IRS-1 phosphorylation at Ser616; these effects were reversed by ERK1/2 inhibition. We further observed that 20-HETE treatment resulted in impaired insulin-stimulated IRS-1 phosphorylation at Tyr632 and subsequent PI3-kinase/Akt activation. Furthermore, 20-HETE treatment blocked insulin-stimulated phosphorylation of eNOS at the stimulatory Ser1177 site, eNOS activation and NO production; these effects were reversed by inhibiting ERK1/2. Treatment of C57BL/6J mice with 20-HETE resulted in ERK1/2 activation and impaired insulin-dependent activation of the IRS-1/PI3K/Akt/eNOS pathway in the aorta. Our data suggest that the 20-HETE activation of IRS-1 phosphorylation at Ser616 is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway.

Human cytomegalovirus infection induces leukotriene B4 and 5-lipoxygenase expression in human placentae and umbilical vein endothelial cells

IntroductionHuman cytomegalovirus (HCMV) can cause congenital infection with risk of neurological disability. Maternal-fetal transmission is associated with placental inflammation. 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of Leukotrienes (LTs), which are proinflammatory mediators. This study investigated the effect of HCMV infection on 5-LO expression and Leukotriene-B4 (LTB4) induction in human placentae and umbilical vein endothelial cells (HUVEC). MethodsSeven placentae from fetuses with congenital HCMV infection and brain damage and six controls were stained with HCMV-immediate-early-antigen (HCMV-IEA) and 5-LO by immunohistochemistry. 5-hydroxyeicosatetraenoic acid (5-HETE) and LTB4 were measured in culture supernatant from ex vivo HCMV-infected placental histocultures by liquid chromatography. In vitro, HCMV infected HUVEC cells were analyzed for 5-LO mRNA and protein expression by real time PCR and immunofluorescence staining. ResultsHCMV-IEA was abundant in all HCMV infected placentae but absent in control placentae. 5-LO expression was higher in endothelial and smooth muscle cells of HCMV-infected placentae, compared to control placentae.HCMV infection induced an up-regulation of LTB4 in ex vivo placental explants with higher levels of LTB4 at 72 h compared to controls (p = 0.002). In vitro, 5-LO transcript and protein expression were significantly induced in HCMV-infected HUVEC, compared to the control cultures (p = 0.036). ConclusionThe presence of HCMV coincided with high 5-LO expression in cells of in vivo HCMV infected placentae. HCMV induced up-regulation of 5-LO in both ex vivo HCMV-infected placental explants and HUVEC. HCMV induced LT-biosynthesis in congenitally infected placentae may have a role in pathogenesis of congenital HCMV disease.

The impact of Interleukin-1β on the structure of human umbilical vein endothelial cells and aquaporin-1 expression

Objective To explore the role of IL-1β in capillary leak syndrome by observing the alterations of AQP-1 expression,apoptosis,and ultrastructural of vascular endothelial cells under the action of IL-1β.Methods Umbilical vein endothelial cells (UVEC) in vitro were randomly allocated into 3 groups:time,concentration,and control.In the time group,UVECs were treated with culture medium containing 20 μg/L IL-1β for3 h(T1),8 h(T2),12 h(T3) and 24 h(T4).In the concentration group,UVECs were treated with culture medium containing 0.2 μg/L(C1),2 μg/L(C2) and 20 μg/L(C3) IL-1β for 24 h.In the control group,UVECs were treated with culture medium without IL-1β for 24 h.The changes of AQP-1 mRNA and protein expression were detected by real-time PCR and Western blot.Apoptosis was detected by flow cytometry,and cell ultrastructural changes were observed by electron microscopy.Results AQP-1 mRNA and protein expression of T1-T4 in the time group and C1-C3 in the concentration group were lower than those of the control group (P ＜ 0.05).The apoptotic rate was increased,and mitochondrial swelling,vacuolar degeneration,karyolysis and necrosis were observed under electron microscopy.These were more pronounced with time or concentration increases.Conclusions IL-1β can cause a decrease of AQP-1 mRNA and protein expression,increase in apoptotic rate and increase in damage to the cells'ultrastructure.This is an important reason for damage to the vascular endothelial barrier and may be associated with capillary leak syndrome. Key words: Capillary leak syndrome; Vascular endothelial barrier; Interleukin-1β ; Aquaporin-1 ; Apoptosis

TNF-α induces CXCL1 chemokine expression and release in human vascular endothelial cells in vitro via two distinct signaling pathways

Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-α has been shown to be upregulated in atherosclerotic lesions and various cancers. The aim of this study was to investigate the mechanisms underlying the TNF-α-induced release of CXCL1 from human vascular endothelial cells in vitro. Human umbilical vein endothelial cells (HUVECs) were treated with different proinflam-matory mediators and growth factors. CXCL1 expression and secretion were determined using RT-PCR and ELISA, respectively. TNF-α-induced cell signaling was assayed with Western blotting. Cell viability/growth was determined using MTT assay. Monocyte migration was measured with transwell migration assay. Among the 17 mediators and growth factors tested, TNF-α, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-α (2, 5 ng/mL) induced CXCL1 release and mRNA expression in the cells in concentration- and time-dependent manners. TNF-α (5 ng/mL) caused activation of JNK, p38 MAPK, PI3K and Akt, whereas pretreatment with JNK inhibitor (SP600125), p38 MAPK inhibitor (SB202190) or PI-3K inhibitor (LY294002) significantly suppressed TNF-α-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-α-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone (up to 500 nmol/L) failed to affect TNF-α-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-α-induced CXCL1 from HUVECs attracted human monocyte migration. TNF-α stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.

Electrospun Gelatin Constructs with Tunable Fiber Orientation Promote Directed Angiogenesis

The field of therapeutic angiogenesis has been predominantly concentrated in modalities that incorporate pro-angiogenic growth factors and/or cells within polymeric constructs that are implanted into the ischemic region. There is growing evidence that construct architecture can significantly affect growth factor activity, cellular viability and differentiation potential. Electrospinning is an attractive but simple scaffold fabrication technique that offers several advantages over traditional fabrication approaches to prepare highly organized structures for therapeutic angiogenesis applications. We recently described the fabrication of nanofibrous scaffolds with aligned fiber orientation that directed cell migration and orientation (i.e.human umbilical vein endothelial cells). Herein we demonstrate the ability of bFGF containing nanofibrous gelatin B scaffolds with controlled fiber orientation to promote capillary formation in vivo. Aligned scaffolds loaded with bFGF induced the highest levels of reperfusion (73% increased in LDPI ratios by day 21 post ischemia induction) in comparison to all other groups including scaffolds with random fiber orientation. Furthermore, the newly formed vasculature, assessed by confocal microscopy, had a parallel alignment along the axis of the scaffold’s fibers. In contrast, no vessel directionality was observed in the animals treated with scaffolds with random fiber orientation in the presence or absence of bFGF.

Effect of HIF-1α on proliferation and apoptosis of human umbilical vein endothelial ECV304 cells exposed to hypoxia

Effect of HIF-1α on proliferation and apoptosis of human umbilical vein endothelial ECV304 cells exposed to hypoxia

Inhibition Of the Rho/Rho-Kinase Pathway Reduces In Vitro Adhesion Of Sickle Cell Anemia Eosinophils To Human Endothelial Cells

