Incubation Of Human Umbilical Vein Endothelial Cells Research Articles

Accumulation of Li+ Ions Triggers Changes in FOS, JUN, EGR1, and MYC Transcription in the LiCl-Treated Human Umbilical Vein Endothelial Cells (HUVEC).

Changes in intracellular concentrations of Na+ and K+ are shown to alter gene expression. Another monovalent cation, Li+, is well known as a medicine for treatment of psychiatric disorders, but mechanism of its action is obscure. Thus, it is important to evaluate the effect of Li+ on gene expression in endothelial cells. Here we studied influence of the increased intracellular Na+ or Li+ concentrations on transcription of Na+i/K+i-sensitive genes. Treatment of the human endothelial cells (HUVEC) with LiCl for 1.5h resulted in accumulation of Li+ in the cells. This was followed by increase in the FOS and EGR1 mRNAs levels and decrease in the JUN and MYC mRNA levels. Treatment of HUVEC with the Na+-ionophore monensin led to accumulation of Na+ and loss of K+ ions. However, monensin had no significant effect on gene expression. Incubation of HUVEC with elevated extracellular NaCl concentration increased intracellular K+ concentration and transcription of the ATF3 gene, while transcription of the JUN gene decreased. These results indicate that Na+ and Li+ ions have different effects on the gene expression profile in the cells that is likely associated with the fact that they affect differently the intracellular monovalent cations ratio.

The Role of Ca2+/PI3K/Akt/eNOS/NO Pathway in Astragaloside IV-Induced Inhibition of Endothelial Inflammation Triggered by Angiotensin II.

Inflammation induced by angiotensin II (Ang II) is a key event in the progression of numerous cardiovascular diseases. Astragaloside IV (AS-IV), a glycoside extracted from Astragalus membranaceus Bunge, has been shown to inhibit Ang II-induced inflammatory responses in vivo. However, the mechanisms underlying the beneficial effects are still unclear. This study investigated whether AS-IV attenuates endothelial inflammation induced by Ang II via the activation of endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) pathway. Human umbilical vein endothelial cells (HUVECs) were cultured in the presence of AS-IV with or without the specific inhibitor of NOS or Ca2+- and phosphatidylinositol 3-kinase (PI3K)/Akt-dependent cascade prior to Ang II exposure. Incubation of HUVECs with AS-IV enhanced NO production and eNOSser1177 phosphorylation. These responses were abrogated by the inhibition of NOS or Ca2+- and PI3K/Akt-dependent pathway. In addition, preincubation of HUVECs with AS-IV inhibited Ang II-induced cytokine and chemokine production, adhesion molecule expression, monocyte adhesion, and nuclear factor kappa B (NF-κB) activation as evidenced by the attenuation of inhibitor of kappa B alpha phosphorylation and subsequent NF-κB DNA binding. These effects of AS-IV were abolished by the suppression of NOS or Ca2+- and PI3K/Akt-dependent cascade. Our findings indicate that AS-IV attenuates inflammatory responses triggered by Ang II possibly via the activation of Ca2+/PI3K/Akt/eNOS/NO pathway in endothelial cells.

KLF4 Protects Endothelial Cells against Senescence by Activating PDGF-BB/PDGFRA Pathway

Aims: To explore the role KLF4 plays in regulating senescence of endothelial cells and the specific mechanism of it. Main Methods: Human umbilical vein endothelial cells (HUVECs) were cultured with glucose or angiotensin (Ang) II to induce senescence. Expressions of KLF4, PDGF-BB, PDGFRA and PDGFRB in normal and senescent HUVECs were assessed by mRNA-seq and protein microarray. KLF4/PDGF-BB/PDGFRA axis then was affected using sh-RNA. Fluorescent dual luciferase reporting system and EMSA were used to determine if KLF4 directly promoted transcription of PDGF-BB/PDGFRA. Senescence of aortic wall in wild type C57BL/6 mice, STZ-induced diabetic mice fed with high-fat diet (HFD) and endothelial cells (ECs)- specific KLF4 knockout mice was assessed by SA-β-Gal staining. Key Findings: STZ-induced diabetes with high-fat diet and absence of endothelial KLF4 led to accelerated senescence of aortic vessel wall in mice. Whole mRNA-seq and protein microarray analysis revealed decreased expression of KLF4, PDGFBB, PDGFRA and PDGFRB in senescent HUVECs. Knock-in of KFL4, PDGF-BB or PDGFRA, but not PDGFRB, led to impaired senescence in glucose treated HUVECs, whereas knock-down of KLF4, PDGF-BB or PDGFRA directly accelerated senescence as revealed by greater proportion of positive SA-β-Gal staining, higher inflammatory factors level, more frequent G2/M cell cycle arrest and typical cellular nuclear senescent morphology. KLF4 directly promoted transcription activity of PDGF-BB/PDGFRA as revealed by fluorescent dual luciferase reporting system and EMSA. Incubation of HUVECs with PDGF-BB and PDGFRs vectors increased KLF4 expression. Significance: In diabetic mice models and glucose/Ang II-induced HUVECs, KLF4 protects endothelial cells against senescence by activating PDGF-BB/PDGFRA pathway

