Primary Bovine Aortic Endothelial Cells Research Articles

Pomegranate (Punica granatum) By-Product Extract Influences the Oxylipids Profile in Primary Bovine Aortic Endothelial Cells in a Model of Oxidative Stress

Aerobic metabolism produces reactive oxygen species (ROS) as a natural by-product that can play a significant role in cell signaling and homeostasis. Excessive and uncontrolled production of ROS, however, can lead to oxidative stress that causes damage to immune cells and is related to several diseases in dairy cattle. Endothelial cells are essential for optimal immune and inflammatory responses but are especially sensitive to the damaging effects of ROS. Accordingly, investigating antioxidant strategies that can mitigate the detrimental impact of ROS on endothelial functions could impact compromised host defenses that lead to increased disease susceptibility. The objective of this study was to test the antioxidant effect of different concentrations (20, 40, 60, 80 μg/ml) of pomegranate by-product extract (PBE) on bovine aortic endothelial cells (BAECs). A model of oxidative stress was developed using in vitro exposure of BAEC to 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) to induce the formation of ROS. The BAEC were then analyzed for cell viability, ROS production, fatty acids profile, and oxylipids formation. The BAECs viability did not change after different concentrations of PBE and remained up to 80% over control; whereas, intracellular ROS showed a reduction passing from 20 to 50% with increasing PBE concentration from 20 to 80 μg/ml, respectively. The PBE extract clearly demonstrated efficacy in reducing the concentrations of pro-inflammatory oxylipids with a concomitant enhancement of anti-inflammatory oxylipids. In particular, the pro-inflammatory 13-hydroxyoctadecadienoic acid and its derived anti-inflammatory 13-hydroperoxoctadecaienoic acid were found lower and higher, respectively, in PBE+AAPH treated cells than AAPH treatment. Data from the present study support in vivo future experimental use of pomegranate by-product extract to study its potential beneficial effect against oxidative stress conditions in dairy cattle.

Characterisation of Novel Angiogenic and Potent Anti-Inflammatory Effects of Micro-Fragmented Adipose Tissue.

Adipose tissue and more specifically micro-fragmented adipose tissue (MFAT) obtained from liposuction has recently been shown to possess interesting medicinal properties whereby its application supports pain reduction and may enhance tissue regeneration particularly in osteoarthritis. Here we have characterised samples of MFAT produced using the Lipogems® International Spa system from eight volunteer individuals in order to understand the critical biological mechanisms through which they act. A variation was found in the MFAT cluster size between individual samples and this translated into a similar variation in the ability of purified mesenchymal stem cells (MSCs) to form colony-forming units. Almost all of the isolated cells were CD105/CD90/CD45+ indicating stemness. An analysis of the secretions of cytokines from MFAT samples in a culture using targeted arrays and an enzyme-linked immunosorbent assay (ELISA) showed a long-term specific and significant expression of proteins associated with anti-inflammation (e.g., interleukin-1 receptor alpha (Il-1Rα) antagonist), pro-regeneration (e.g., hepatocyte growth factor), anti-scarring and pro-angiogenesis (e.g., transforming growth factor beta 1 and 2 (TGFβ1/2) and anti-bacterial (e.g., chemokine C-X-C motif ligand-9 (CXCL-9). Angiogenesis and angiogenic signalling were notably increased in primary bovine aortic endothelial cells (BAEC) to a different extent in each individual sample of the conditioned medium whilst a direct capacity of the conditioned medium to block inflammation induced by lipopolysaccharides was shown. This work characterises the biological mechanisms through which a strong, long-lasting, and potentially beneficial effect can be observed regarding pain reduction, protection and regeneration in osteoarthritic joints treated with MFAT.

RNA-Seq Analyses Reveal That Endothelial Activation and Fibrosis Are Induced Early and Progressively by Besnoitia besnoiti Host Cell Invasion and Proliferation.

The pathogenesis of bovine besnoitiosis and the molecular bases that govern disease progression remain to be elucidated. Thus, we have employed an in vitro model of infection based on primary bovine aortic endothelial cells (BAEC), target cells during the acute infection. Host-parasite interactions were investigated by RNA-Seq at two post-infection (pi) time points: 12 hpi, when tachyzoites have already invaded host cells, and 32 hpi, when tachyzoites have replicated for at least two generations. Additionally, the gene expression profile of B. besnoiti tachyzoites was studied at both pi time points. Up to 446 differentially expressed B. taurus genes (DEGs) were found in BAEC between both pi time points: 249 DEGs were up-regulated and 197 DEGs were down-regulated at 32 hpi. Upregulation of different genes encoding cytokines, chemokines, leukocyte adhesion molecules predominantly at 12 hpi implies an activation of endothelial cells, whilst upregulation of genes involved in angiogenesis and extracellular matrix organization was detected at both time points. NF-κB and TNF-α signaling pathways appeared to be mainly modulated upon infection, coordinating the expression of several effector proteins with proinflammatory and pro-fibrotic phenotypes. These mediators are thought to be responsible for macrophage recruitment setting the basis for chronic inflammation and fibrosis characteristic of chronic besnoitiosis. Angiogenesis regulation also predominated, and this multistep process was evidenced by the upregulation of markers involved in both early (e.g., growth factors and matrix metalloproteinases) and late steps (e.g., integrins and vasohibin). Besnoitia besnoiti ortholog genes present in other Toxoplasmatinae members and involved in the lytic cycle have shown to be differentially expressed among the two time points studied, with a higher expression at 32 hpi (e.g., ROP40, ROP5B, MIC1, MIC10). This study gives molecular clues on B. besnoiti- BAECs interaction and shows the progression of type II endothelial cell activation upon parasite invasion and proliferation.