IntroductionSickle cell anemia (SCA) individuals present a chronic inflammatory condition that contributes to the recurrent episodes of vaso-occlusion. This process is a multi-step process that involves different cell types such as red blood cells, reticulocytes, activated endothelial cells, platelets and leukocytes. Endothelial dysfunction contributes to the vaso-occlusion process and leads to inflammation. Leukocytes may have a propagating and, possibly, initiating role in SCA vaso-occlusion. In vivo and in vitro studies using animal models show that neutrophils play a key role in vaso-occlusion; however, eosinophils (EOs) may also participate in this phenomenon in humans. We have previously shown that SCA eosinophils demonstrating greater adhesion to fibronectin. A small GTPase, Rho, and its target molecule, Rho-kinase, play an important role in the cell functions, including contractility, chemotaxis, adhesion, and migration. Y-27632 and NSC23766 are Rho-kinase inhibitors, that reduce leukocyte infiltration in several models of EOs inflammation, such as endotoxic liver and lung injury. The objective of this study was to investigate the effect of Rho kinase pathway on the in vitro adhesion of sickle EOs to activated human endothelial cell layers (umbilical vein endothelial cells, HUVEC). MethodsEosinophils were isolated from peripheral blood of healthy controls (n=5), steady-state SCA patients (SCA, n=7) and SCA patients on HU therapy (SCAHU, n=5), using Percoll gradient separation, followed by immunomagnetic sorting. EO adhesion (1x106 cell/ml in Ham's F12 K) to cultured HUVEC grown to confluence was assessed using static adhesion assays. HUVEC cells were treated, or not, with 100 µM NSC23766 or Y-27632 for 1h before incubation with an inflammatory stimulus (10nM TNF-alpha – 3 h), subsequently Eos were added to the cell layer (1 hour, 37°C, 5%CO2). For adhesion under flow conditions, biochips with microchannels coated with HUVEC were pre treated or not with Y-27632 or NSC23766 per 1 h before the TNF-alphastimulus. Purified eosinophils was added to HUVEC-coated microchannels at a flow rate of 0.5 dynes/cm2. Images of adherent eosinophils were recorded using real-time video microscopy and analysed using Duco Cell software. ResultsEOs from SCA patients demonstrated a significantly greater adhesion to HUVEC than healthy controls (14.42 ± 1.8% vs 9.35 ± 1.7 %; p<0.02); no significant difference was observed between the SCAHU group, compared to the healthy control and SCA group (10.5 ± 1.6%). Pre treatment of HUVEC with TNF-alpha significantly augmented the adhesion of healthy control, SCA, and SCAHU eosinophils to HUVEC (16.1 ± 3.2%, 33.1 ± 5.1%, 17.9 ± 3.5%, P<0.01, respectively). However, this adhesion was significantly higher for SCA EOs compared to healthy control and SCAHU EOs (p<0.05). Interestingly, when the HUVEC was pre-treated with NSC23766 then stimulated with TNF-alpha, adhesion of EOs from SCA and SCAHU was significantly reduced (22.9 ± 5,6%, 17.2 ± 6.1%, respectively, p<0.05). However, when HUVEC was pre-treated with Y-27632 no significant difference was found. In addition, EOs from SCA patients demonstrated greater adhesion to HUVEC under flow conditions when compared to healthy control and SCAHU. EOs when the HUVEC was protect with Rho inhibitors (Y-27632 and NSC23766) before stimulation with TNF-alpha, EOs adhesion under physiological shear stress was diminished in all groups investigated (healthy control, SCA and SCAHU). ConclusionRho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion, in an in vitro model. Therapy with such Rho-kinase inhibitors could be used in patients with SCA, with beneficial effects on the vascular occlusion process.Financial Support: FAPESP/CNPq/INCTS Disclosures:No relevant conflicts of interest to declare.

Identification and function analysis of a novel transcript 630 in acute T-cell lymphoblastic leukemia Jurkat cell lines

Objective To identify and analyze a novel transcript 630 in acute T-cell lymphoblastic leukemia cell (T-ALL) Jurkat cell lines. Methods RNA-sequencing was applied to transcriptome profiling. The novel transcript 630 was identified by reverse transcription polymerase chain reaction (RT-PCR) combined with sequencing. The expression level of the novel transcript was detected in some tumor cell lines and normal cells. The sequences for small interfering RNA (siRNA) against 630 were designed and synthesized. Then, the effects of siRNA were determined by real-time PCR. Apoptotic cells and cell cycle analysis were investigated with the flow cytometry after siRNA-mediated knockdown of 630. Results The novel transcript 630 was identified. High expression level of 630 was detected in Jurkat E6-1, HL60 and human umbilical vein endothelial(HUVE) cells. The knockdown of transcript 630 induced a decrease of cellular proliferation. Conclusions The novel transcript 630 is one of the novel transcripts in Jurkat cells. The decreased expression level of transcript 630 can suppress the proliferation of leukemia cells. The novel transcript 630 might contribute to the treatment of T-ALL. Key words: Leukemia; Jurkat cell; Transcriptome

Pericyte Regulation of Vascular Remodeling Through the CXC Receptor 3

To understand the role, if any, played by pericytes in the regulation of newly formed vessels during angiogenesis. In this study, we investigate whether pericytes regulate the number of nascent endothelial tubes. Using an in vitro angiogenesis assay (Matrigel assay), we demonstrate that pericytes can inhibit vessel formation and induce vessel dissociation via CXCR3-induced involution of the endothelial cells. In a coculture Matrigel assay for cord formation, pericytes prevented endothelial cord formation of human dermal microvascular endothelial cells but not umbilical vein endothelial cells. Blockade of endothelial CXCR3 function or expression inhibited the repressing effect of the pericytes. We further show that pericytes are also able to induce regression of newly formed microvascular cords through CXCR3 activation of calpain. When CXCR3 function was inhibited by a neutralizing antibody or downregulated by siRNA, cord regression mediated by pericytes was abolished. We show for the first time that pericytes regulate angiogenic vessel formation, and that this is mediated through CXCR3 expressed on endothelial cells. This suggests a role for pericytes in the pruning of immature vessels overproduced during wound repair.

