Lysosomal destabilization activates the NLRP3 inflammasome in human umbilical vein endothelial cells (HUVECs)

Inflammation is a crucial component in the pathogenesis of many vascular diseases, such as atherosclerosis and diabetes. Inflammasomes are intracellular signalling complexes whose activation promotes inflammation. Nucleotide-binding domain and Leucine-rich repeat Receptor containing a Pyrin domain 3 (NLRP3) is a pattern recognition receptor (PRR) forming the best-known inflammasome. Disturbances in NLRP3 have been associated with multiple diseases. The purpose of this study was to explore the lysosomal destabilization-related NLRP3 inflammasome signaling pathway in human endothelial cells. In order to prime and activate NLRP3, human umbilical vein cells (HUVECs) were exposed to TNF-α and the lysosomal destructive agent Leusine-Leusine-O-Methylesther (Leu-Leu-OMe), respectively. A caspase-1 inhibitor was used to block caspase-1’s enzymatic function and an interleukin 1 receptor antagonist (IL-1RA) to prevent any possible secondary effects of IL-1β. Leu-Leu-OMe increased the expression of NLRP3, IL-1β, and IL-18 in HUVECs. Exposure to Leu-Leu-OMe significantly promoted the production of IL-6 and IL-8 in primed HUVECs; this effect was prevented by the pre-treatment of cells with an IL-1RA. Our results suggest that lysosomal destabilization activates the NLRP3 inflammasome pathway that promotes the production of IL-6 and IL-8 in an autocrine manner in HUVEC cells.     

Human umbilical vein endothelial cells (HUVECs) show Ca(2+) mobilization as well as Ca(2+) influx upon hypoxia

Bleb formation is an early event of cellular damage observed in a variety of cell types upon hypoxia. Although we previously found that the [Ca(2+)](i) rise before bleb formation only at the same loci of HUVECs upon hypoxia (localized [Ca(2+)](i) rise), the mode of the [Ca(2+)](i) rise remains ill-defined. In order to clarify the mechanisms causing the localized [Ca(2+)](i) rise in hypoxia challenged HUVECs, we studied the effects of several Ca(2+) channel blockers or a Ca(2+) chelator, EGTA, which reduces extracellular Ca(2+) concentration on the hypoxia-induced localized [Ca(2+)](i) rise and bleb formation by employing a confocal laser scanning microscopy (CLSM). After the initiation of hypoxia, [Ca(2+)](i) rose gradually in a localized fashion up to 15 min, which was associated with bleb formation at the same loci. The maximal [Ca(2+)](i) rise was 435 +/- 84 nM at the loci of bleb formation. Ca(2+) channel blockers including Ni(2+) (non-specific, 1 mM), nifedipine (L type, 10 microM), nicardipine (L + T type, 10 microM), and cilnidipine (L + N type, 10 microM) did not inhibit either the localized [Ca(2+)](i) rise or bleb formation. Although both the localized [Ca(2+)](i) rise and bleb formation were inhibited by lowering extracellular Ca(2+) concentration below 100 nM, a diffuse [Ca(2+)](i) rise through the cytoplasm remained without bleb formation, which was inhibited by a phospholipase C (PLC) inhibitor, U73122. In conclusion, hypoxia causes both the Ca(2+) mobilization and the Ca(2+) influx in HUVECs and the Ca(2+) influx through unknown Ca(2+) channels is responsible for the localized [Ca(2+)](i) rise integral to bleb formation.     

Human umbilical artery endothelial cells from Large-for-Gestational-Age newborn have increased antioxidant efficiency and gene expression

Obesity is a public health problem worldwide, and especially in women in reproductive age where more than one in three have obesity. Maternal obesity is associated with an increased maternal, placental, and newborn oxidative stress, which has been proposed as a central factor in vascular dysfunction in large-for-gestational-age (LGA) newborn. However, cellular and molecular mechanisms behind this effect have not been elucidated. Untreated human umbilical artery endothelial cells (HUAEC) from LGA (LGA-HUAEC) presented higher O₂⁻ levels, superoxide dismutase activity and heme oxygenase 1 messenger RNA (mRNA) levels, paralleled by reduced GSH:GSSG ratio and NRF2 mRNA levels. In response to an oxidative challenge (hydrogen peroxide), only HUAEC from LGA exhibited an enhanced Glutathione Peroxidase 1 (GPX1) expression, as well as a more efficient antioxidant machinery measured by the biosensor probe, HyPer. An open state of chromatin in the TSS region of GPX1 in LGA-HUAEC was evidenced by the DNase-HS assay. Altogether, our data indicate that LGA-HUAEC have an altered cellular and molecular antioxidant system. We propose that a chronic pro-oxidant intrauterine milieu, as evidenced in pregestational obesity, could induce a more efficient antioxidant system in fetal vascular cells, which could be maintained by epigenetic mechanism during postnatal life.     