Improvement of endothelial function by Gunnera tinctoria extract with antioxidant properties

BackgroundGunnera tinctoria has been collected by Mapuche-Pewenche people for food and medicinal purposes. The high polyphenol content of methanolic extract from G. tinctoria leaves with chemical constituents such as ellagic acid and quercetin derivatives suggests its application to prevent endothelial dysfunction and oxidative stress. The aim of this study was to provide evidence of the protective effect of this extract on endothelial function by reducing oxidative stress induced by high d-glucose and H2O2, as well as by stimulating nitric oxide (NO) levels in human umbilical vein endothelial cells (HUVECs).ResultsA methanolic extract with a high content of polyphenols (520 ± 30 mg gallic acid equivalents/g dry extract) was obtained from G. tinctoria leaves. Its main constituent was ellagic acid. The results of Ferric reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays of the extract confirmed its antioxidant activity by inhibition pathway of radical species. The incubation of HUVECs with the extract decreased the apoptosis and reactive oxygen species (ROS) synthesis induced by high extracellular concentration of d-glucose or hydrogen peroxide. The extract increased endothelial NO levels and reduced vasoconstriction in human placental vessels.ConclusionsThis study provides evidence about the antioxidant and endothelial protective properties of methanolic G. tinctoria leaf extract. The extract improves the availability of NO in HUVECs, inhibiting the production of ROS and vasoconstriction.

Effects of physiochemical characteristic of nano-sized TiO2 on the adhesion of monocytes to endothelial cells

Nano-sized titanium dioxide (TiO2) particles are produced on a large scale and are widely used. However, the effects of exposure to nano-sized TiO2 particles on the cardiovascular system remain elusive. The present study investigated how the crystal structure and surface characteristics of nano-sized TiO2 particles affect the adhesion of monocytes to endothelial cells, which is an essential process in atherosclerogenesis. Human umbilical vein endothelial cells (HUVECs) and human monocytic leukemia cells (THP-1) were exposed to anatase (NM102), rutile hydrophobic (NM103), and rutile hydrophilic (NM104) TiO2 nanoparticles. Incubation of HUVECs with NM102, NM103, or NM104 at 100 μg/ml significantly reduced cell viability, and cell viability was also significantly reduced after exposure to 75 μg/ml of the anatase particles (NM102). Brief exposure to NM102 and NM103 increased the level of intercellular adhesion molecule-1 (ICAM-1) in HUVECs. Exposure to NM102 also upregulated the expression of lymphocyte function-associated antigen-1 (LFA-1) in THP-1 cells, resulting in enhanced adhesion of these cells to HUVECs. The amount of titanium uptake of NM102 in cells was higher than that of NM103 or NM104, as evidenced by inductively coupled plasma optical emission spectroscopy (ICP-OES). To examine the effects of long-term exposure to nano-sized TiO2 on the process of atherosclerosis, apolipoprotein E deficient (ApoE−/−) mice were exposed to TiO2 NPs (NM102, NM103, or NM104 at 10 or 40 μg/mouse) by pharyngeal aspiration once every other week for 10 weeks. As the results, NM102 (40 μg/mouse) increased plaque formation in the aorta, and this increase correlated with a significant upregulation of ICAM-1 and F4/80 expression in the aorta. Anatase, but not rutile, TiO2 nanoparticles promoted the adhesion of monocytes to endothelial cells, and enhanced atherosclerogenesis in a susceptible animal model.

Cannabidiol Promotes Endothelial Cell Survival by Heme Oxygenase-1-Mediated Autophagy.