Exposing endothelial cells to tumor necrosis factor-α and peripheral blood mononuclear cells damage endothelial integrity via interleukin-1ß by degradation of vascular endothelial-cadherin

We demonstrated previously that the administration of tumor necrosis factor alpha (TNF-α) for the treatment of solid tumors enhanced the response to chemotherapy by augmenting intratumoral drug accumulation. TNF-α changes the integrity of the endothelial cell monolayer in combination with interferon gamma (IFN-γ), which is further enhanced by the addition of peripheral blood mononuclear cells (PBMCs). The improved effect of PBMCs was mostly induced by the endogenous production of interleukin-1beta (IL-1ß) after TNF-α stimulation. In the current study, we demonstrate that exposing endothelial cells to TNF-α and PBMCs mediates the loss of vascular endothelial (VE)-cadherin, an important adherens junction protein for maintaining endothelial integrity, through endogenous IL-1ß. This loss increases permeability of the endothelial layer, thereby explaining the augmented passage of chemotherapeutics into the tumor. Human umbilical vein endothelial cells were exposed to TNF-α, IFN-γ, PBMCs, or IL-1ß, and the effects on the endothelial integrity were assessed by morphological changes and permeability changes with the use of fluorescein isothiocyanate-labeled bovine serum albumin flux. The loss of VE-cadherin was assessed using immunofluorescence, western blotting, and polymerase chain reaction. Incubating endothelial cells with TNF-α, IFN-γ, and PBMCs increased cell elongation, gap formation, and subsequently the permeability of fluorescein isothiocyanate-labeled bovine serum albumin compared with control or TNF-α and IFN-γ-treated cells (P < .05). When PBMCs were replaced with IL-1ß, identical changes were observed. These changes in integrity were associated with a loss of VE-cadherin at the membrane. We conclude that VE-cadherin is lost at the membrane when endothelial cells are exposed to TNF-α, IFN-γ, and PBMCs, which results in loss of integrity. IL-1ß can mimic the effects of PBMCs, indicating a dominant role of endogenously produced IL-1ß in this process.

Periodontal Ligament Stem Cells Possess the Characteristics of Pericytes

Periodontal ligament (PDL) contributes to maintaining homeostasis in periodontal tissues by supplying stem/progenitor cells. It has long been suggested that PDL stem cells/progenitors are located around blood vessels. Recently mesenchymal stem cells (MSCs) have been isolated and cultured from PDL in vitro, although the location of the stem cells in PDL is unclear. The purpose of this study is to test the characteristics of human PDL stem cells (PDLSCs) and examine their similarity to related vascular cell types, such as pericytes and endothelial cells. PDLSCs were obtained from healthy extracted teeth using the collagenase/dispase enzyme digestion method. MSC and pericyte characteristics of PDLSCs were examined by cell surface marker expression using flow cytometry. The expression of pericyte markers was tested using immunohistochemistry. Pericyte-like functions of PDLSCs were examined in co-culture of PDLSCs and umbilical vein endothelial cells on a gel matrix. Cultured PDLSCs were positive for both MSC markers and pericyte markers, including cluster of differentiation 146 (CD146), neural/glial antigen 2 (NG2), and CD140b. When pericyte marker expression was explored in rat periodontal tissue sections, CD146- and NG2-positive signals were observed in the perivascular area of the PDL. Further, when the cells were cultured with human umbilical cord endothelial cells under conditions for forming capillary-like structures in vitro, PDLSCs localized adjacent to endothelial cells and contributed to the stability of the capillary-like structure. PDLSCs possess pericyte-like characteristics and may localize as pericytes in the PDL. These data provide useful information for stem cell biology in periodontal research and stem cell-based periodontal therapy.