Lytic cycle of Besnoitia besnoiti tachyzoites displays similar features in primary bovine endothelial cells and fibroblasts

BackgroundBovine besnoitiosis, caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that continues to spread in Europe in the absence of control tools. In this scenario, in vitro culture systems are valuable tools to carry out drug screenings and to unravel host-parasite interactions. However, studies performed in bovine target cells are scarce.MethodsThe objective of the present study was to obtain primary bovine aortic endothelial cells (BAECs) and fibroblast cell cultures, target cells during the acute and the chronic stage of the disease, respectively, from healthy bovine donors. Afterwards, expression of surface (CD31, CD34 and CD44) and intracellular markers (vimentin and cytokeratin) was studied to characterize cell populations by flow cytometry. Next, the lytic cycle of B. besnoiti tachyzoites was studied in both target cells. Invasion rates (IRs) were determined by immunofluorescence at several time points post-infection, and proliferation kinetics were studied by quantitative PCR (qPCR). Finally, the influence of bovine viral diarrhea virus (BVDV) co-infection on the host cell machinery, and consequently on B. besnoiti invasion and proliferation, was investigated in BAECs.ResultsMorphology and cytometry results confirmed the endothelial and fibroblast origins. CD31 was the surface marker that best discriminated between BAECs and fibroblasts, since fibroblasts lacked CD31 labelling. Expression of CD34 was weak in low-passage BAECs and absent in high-passage BAECs and fibroblasts. Positive labelling for CD44, vimentin and cytokeratin was observed in both BAECs and fibroblasts. Regarding the lytic cycle of the parasite, although low invasion rates (approximately 3–4%) were found in both cell culture systems, more invasion was observed in BAECs at 24 and 72 hpi. The proliferation kinetics did not differ between BAECs and fibroblasts. BVDV infection favoured early Besnoitia invasion but there was no difference in tachyzoite yields observed in BVDV-BAECs compared to BAECs.ConclusionsWe have generated and characterized two novel standardized in vitro models for Besnoitia besnoiti infection based on bovine primary target BAECs and fibroblasts, and have shown the relevance of BVDV coinfections, which should be considered in further studies with other cattle pathogens.

Widespread basal cytochrome P450 expression in extrahepatic bovine tissues and isolated cells

Periparturient cattle face increased risk of both metabolic and infectious diseases. Factors contributing to this predisposition include oxidized polyunsaturated fatty acids, also known as oxylipids, whose production is altered during the periparturient period and in diseased cattle. Alterations in the production of oxylipids derived from cytochrome P450 (CYP450) enzymes are over-represented during times of increased disease risk and clinical disease, such as mastitis. Many of these same CYP450 enzymes additionally regulate metabolism of fat-soluble vitamins, such as A, D, and E. These vitamins are essential to maintaining immune health, yet circulating concentrations are diminished near calving. Despite this, a relatively small amount of research has focused on the roles of CYP450 enzymes outside of the liver. The aim of this paper is to describe the relative gene expression of 11 CYP450 in bovine tissues and common in vitro bovine cell models. Eight tissue samples were collected from 3 healthy dairy cows after euthanasia. In vitro samples included primary bovine aortic and mammary endothelial cells and immortalized bovine kidney and mammary epithelial cells. Quantitative real-time-PCR was carried out to assess basal transcript expression of CYP450 enzymes. Surprisingly, CYP450 mRNA was widely expressed in all tissue samples, with predominance in the liver. In vitro CYP450 expression was less robust, with several cell types lacking expression of specific CYP450 enzymes altogether. Overall, cell culture models did not reflect expression of tissue CYP450. However, when CYP450 were organized by activity, certain cell types consistently expressed specific functional groups. These data reveal the widespread expression of CYP450 in individual organs of healthy dairy cows. Widespread expression helps to explain previous evidence of significant changes in CYP450-mediated oxylipid production and fat-soluble vitamin metabolism in organ microenvironments during periods of oxidative stress or disease. As such, these data provide a foundation for targeted functional experiments aimed at understanding the activities of specific CYP450 and associated therapeutic potential during times of increased disease risk.

Foam cell-derived 4-hydroxynonenal induces endothelial cell senescence in a TXNIP-dependent manner.