Cannabidiol (CBD), a non-psychoactive cannabinoid, has been reported to mediate antioxidant, anti-inflammatory, and anti-angiogenic effects in endothelial cells. This study investigated the influence of CBD on the expression of heme oxygenase-1 (HO-1) and its functional role in regulating metabolic, autophagic, and apoptotic processes of human umbilical vein endothelial cells (HUVEC). Concentrations up to 10 µM CBD showed a concentration-dependent increase of HO-1 mRNA and protein and an increase of the HO-1-regulating transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). CBD-induced HO-1 expression was not decreased by antagonists of cannabinoid-activated receptors (CB1, CB2, transient receptor potential vanilloid 1), but by the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC). The incubation of HUVEC with 6 µM CBD resulted in increased metabolic activity, while 10 µM CBD caused decreased metabolic activity and an induction of apoptosis, as demonstrated by enhanced caspase-3 cleavage. In addition, CBD triggered a concentration-dependent increase of the autophagy marker LC3A/B-II. Both CBD-induced LC3A/B-II levels and caspase-3 cleavage were reduced by NAC. The inhibition of autophagy by bafilomycin A1 led to apoptosis induction by 6 µM CBD and a further increase of the proapoptotic effect of 10 µM CBD. On the other hand, the inhibition of HO-1 activity with tin protoporphyrin IX (SnPPIX) or knockdown of HO-1 expression by Nrf2 siRNA was associated with a decrease in CBD-mediated autophagy and apoptosis. In summary, our data show for the first time ROS-mediated HO-1 expression in endothelial cells as a mechanism by which CBD mediates protective autophagy, which at higher CBD concentrations, however, can no longer prevent cell death inducing apoptosis.

Ouabain and Marinobufagenin: Physiological Effects on Human Epithelial and Endothelial Cells.

Long-term study on the identification of Na,K-ATPase endogenous inhibitors in mammalian tissues has resulted in the discovery of ouabain, marinobufagenin (MBG), and other cardiotonic steroids (CTS) in the blood plasma. Production of ouabain and MBG is increased in essential hypertension and other diseases associated with hypervolemia. Here, we compared the effects of ouabain and MBG on the Na,K-ATPase activity (measured as the transport of Na+, K+, and Rb+ ions) and proliferation and death of human renal epithelial cells (HRECs) and human umbilical vein endothelial cells (HUVEC) expressing α1-Na,K-ATPase. Ouabain concentration that provided the half-maximal inhibition of the Rb+ influx (IC50) into HRECs and HUVECs was 0.07 μM. In both types of cells, the IC50 values for MBG were 10 times higher than for ouabain. Incubation of HREC and HUVEC with 0.001-0.01 μM ouabain for 30 h resulted in 40% increase in the [3H]thymidine incorporation into DNA; further elevation of ouabain concentration to 0.1 μM completely suppressed DNA synthesis. MBG at the concentration of 0.1 μM activated DNA synthesis by 25% in HRECs, but not in HUVECs; 1 μM MBG completely inhibited DNA synthesis in HRECs and by 50% in HUVECs. In contrast to HRECs, incubation of HUVECs in the serum-free medium induced apoptosis, which was almost completely suppressed by ouabain and MBG at the concentrations of 0.1 and 3 μM, respectively. Based on these data, we can conclude that (i) the effect of MBG at the concentrations detected in the blood plasma (<0.01 μM) on HRECs and HUVECs was not due to the changes in the [Na+]i/[K+]i ratio; (ii) the effect of physiological concentrations of ouabain on these cells might be mediated by the activation of Na,K-ATPase, leading to cell proliferation.

Galectin-3 aggravates ox-LDL-induced endothelial dysfunction through LOX-1 mediated signaling pathway.