Activation of the Unfolded Protein Response in Vascular Endothelial Cells of Nondiabetic Obese Adults

Activation of the unfolded protein response (UPR) is emerging as an important molecular signature of cardiometabolic diseases associated with obesity. However, despite the well-established role of the vascular endothelium in obesity-related cardiometabolic dysfunction, it is unclear whether the UPR is activated in endothelial cells of obese adults. The objective of the study was to determine whether markers of UPR activation are increased in endothelial cells (ECs) of nondiabetic obese adults with impaired endothelial function. Endothelial cells were obtained from antecubital veins of the nondiabetic obese adults [body mass index (BMI) ≥ 30 kg/m(2), n = 12] with impaired endothelial function and from their nonobese peers (BMI < 30 kg/m(2), n = 14). UPR activation via expression (quantitative immunofluorescence) of the proximal UPR sensors, inositol-requiring endoplasmic reticulum (ER)-to-nucleus signaling protein 1 (IRE1), RNA-dependent protein kinase-like ER eukaryotic initiation factor-2α kinase (PERK), and activating transcription factor 6 (ATF6), were the main outcome variables. IRE1 expression was greater in obese vs nonobese individuals (0.84 ± 0.09 vs 0.47 ± 0.02 IRE1 intensity/human umbilical vein EC (HUVEC) intensity (n = 10/8, P < .01). Obese individuals also had greater EC activation of UPR stress sensors PERK and ATF6, indicated by increased expression of phosphorylated PERK [p-PERK; 0.49 ± 0.05 vs 0.36 ± 0.03, p-PERK (threonine 981) intensity/HUVEC intensity, n = 10 men, 13 women, P < .05] and nuclear localization of ATF6 (0.38 ± 0.05 vs 0.23 ± 0.02, nuclear ATF6 intensity/HUVEC intensity, n = 5 men, 9 women, P < .01), respectively. Stepwise linear regression analysis revealed that indices of body fat (BMI and waist circumference) were the strongest independent predictors of all 3 UPR mediators, explaining between 18% and 59% of the variance in endothelial cell expression of IRE1, p-PERK, and nuclear ATF6 localization. These results provide novel evidence for UPR activation in the endothelial cells of nondiabetic obese adults with vascular endothelial dysfunction.

Effects of NO on the expression and function of bradykinin type 2 receptors. Role for angioedema?

Purpose: The pathophysiology of angioedema induced by angiotensin-converting enzyme inhibitors (ACEi) is not well understood. While activation of bradykinin (BK) type 2 receptors (B2) has been established, the triggering events remain obscure. In this study we investigated effect of NO as one product of B2 stimulation. Methods: Brain (bEND3), human umbilical vein (HUVEC) and porcine aortic endothelial cells (PAEC) were incubated with the NO-donors DEA/NO (10μM), DETA/NO (100μM) and SNAP (1-100μM) for 3, 6 and 12 h. B2 expression was assessed by RT-PCR and westernblot using monoclonal B2 antibodies staining either glycosylated or unglycosylated B2. Function of B2 was determined by monitoring iCa2+ upon activation with BK. Likewise, B2 protein levels were evaluated in aorta, heart and lung of (1) two different eNOS-/- strains, (2) mice with endothelial-specific overexpression of eNOS (eNOS-tg), (3) in C57Bl/6 mice treated with NOS inhibitor L-nitroarginine (L-NA). Here, B2 function was evaluated in aortic rings. Results: After 3h incubation with DEA/NO of bEND3, B2-mRNA was unchanged (160±32%, n=6, P>0.05). Likewise, B2 protein levels remained stable (98±13.6%, n=8, P>0.05). Similar results were obtained in PAECs and HUVECs and after extended incubation time and increased concentration. There was also a similar rise of iCa2+ in response to 0.01-100 μM BK in bEND3 treated with DEA/NO or vehicle as demonstrated by almost identical flourescence signals. These results obtained in cell lines and primary cultured PAEC were confirmed using transgenic mice with different levels of vascular NO-bioavailability. In aorta, neither the complete lack of endothelial NO in eNOS-/- (93±26%, n=5, P>0.05), nor overexpression in eNOS-tg (112±13.7%, n=10, P>0.05) changed B2 protein expression and a similar observation was made in C56Bl/6 treated with L-NA (110±19.4%, n=5, P>0.05). Aortic constrictor responses to 10 μM BK (related to maximal constriction to KCl) were identical in C57Bl/6 (24.3±2.7%, n=7) and eNOS-tg (30±11.8%, n=4, P>0.05). However, B2 signaling involved generation of NO as constrictor responses were significantly increased following L-NA in both C57BL/6 (64±18.3%, n=4) and eNOS-tg (67±3.6%, n=4) and a similar constriction was observed in eNOS-/- without (61±10.9%, n=7) and with L-NA (61±14.6%, n=6). None of these responses differed significantly. Conclusion: These data demonstrate that NO does not regulate vascular B2 gene expression and function suggesting that changes of B2 expression by this product of B2 activation are unlikely involved in triggering ACEi-induced angioedema.