Differential of sex steroids of prostaglandin secretion by male and female cultured piglet endothelial cells

The effect of sex steroids, 17β-estradiol and testosterone, on the production of 6-keto-prostaglandin F_1α, prostaglandin F_2α and prostaglandin E₂ was studied in cultures of piglet aorta endothelial cells. In cells isolated from female animals both steroids stimulated the secretion of prostaglandins. In contrast, sex steroids did not affect prostaglandin synthesis by endothelial cells taken from male animals. In addition, female endothelial cells convert testosterone into Estriol, estrone and estradiol. estradiol-induced stimulation of prostacyclin production may explain in part the beneficial role generally attributed to naturally occuring estrogens in cardiovascular diseases.     

Serum starvation induces sexual dimorphisms in secreted proteins of human umbilical vein endothelial cells (HUVECs) from twin pairs

There is growing evidence for sex and gender differences in the clinical manifestation and outcomes of human diseases. Human primary endothelial cells represent a useful cardiovascular model to study sexual dimorphisms at the cellular level. Here, we analyzed sexual dimorphisms of the secretome after serum starvation using human umbilical vein endothelial cells (HUVECs) from twin pairs of the opposite sex to minimize the impact of varying genetic background. HUVECs were starved for 5 and 16 h, respectively, and proteins of the cell culture supernatants were analyzed by tandem mass spectrometry. Altogether, 960 extracellular proteins were identified of which 683 were amendable to stringent quantification. Significant alterations were observed for 455 proteins between long-term and short-term starvation and the majority were similar in both sexes. Only 5 proteins showed significant sex-specific regulation between long-versus short-term starvation. Furthermore, 19 unique proteins with significant sexual dimorphisms at the same time points of serum starvation were observed. A larger number of proteins, for example tissue factor inhibitor 2 (TFPI2), displayed higher levels in the supernatants of females compared to male cells after long term serum starvation that might point to higher adaptation capacity of female cells. The overall results demonstrate that male and female cells differ in their secretome.     

Human fetal placental endothelial cells have a mature arterial and a juvenile venous phenotype with adipogenic and osteogenic differentiation potential

Growing interest in the sources of origin of blood vessel related diseases has led to an increasing knowledge about the heterogeneity and plasticity of endothelial cells lining arteries and veins. So far, most of these studies were performed on animal models. Here, we hypothesized that the plasticity of human fetal endothelial cells depends on their vascular bed of origin i.e. vein or artery and further that the differences between arterial and venous endothelial cells would extend to phenotype and genotype. We established a method for the isolation of fetal arterial and venous endothelial cells from the human placenta and studied the characteristics of both cell types. Human placental arterial endothelial cells (HPAEC) and human placental venous endothelial cells (HPVEC) express classical endothelial markers and differ in their phenotypic, genotypic, and functional characteristics: HPAEC are polygonal cells with a smooth surface growing in loose arrangements and forming monolayers with classical endothelial cobblestone morphology. They express artery-related genes (hey-2, connexin 40, depp) and more endothelial-associated genes than HPVEC. Functional testing demonstrated that vascular endothelial growth factors (VEGFs) induce a higher proliferative response on HPAEC, whereas placental growth factors (PlGFs) are only effective on HPVEC. HPVEC are spindle-shaped cells with numerous microvilli at their surface. They grow closely apposed to each other, form fibroblastoid swirling patterns at confluence and have shorter generation and population doubling times than HPAEC. HPVEC overexpress development-associated genes (gremlin, mesenchyme homeobox 2, stem cell protein DSC54) and show an enhanced differentiation potential into adipocytes and osteoblasts in contrast to HPAEC. These data provide collective evidence for a juvenile venous and a more mature arterial phenotype of human fetal endothelial cells. The high plasticity of the fetal venous endothelial cells may reflect their role as tissue-resident endothelial progenitors during embryonic development with a possible benefit for regenerative cell therapy.     

Differential effects of sex steroids on prostaglandin secretion by male and female cultured piglet endothelial cells

The effect of sex steroids, 17 beta-estradiol and testosterone, on the production of 6-keto-prostaglandin F1 alpha, prostaglandin F2 alpha and prostaglandin E2 was studied in cultures of piglet aorta endothelial cells. In cells isolated from female animals both steroids stimulated the secretion of prostaglandins. In contrast, sex steroids did not affect prostaglandin synthesis by endothelial cells taken from male animals. In addition, female endothelial cells convert testosterone into estriol, estrone and estradiol. Estradiol-induced stimulation of prostacyclin production may explain in part the beneficial role generally attributed to naturally occurring estrogens in cardiovascular diseases.     