Galectin-3, a biomarker linking oxidative stress and inflammation, participates in different mechanisms related to atherothrombosis, such as inflammation, proliferation, or macrophage chemotaxis. Accumulating evidence indicates that galectin-3 may also promote atherogenesis through inducing endothelial dysfunction. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1), a receptor for oxLDL uptake, contributes to oxLDL-induced endothelial dysfunction. Whether galectin-3 induces endothelial dysfunction through modulation of LOX-1-mediated signaling remains unclear. In the present study, we explored the mechanisms underlying galectin-3 enhanced cytotoxicity of oxLDL in human umbilical vein endothelial cells (HUVECs) and the role of LOX-1. Incubation of HUVECs with galectin-3 increased the expression of LOX-1 in RNA and protein levels. In addition, the expression of LOX-1 induced by oxLDL was promoted by galectin-3. However, pretreatment of LOX-1 antibody reduced LOX-1 mRNA expression level in cells with oxLDL plus galectin-3 incubation. Compared to cells treated with oxLDL alone, reactive oxygen species (ROS) generation via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and subsequent activation of p38 mitogen-activated protein kinases followed by nuclear factor kappa B (NF-κB) activation and related inflammatory responses including adhesion molecule expression, adhesiveness of monocytic cells, and IL-8 release were also aggravated in cells treated with galectin-3 combined with oxLDL. Compared to cells treated with galectin-3 plus oxLDL group. We found that LOX-1 antibody mitigated NADPH oxidase activity, p-38 up-regulation, NF-κB activation, and proinflammatory responses in cells treated with galectin-3 combined with oxLDL. We conclude that galectin-3 enhances endothelial LOX-1 expression and propose a new mechanism by which galectin-3 may promote endothelial dysfunction by inducing inflammation via LOX-1/ROS/p38/NF-κB-mediated signaling pathway.

Differential Regulation of sFlt‐1 in response to VEGF Family‐Based Therapeutics In Endothelial Cells

Preeclampsia (PE) is a hypertensive disorder of pregnancy which affects ~5–7% of all pregnancies in the United States. Hallmarked by new‐onset hypertension and organ damage, it is a leading cause of premature birth and maternal/fetal perinatal morbidity. While the cause of PE remains elusive, placental dysfunction due to chronic ischemia is believed to be a central regulator of the disorder. One of the factors known to be released from the ischemic placenta is the soluble form of the VEGF receptor Flt‐1 (sFlt‐1), which is released into the maternal circulation and is believed to be a major contributor to the maternal endothelial dysfunction seen in PE. It has been hypothesized that restoring angiogenic balance by way of administration of VEGF family members could be beneficial in PE. To that end, we have recently described several novel VEGF family‐based chimeric proteins with enhanced bioavailability and improved pharmacokinetic profiles. Recently, we have demonstrated that in vivo administration of a VEGFA‐based chimeric therapeutic protein was able to relieve the symptoms of PE in a rodent model, but resulted in adverse effects at dose‐limiting toxicity; in particular a marked increase in the circulating levels of sFlt‐1. Here we hypothesize that chimeric therapeutics based on the VEGF family members Placental Growth Factor (PlGF) and VEGFB will cause reduced production of sFlt‐1 from placental and vascular cells when compared to their VEGFA counterpart. To that end, we utilized cultured rodent placental explants, cultured BeWo cells derived from placental syncytiotrophoblasts, and primary Human Umbilical Vein Endothelial Cells (HUVEC) cells to determine the relative effects of the VEGF chimeras on sFlt‐1 production. Incubation of HUVECs with chimeric VEGFA caused a significant increase in released sFlt‐1 (6593±199 vs 14547±364 pg/ml, p&lt;0.0001), while there was no induction with either chimeric PlGF (6830±88pg/ml) or VEGFB (7217±71pg/ml). To further elucidate the effects of chimeric VEGFA on HUVEC sFlt‐1 production, we examined three splice variants of sFlt‐1 as well as the full length Flt‐1 by qRT‐PCR. In response to VEGF, the β‐Actin normalized expression increased in sFlt v2 (100±34% vs 401±36%, p&lt;0.0001), a trend to increase full length Flt‐1 message (100±34% vs 177±20%, p=−0.11) and sFlt‐1 v3 (123±8%, p=0.16) with no effect on sFlt v4 (100±34% vs 68±8%, p=0.36). This VEGF induced sFlt‐1 release was significantly attenuated with either a dual VEGFR1/VEGFR2 antagonist or a VEGR2 specific antagonist, but not a neuropilin‐1 selective antagonist, suggesting the receptor responsible for the release of sFlt‐1 in response to VEGF is the VEGFR2 receptor. In conclusion we conclude that the significant in vivo increase in sFlt‐1 caused by chronic chimeric VEGF administration is due to increased endothelial production of sFlt‐1. This is associated with increase production of the sFlt‐1 v2 splice variant, and is likely due to activation of the VEGFR2 receptor.Support or Funding InformationThis work was funded in part by NIH grants P01HL51971, P20GM104357, T32HL105324, R00HL11677, R25 HL121042‐01, and R01HL 137791.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Didymin prevents hyperglycemia-induced human umbilical endothelial cells dysfunction and death

Although didymin, a flavonoid-O-glycosides compound naturally found in the citrus fruits, has been reported to be a potent anticancer agent in the prevention of various cancers, its role in the prevention of cardiovascular complications is unclear. Most importantly, its effect in the prevention of endothelial dysfunction, a pathological process involved in the atherogenesis, is unknown. We have examined the efficacy of didymin in preventing the high glucose (HG; 25 mM)-induced human umbilical vein endothelial cells (HUVECs) dysfunction. Our results indicate that incubation of HUVECs with HG resulted in the loss of cell viability, and pre-incubation of didymin prevented it. Further, didymin prevented the HG-induced generation of reactive oxygen species (ROS) as well as lipid peroxidation product, malondialdehyde. Pretreatment of HUVECs with didymin also prevented the HG-induced decrease in eNOS and increase in iNOS expressions. Further, didymin prevented the HG-induced monocytes cell adhesion to endothelial cells, expressions of ICAM-1 and VCAM-1 and activation of NF-κB. Didymin also prevented the release of various inflammatory cytokines and chemokines in HG-treated HUVECs. In conclusion, our results demonstrate that didymin with its anti-oxidative and anti-inflammatory actions prevents hyperglycemia-induced endothelial dysfunction and death. Thus, it could be developed as a potential natural therapeutic agent for the prevention of cardiovascular complications in diabetes.

Maternal insulin therapy does not restore foetoplacental endothelial dysfunction in gestational diabetes mellitus

Pregnant women diagnosed with gestational diabetes mellitus subjected to diet (GDMd) that do not reach normal glycaemia are passed to insulin therapy (GDMi). GDMd associates with increased human cationic amino acid transporter 1 (hCAT-1)-mediated transport of L-arginine and nitric oxide synthase (NOS) activity in foetoplacental vasculature, a phenomenon reversed by exogenous insulin. Whether insulin therapy results in reversal of the GDMd effect on the foetoplacental vasculature is unknown. We assayed whether insulin therapy normalizes GDMd-associated foetoplacental endothelial dysfunction. Primary cultures of human umbilical vein endothelial cells (HUVECs) from GDMi pregnancies were used to assay L-arginine transport kinetics, NOS activity, p44/42mapk and protein kinase B/Akt activation, and umbilical vein rings reactivity. HUVECs from GDMi or GDMd show increased hCAT-1 expression and maximal transport capacity, NOS activity, and eNOS, and p44/42mapk, but not Akt activator phosphorylation. Dilation in response to insulin or calcitonin-gene related peptide was impaired in umbilical vein rings from GDMi and GDMd pregnancies. Incubation of HUVECs in vitro with insulin (1 nmol/L) restored hCAT-1 and eNOS expression and activity, and eNOS and p44/42mapk activator phosphorylation. Thus, maternal insulin therapy does not seem to reverse GDMd-associated alterations in human foetoplacental vasculature.

Eicosapentaenoic Acid-Enriched High-Density Lipoproteins Exhibit Anti-Atherogenic Properties

It has previously been reported that oral administration of purified eicosapentaenoic acid (EPA) generates EPA-rich high-density lipoprotein (HDL) particles with a variety of anti-inflammatory properties. In this study, the mechanism underlying the anti-atherogenic effects of EPA-rich HDL using reconstituted HDL (rHDL) was investigated.Methods and Results:rHDL was generated by the sodium cholate dialysis method, using apolipoprotein A-1 protein, cholesterol, and various concentrations of EPA-phosphatidylcholine (PC) or egg-PC. Increased EPA-PC contents in rHDL resulted in decreased particle size. Next, the effects of rHDL containing various amounts (0-100% of total PC) of EPA-PC on vascular cell adhesion molecule-1 (VCAM-1) expression in human umbilical vein endothelial cells (HUVECs) was examined. Cytokine-stimulated VCAM-1 expression was inhibited in a dose-dependent manner based on the amount of EPA-PC in rHDL. Surprisingly, the incubation of HUVECs with EPA-rich rHDL resulted in the production of resolvin E3 (RvE3), an anti-inflammatory metabolite derived from EPA. Incubation with EPA-PC alone did not adequately induce RvE3 production, suggesting that RvE3 production requires an endothelial cell-HDL interaction. The increased anti-inflammatory effects of EPA-rich HDL may be explained by EPA itself and RvE3 production. Furthermore, the increase in EPA-PC content enhanced cholesterol efflux. The EPA-enriched HDL particles exhibit cardioprotective properties via the production of anti-inflammatory lipid metabolites and the increase in cholesterol efflux.