Xanthones as inhibitors of microsomal lipid peroxidation and TNF-alpha induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs)

Xanthones bearing different functionalities, namely 1-hydroxyxanthone (1), 3-hydroxyxanthone (2), 1,4-dihydroxyxanthone (3), 2,6-dihydroxyxanthone (4), 1,2-diacetoxyxanthone (5), 2,6-diacetoxyxanthone (6), 3-methoxyxanthone (7), 1,3,7-trimethoxyxanthone (8) and 1,5-dihydroxy-6-methoxyxanthone (9) were synthesised and examined for their effect on nicotinamide adenine dinucleotide phosphate (NADPH)-catalysed liver microsomal lipid peroxidation and on tumour necrosis factor-alpha (TNF-alpha) induced expression of intercellular adhesion moledule-1 (ICAM-1) on endothelial cells, with a view to establish structure-activity relationship. Hydroxy- and acetoxyxanthones showed potent inhibitory effects on NADPH-catalysed lipid peroxidation and TNF-alpha induced expression of ICAM-1 on endothelial cells.     

Novel thiocoumarins as inhibitors of TNF-alpha induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs) and microsomal lipid peroxidation

Different coumarin/thiocoumarin derivatives, that is, 7-hydroxy-4-methylcoumarin, 7,8-dihydroxy-4-methylcoumarin, 7-acetoxy-4-methylcoumarin, 7,8-diacetoxy-4-methylcoumarin, 7-hydroxy-4-methylthiocoumarin, 7,8-dihydroxy-4-methylthiocoumarin, 7-acetoxy-4-methylthiocoumarin and 7,8-diacetoxy-4-methylthiocoumarin were synthesized and evaluated for their effects on TNF-alpha induced expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and on NADPH-catalyzed rat liver microsomal lipid peroxidation with a view to identify modulators for expression of cell adhesion molecules and to establish structure-activity relationship. We found that dihydroxy and diacetoxy derivatives of thiocoumarin were more potent in comparison to the corresponding coumarin derivatives in inhibiting TNF-alpha-induced expression of ICAM-1. However, coumarin derivatives were found to be more potent in comparison to the corresponding thiocoumarins in inhibiting microsomal lipid peroxidation. We have also tested the intermediate compounds 7,8-dibenzyloxy-4-methylcoumarin and 7,8-dibenzyloxy-4-methylthiocoumarin for their inhibitory activity on TNF-alpha-induced ICMA-1 expression. We found that dibenzyloxy-4-methylthiocoumarin is better than dibenzyloxy-4-methylcoumarin. The mechanisms underlying the observed activities of coumarins and thiocoumarins have been discussed with reference to their structures. Such structure-function relationship studies may help in developing molecules with better anti-inflammatory and anti-oxidant activities.     

Gradients in cytoplasmic calcium concentration ([Ca2+]i) in migrating human umbilical vein endothelial cells (HUVECs) stimulated by shear-stress

Using a parallel-plate flow-chamber and confocal laser scanning microscopy (CLSM), we studied the distribution and temporal changes in intracellular Ca2+ concentration ([Ca2+]i) in migrating HUVECs stimulated by shear-stress. In the presence or absence of ATP, shear-stress (10 dyne/cm2) caused morphological change and migration of individual HUVECs in the random direction. After 120 minute exposure to shear-stress, 70% of the cells migrated in the direction of flow, whereas, as many as 30% of the cells migrated to the upstream against flow. A nonspecific plasma membrane Ca2+ channel blocker, Ni2+, abolished such responses markedly, suggesting that Ca2+ influx may be essential for shear-stress dependent morphological change and migration of HUVECs. Analysis of [Ca2+]i distribution revealed the appearance of localized [Ca2+]i elevation inside lamellipodium formed in the direction of cell migration. The localized rise in [Ca2+]i might be closely related with morphological change to regulate the direction of cell migration induced by shear-stress.     

Isolation of human umbilical vein endothelial cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.     