C1q/TNF-Related Protein-9 Ameliorates Ox-LDL-Induced Endothelial Dysfunction via PGC-1α/AMPK-Mediated Antioxidant Enzyme Induction.

Oxidized low-density lipoprotein (ox-LDL) accumulation is one of the critical determinants in endothelial dysfunction in many cardiovascular diseases such as atherosclerosis. C1q/TNF-related protein 9 (CTRP9) is identified to be an adipocytokine with cardioprotective properties. However, the potential roles of CTRP9 in endothelial function remain largely elusive. In the present study, the effects of CTRP9 on the proliferation, apoptosis, migration, angiogenesis, nitric oxide (NO) production and oxidative stress in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL were investigated. We observed that treatment with ox-LDL inhibited the proliferation, migration, angiogenesis and the generation of NO, while stimulated the apoptosis and reactive oxygen species (ROS) production in HUVECs. Incubation of HUVECs with CTRP9 rescued ox-LDL-induced endothelial injury. CTRP9 treatment reversed ox-LDL-evoked decreases in antioxidant enzymes including heme oxygenase-1 (HO-1), nicotinamide adenine dinucleotide phosphate (NAD(P)H) dehydrogenase quinone 1, and glutamate-cysteine ligase (GCL), as well as endothelial nitric oxide synthase (eNOS). Furthermore, CTRP9 induced activation of peroxisome proliferator-activated receptor γ co-activator 1α (PGC1-α) and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK). Of interest, AMPK inhibition or PGC1-α silencing abolished CTRP9-mediated antioxidant enzymes levels, eNOS expressions, and endothelial protective effects. Collectively, we provided the first evidence that CTRP9 attenuated ox-LDL-induced endothelial injury by antioxidant enzyme inductions dependent on PGC-1α/AMPK activation.

Vascular Endothelial Growth Factor Augments Arginine Transport and Nitric Oxide Generation via a KDR Receptor Signaling Pathway

Background/Aims: Vascular endothelial growth factor (VEGF) is an endothelium-specific peptide that stimulates angiogenesis via two receptor tyrosine kinases, Flt-1 and KDR. Endothelial nitric oxide synthase (eNOS) plays a major role in VEGF signaling. Delivery of arginine to membrane bound eNOS by the cationic amino acid transporter-1 (CAT-1) has been shown to modulate eNOS activity. The current studies were designed to test the hypothesis that VEGF enhances eNOS activity via modulation of arginine transport by CAT-1. Methods: Using radio-labeled arginine, {[³H] L-arginine} uptake was determined in human umbilical vein endothelial cells (HUVEC) following incubation with VEGF with and without silencing the VEGF receptors Flt-1 or KDR. Subsequently, western blotting for CAT-1, PKCα, ERK 1/2, JNK, and their phosphorylated forms were performed. NO generation was measured by the Griess reaction. Results: VEGF (50 and 100 ng/ml) significantly augmented endothelial arginine transport in a time dependent manner, an effect which was prevented by Sunitinib (2 µM), a multi targeted receptor tyrosine kinase inhibitor. The increase in arginine transport velocities by VEGF was not affected by silencing Flt-1 while silencing KDR abrogated VEGF effect. Furthermore, incubating cells with 50 and 100 ng of VEGF for 30 minutes significantly augmented CAT-1 abundance. The expression of PKC-α, JNK, and ERK1/2 and their phosphorylated forms were unchanged following incubation of HUVEC with VEGF. The concentration of NO2/NO3 following incubation with VEGF was significantly higher than from untreated cells. This increase was significantly attenuated by silencing KDR. Conclusions: VEGF increases arginine transport via modulation of CAT-1 in endothelial cells. This effect is exclusively dependent on KDR rather than Flt-1.

Angiotensin-converting enzyme inhibitors attenuate propofol-induced pro-oxidative and antifibrinolytic effect in human endothelial cells.