Discrimination of female sex pheromone by male Trichoplusia ni (Hubner)

The olfactory discrimination process of male cabbage loopermoths, Trichoplusa ni (Hübner), was assessed by measuringtheir response to one of two emission sources within a windtunnel. The males discriminated between (1) Z7–12:Ac concentrations;(2) Z7–12:Ac alone and the volatile emission from excisedfemale sex pheromone glands; and (3) Z7–12:Ac and theemission from a mixture of six synthetic pheromone componentsthat mimics the volatile emissions of a female gland. Althoughmales could discriminate between a freshly excised female sexpheromone gland and 7.4x 10^–11 M Z7–12:Ac, theycould not discriminate between a gland and 78.5x10^–11M Z7–12:Ac. Males also could not discriminate betweenthe mixture of six volatile compounds and 28.7x10^–11 Mof Z7–12:Ac. The data show that male cabbage looper mothshave difficulty discriminating between Z7–12:Ac aloneand in mixtures with other female-emitted volatile compounds.     

Palmitate and oleate have distinct effects on the inflammatory phenotype of human endothelial cells

Free fatty acids may create a state of continuous and progressive damaging to the vascular wall manifested by endothelial dysfunction. In this study we determine the mechanisms by which fatty acids palmitate (C16:0) and oleate (C18:1) affect intracellular long chain acyl-CoA (LCAC) content, energy metabolism, cell survival and proliferation and activation of NF-kappaB in cultured endothelial cells. A 48-h exposure of human umbilical vein endothelial cells (HUVEC) to 0.5 mM palmitate or 0.5 mM oleate increased total long chain acyl-CoA (LCAC) content 1.7 and 2 fold, respectively and decreased ATP(total)/ADP(total) ratio by 26+/-5% (mean+/-SEM) and 15+/-2%, respectively, which was prevented by the acyl-CoA synthetase inhibitor triacsin C. Furthermore, palmitate inhibited cell proliferation by 34+/-5%, while oleate stimulated it by 12+/-2%. alpha-Tocopherol fully and triacsin C partially abolished the effect of palmitate on cell proliferation. Palmitate and oleate increased caspase-3 activity 3.2 and 1.4 fold, respectively. Palmitate-induced caspase-3 activation was prevented by triacsin C and slightly reduced by alpha-tocopherol and by the de novo ceramide synthesis inhibitor fumonisin B(1). Both fatty acids induced antioxidant-sensitive nuclear translocation of NF-kappaB after 72 h, but not after 48 h. In conclusion, we showed that fatty acids influence different aspects of HUVEC function resulting in amongst other activation of apoptotic and inflammatory pathways. Our results indicate that the effects depend on the fatty acid type and may be related to accumulation of LCAC.     

雄性棉铃虫和烟青虫对雌性信息素的触角电生理反应

利用触角电位图(Electroantennogram,EAG)技术,比较研究了二近缘种棉铃虫、烟青虫对其性信息素主要成分Z—11—16：Ald、Z—9—16：Ald的触角电生理反应。剂量反应曲线表明,对Z—11—16：Ald,棉铃虫和烟青虫均有明显的EAG反应,且随浓度的增加而增强,但棉铃虫比烟青虫的反应较强;对Z—9—16：Ald,烟青虫有很强的EAG反应,棉铃虫的反应则很弱;对Z—11—16：Ald和Z—9—16：Ald以97：3和7：93比例形成的混合物,棉铃虫、烟青虫均有EAG反应,但二者之间没有显著差异[动物学报49(6)：795～799,2003]。     

Discrimination of mixtures of female sex pheromone components by male Trichoplusia ni (Hubner)

Pheromone mixture discrimination by male cabbage loopers, Trichoplusiani (Hübner), was assessed in a wind tunnel by responsedifferences to paired stimuli. They discriminate differencesbetween mixtures that contain three and all six of the pheromonecomponents that are emitted by virgin females. The ability todiscriminate between these two mixtures is perplexing becauseonly three antennal pheromone specialist neurons respond atnatural stimulus intensities. The cabbage looper also discriminatedbetween mixtures whose component ratios differed slightly fromthat of the female. There was some interaction between the stimulusintensity, the distance between the stimulus sources, and possiblythe extent of the perceptual difference. The mixtures of pheromonecomponents failed to enhance the amount of upwind flight responseover that of Z7–12:Ac alone. Other evidence argues thatthe instantaneous release of pheromone may exceed time-averagedmeasures.     