Introduction:The aim of this study was to investigate the effects of plasma and tissue angiotensin-converting enzyme inhibitors (ACE-Is) against propofol-induced endothelial dysfunction and to elucidate the involved mechanisms in vitro.Materials and methods:We examined the effects of propofol (50 μM), quinaprilat and enalaprilat (10−5 M) on fibrinolysis (t-PA, PAI-1, TAFI antigen levels), oxidative stress parameters (H2O2 and MDA antigen levels and SOD and NADPH oxidase mRNA levels) and nitric oxide bioavailability (NO2/NO3 concentration and NOS expression at the level of mRNA) in human umbilical vein endothelial cells (HUVECs).Results:We found that both ACE-Is promoted similar endothelial fibrinolytic properties and decreased oxidative stress in vitro. Propofol alone increased the release of antifibrinolytic and pro-oxidative factors from the endothelium and increased mRNA iNOS expression. We also found that the incubation of HUVECs in the presence of propofol following ACE-Is pre-incubation caused weakness of the antifibrinolytic and pro-oxidative potential of propofol and this effect was similar after both ACE-Is.Conclusions:This observation suggests that the studied ACE-Is exerted protective effects against endothelial cell dysfunction caused by propofol, independently of hemodynamics.

Transfer of microRNA-486-5p from human endothelial colony forming cell–derived exosomes reduces ischemic kidney injury

Administration of human cord blood endothelial colony-forming cells (ECFCs) or their exosomes protects mice against kidney ischemia/reperfusion injury. Here we studied the microRNA (miRNA) content of ECFC exosomes and the role of miRNA transfer in kidney and endothelial cell protection. ECFC exosomes were enriched in miR-486-5p, which targets the phosphatase and tensin homolog (PTEN) and the Akt pathway. In cultured endothelial cells exposed to hypoxia, incubation with ECFC exosomes increased miR-486-5p, decreased PTEN, and stimulated Aktphosphorylation. Exposure of hypoxic endothelial cellsto conditioned medium from ECFCs pretreated with anti-miR-486-5p blocked increases in miR-486-5p and phosphorylated Akt, restored expression of PTEN, and enhanced apoptosis. Coculture of endothelial cells with ECFCs enhanced endothelial miR-486-5p levels. Targeting of PTEN by miR-486-5p was observed in endothelial cells, and PTEN knockdown blocked apoptosis. In mice with ischemic kidney injury, infusion of ECFC exosomes induced potent functional and histologic protection, associated with increased kidney miR-486-5p levels, decreased PTEN, and activation of Akt. Infusion of exosomes from ECFCs transfected with anti-miR-486-5p had no protective effect. Thus, delivery of ECFC exosomes reduces ischemic kidney injury via transfer of miR-486-5p targeting PTEN. Exosomes enriched in miR-486-5p could represent a therapeutic tool in acute kidney injury.

ADAMTS13 and its variants promote angiogenesis via upregulation of VEGF and VEGFR2.

Severe plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. The goals of this study were to understand the angiogenic pathways ADAMTS13 activates and to identify the important structural components of ADAMTS13 that stimulate angiogenesis. Incubation of human umbilical vein endothelial cells (HUVEC) with 150 ng/mL (1 nM) of recombinant human ADAMTS13 induced VEGF expression by 53 % and increased VEGF mRNA by over sixfold, both within 10 min; the measured VEGF levels steadily decreased over 2 h, as shown by Western blot and ELISA. Phosphorylation of VEGFR2 was significantly enhanced in HUVEC after incubation with ADAMTS13 (1 nM). Structure-function analysis showed that an ADAMTS13 variant containing thrombospondin type 1 (TSP1) 2-8 repeats (TSP1 2-8), TSP1 2-8 plus CUB domains (TSP1 2-8 plus CUB), or TSP1 5-8 repeats plus CUB domains (TSP1 5-8 plus CUB) increased HUVEC proliferation by 41-54 % as compared to the EBM-2 controls. Chemotaxis assays further demonstrated that the TSP1 domains of ADAMTS13 increased HUVEC migration by 2.65-fold. Incubation of HUVEC with both ADAMTS13 variants containing TSP1 repeats and anti-VEGF IgG abrogated the enhanced effect of ADAMTS13 on proliferation, migration, and VEGFR2 phosphorylation. In conclusion, ADAMTS13-induced endothelial cell angiogenesis occurs via the upregulation of VEGF and phosphorylation of VEGFR2. This angiogenic activity depends on the C-terminal TSP1 repeats of ADAMTS13.