Human umbilical cord perivascular cells (HUCPVC)

Human bone marrow mesenchymal stem cells (hBM-MSC) have recently been employed in the clinical treatment of challenging skin defects. We have described an MSC population that can be easily harvested from human umbilical cord perivascular tissue, human umbilical cord perivascular cells (HUCPVC), which exhibit a higher proliferative rate and frequency than hBM-MSC. Our objective was to establish whether HUCPVC could promote healing of full thickness murine skin defects, and thus find utility as a cell source for dermal repair. To this end, bilateral full thickness defects were created on the dorsum of Balb/c nude mice. Fibrin was used as a delivery vehicle for 1 x 106 PKH-67 labeled HUCPVC with contralateral controls receiving fibrin only. Epifluorescent and brightfield microscopic evaluation of the wound site was carried out at 3 and 7 days while mechanical testing of wounds was carried out at 3, 7, and 10 days. Our results show that by 3 days, marked contraction of the wound was observed in the fibrin controls whilst the HUCPVC samples exhibited neither collapse nor contraction of the defect, and the dermal repair tissue was considerably thicker and more organized. By 7 days, complete re-epithelialization of the HUCPVC wounds was observed whilst in the controls re-epithelialization was limited to the wound margins. Wound strength was significantly increased in the HUCPVC treatment group by 3 and 7 days but no statistical difference was seen at 10 days. We conclude that HUCPVCs accelerate early wound healing in full thickness skin defects and thus represent a putative source of human MSCs for use in dermal tissue engineering.     

The novel exchange protein activated by cyclic AMP 1 (EPAC1) agonist,I942, regulates inflammatory gene expression in human umbilical vascular endothelial cells (HUVECs)

Exchange protein activated by cyclic AMP (EPAC1) suppresses multiple inflammatory actions in vascular endothelial cells (VECs), partly due to its ability to induce expression of the suppressor of cytokine signalling 3 (SOCS3) gene, the protein product of which inhibits interleukin 6 (IL6) signalling through the JAK/STAT3 pathway. Here, for the first time, we use the non-cyclic nucleotide EPAC1 agonist, I942, to determine its actions on cellular EPAC1 activity and cyclic AMP-regulated gene expression in VECs. We demonstrate that I942 promotes EPAC1 and Rap1 activation in HEK293T cells and induces SOCS3 expression and suppresses IL6-stimulated JAK/STAT3 signalling in HUVECs. SOCS3 induction by I942 in HUVECs was blocked by the EPAC1 antagonist, ESI-09, and EPAC1 siRNA, but not by the broad-spectrum protein kinase A (PKA) inhibitor, H89, indicating that I942 regulates SOCS3 gene expression through EPAC1. RNA sequencing was carried out to further identify I942-regulated genes in HUVECs. This identified 425 I942-regulated genes that were also regulated by the EPAC1-selective cyclic AMP analogue, 007, and the cyclic AMP-elevating agents, forskolin and rolipram (F/R). The majority of genes identified were suppressed by I942, 007 and F/R treatment and many were involved in the control of key vascular functions, including the gene for the cell adhesion molecule, VCAM1. I942 and 007 also inhibited IL6-induced expression of VCAM1 at the protein level and blocked VCAM1-dependent monocyte adhesion to HUVECs. Overall, I942 represents the first non-cyclic nucleotide EPAC1 agonist in cells with the ability to suppress IL6 signalling and inflammatory gene expression in VECs.     

Survival rates in a natural population of the damselfly Ceriagrion tenellum: effects of sex and female phenotype

1. Ceriagrion tenellum females show genetic colour polymorphism. Androchrome (erythrogastrum) females are brightly (male‐like) coloured while gynochrome females (typica and melanogastrum) show cryptic colouration. 2. Several hypotheses have been proposed to explain the existence of more than one female morph in damselfly populations. The reproductive isolation and intraspecific mimicry hypotheses predict greater survival of gynochrome females, while the density dependent hypothesis predicts no differential survival between morphs. 3. Mature males had greater recapture probability than females while the survival probability was similar for both sexes. Survival and recapture rates were similar for androchrome and gynochrome females. 4. Gynochrome females showed greater mortality or migration rate than androchrome females during the pre‐reproductive period. This result is not predicted by the above hypotheses or by the null hypothesis that colour polymorphism is only maintained by random factors: founder effects, genetic drift, and migration.     

Expression of MUC1 mucin in human umbilical vein endothelial cells (HUVEC)

Mucin 1 (MUC1) is a membrane-bound glycoprotein that is expressed by various epithelial cell types. MUC1 functions include modulation of cell adhesion, signal transduction, lubrication and hydration of epithelial surfaces, and their protection from infection. In this study we demonstrated that MUC1 is expressed in human umbilical vein endothelial cells (HUVECs) and could be released/shed from cellular membrane. MUC1 presence in these cells was verified using three methods: Western blotting, flow cytometry and metabolic labeling. We also showed that mucin expression is stimulated by proinflammatory cytokines: about a 2-fold increase was observed after TNF-α treatment and lower after IFN-γ alone and in combination with TNF-α treatment. It can be assumed that the presence of MUC1 in endothelial cells may have an important role in the interactions with different cell types in physiological and pathological processes.