Role of tumour necrosis factor receptor-1 and nuclear factor-κB in production of TNF-α-induced pro-inflammatory microparticles in endothelial cells.

Tumour necrosis factor-α (TNF-α) is upregulated in many inflammatory diseases and is also a potent agent for microparticle (MP) generation. Here, we describe an essential role of TNF-α in the production of endothelial cell-derived microparticles (EMPs) in vivo and the function of TNF-α-induced EMPs in endothelial cells. We found that TNF-α rapidly increased blood levels of EMPs in mice. Treatment of human umbilical vein endothelial cells (HUVECs) with TNF-α also induced EMP formation in a time-dependent manner. Silencing of TNF receptor (TNFR)-1 or inhibition of the nuclear factor-κB (NF-κB) in HUVECs impaired the production of TNF-α-induced EMP. Incubation of HUVECs with PKH-67-stained EMPs showed that endothelial cells readily engulfed EMPs, and the engulfed TNF-α-induced EMPs promoted the expression of pro-apoptotic molecules and upregulated intercellular adhesion molecule-1 level on the cell surface, which led to monocyte adhesion. Collectively, our findings indicate that the generation of TNF-α-induced EMPs was mediated by TNFR1 or NF-κB and that EMPs can contribute to apoptosis and inflammation of endothelial cells.

Red grape berry-cultured cells reduce blood pressure in rats with metabolic-like syndrome

Cumulative evidence suggests that moderate red wine consumption protects the cardiovascular system. The effect of cultured cells derived from red grape berry (RGC) on blood pressure (BP) has not been investigated. We therefore studied the antihypertensive effects of oral consumption of RGC in experimental rat model of metabolic-like syndrome and assessed its effect on human umbilical vein endothelial cells (HUVECs). Forty male Sprague-Dawley rats were fed for 5 weeks with either a high fructose diet (HFD) (n = 10) or HFD supplemented, during the last 2 weeks, with different doses (200, 400 and 800 mg/kg/day) of RGC suspended in their food (n = 30). BP, plasma triglycerides, insulin and adiponectin levels were measured at the beginning and after 3 and 5 weeks of diet. RGC effect on vasodilatation was evaluated by its ability to affect endothelin-1 (ET-1) production and endothelial nitric oxide synthase (eNOS) expression in HUVECs. BP, plasma triglycerides, insulin and adiponectin increased significantly in rats fed with a HFD. The increase in BP, plasma triglycerides and insulin was attenuated by RGC supplementation. Incubation of HUVECs with RGC demonstrated a concentration-dependent inhibition of ET-1 secretion and increase in the level of eNOS, signaling a positive effect of RGC on vasodilatation. In rats with metabolic-like syndrome, RGC decreased BP and improved metabolic parameters. These beneficial effects may be mediated by the cell constituents, highly rich with polyphenols and resveratrol, reside in their natural state.

Acetylcholinesterase inhibitors promote angiogenesis in chick chorioallantoic membrane and inhibit apoptosis of endothelial cells.

Alzheimer's disease (AD) is one of the most common causes of dementia in the elderly. Recently, a great attention has been paid to the possible role of vascular changes in the pathogenesis of AD. Reduced microvascular density and degeneration of the endothelium are of structural cerebrovascular changes in AD. Acetylcholinesterase (AChE) inhibitors are widely used for the improvement of AD symptoms. Until now, however, the effects of AChE inhibitors on vascular changes including angiogenesis and endothelial cell apoptosis are not fully understood. In the present work, the effects of three AChE inhibitors (donepezil, rivastigmine, and galantamine) were tested on H2O2-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and on angiogenesis in chicken chorioallantoic membrane model. Incubation of HUVEC with H2O2 led to a significant decrease in cell viability and an increase in the percentage of apoptotic cells. The tested drugs, at concentrations of 1–100 μM, significantly inhibited the H2O2-induced toxicity. Also, all donepezil, rivastigmine and galantamine significantly increased the number of vessels in the chorioallantoic membrane when injected into fertilized eggs. In conclusion, AChE inhibitors possess angiogenesis-accelerating properties and have antiapoptotic effects on endothelial cells. These effects of AChE inhibitors may be involved in their beneficial effects on AD.