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co-culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP-1 monocyte-derived foam cells, were analysed for the induction of senescence. Senescence associated β-galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4-hydroxnonenal (4-HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4-HNE in the co-culture medium blunted this effect. Furthermore, both foam cells and 4-HNE increased the expression of the pro-oxidant thioredoxin-interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell-induced senescence. Previous studies showed that peroxisome proliferator-activated receptor (PPAR)δ was activated by 4-hydroalkenals, such as 4-HNE. Pharmacological interventions supported the involvement of the 4-HNE-PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell-released 4-HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.

Detailed morphological characterisation of Hendra virus infection of different cell types using super-resolution and conventional imaging.

BackgroundHendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells.MethodsA range of primary cells and immortalised cell lines were infected with HeV, fixed at various time points post-infection, labelled for HeV proteins and imaged by confocal, super-resolution and transmission electron microscopy.ResultsSignificant differences were noted in viral protein distribution depending on the infected cell type. At 8 hpi HeV G protein was detected in the endoplasmic reticulum and M protein was seen predominantly in the nucleus in all cells tested. At 18 hpi, HeV-infected Vero cells showed M and G proteins throughout the cell and in transmission electron microscope (TEM) sections, in pleomorphic virus-like structures. In HeV infected MDBK, A549 and HeLa cells, HeV M protein was seen predominantly in the nucleus with G protein at the membrane. In HeV-infected primary bovine and porcine aortic endothelial cells and two bat-derived cell lines, HeV M protein was not seen at such high levels in the nucleus at any time point tested (8,12, 18, 24, 48 hpi) but was observed predominantly at the cell surface in a punctate pattern co-localised with G protein. These HeV M and G positive structures were confirmed as round HeV virions by TEM and super-resolution (SR) microscopy. SR imaging demonstrated for the first time sub-virion imaging of paramyxovirus proteins and the respective localisation of HeV G, M and N proteins within virions.ConclusionThese findings provide novel insights into the structure of HeV and show that for HeV imaging studies the choice of tissue culture cells may affect the experimental results. The results also indicate that HeV should be considered a predominantly round virus with a mean diameter of approximately 280 nm by TEM and 310 nm by SR imaging.

Stress‐activated signaling in endothelial cells (609.21)

Endothelial cells (ECs) form the interior layer of blood vessels, creating a selectively permeable barrier. Damage to this monolayer triggers inflammatory responses which may eventually lead to vascular disease, making it relevant to characterize EC reactions to inflammatory stress. Pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) activates several pathways in target cells, which interact to determine cell fate. Binding of TNF‐α to its receptor causes activation of certain caspases, advancing apoptosis. c‐jun N‐terminal kinase (JNK) is activated as well, regulating transcription factor activity and possibly promoting an apoptotic response. Also activated is nuclear factor κ‐light‐chain‐enhancer of activated B‐cells (NFκB), a transcription factor which has pro‐survival effects, partially due to interference with both JNK and caspases. ECs encounter TNF‐α as it travels in the blood, however, while most cells are rarely exposed. Thus, we explored whether EC response to TNF‐α is similar to that of other cell types, using immunofluorescence microscopy and Western blotting to analyze TNF‐α‐induced JNK, NFκB, and caspase‐3 activity in primary bovine aortic ECs (BAOECs). NFκB is usually kept in the cytoplasm by an inhibitor; TNF‐α‐binding leads to inhibitor degradation and allows NFκB to move to the nucleus. Upon TNF‐α treatment (10 ng/mL), this translocation begins by 30 minutes, maximizes from 60 to 120 minutes, and declines thereafter. JNK activation is transient, with p‐JNK levels peaking after 30 minutes before decreasing. Excitation of BAOECs with high TNF‐α levels (50 ng/mL for 20 hours) induces caspase‐3 activation, suggesting significant apoptosis. This reaction profile resembles that of other cell types, though it is possible that there are important differences in pathway cross‐talk and communication.Grant Funding Source: supported in part by NIH grant HL54269

Layered long-term co-culture of hepatocytes and endothelial cells on a transwell membrane: toward engineering the liver sinusoid

This paper presents a novel liver model that mimics the liver sinusoid where most liver activities occur. A key aspect of our current liver model is a layered co-culture of primary rat hepatocytes (PRHs) and primary rat liver sinusoidal endothelial cells (LSECs) or bovine aortic endothelial cells (BAECs) on a transwell membrane. When a layered co-culture was attempted with a thin Matrigel layer placed between hepatocytes and endothelial cells to mimic the space of Disse, the cells did not form completely separated monolayers. However, when hepatocytes and endothelial cells were cultured on the opposite sides of a transwell membrane, PRHs co-cultured with LSECs or BAECs maintained their viability and normal morphology for 39 and 57 days, respectively. We assessed the presence of hepatocyte-specific differentiation markers to verify that PRHs remained differentiated in the long-term co-culture and analyzed hepatocyte function by monitoring urea synthesis. We also noted that the expression of cytochrome P-450 remained similar in the co-cultured system from day 1 to day 48. Thus, our novel liver model system demonstrated that primary hepatocytes can be cultured for extended times and retain their hepatocyte-specific functions when layered with endothelial cells.

Effects of the green tea polyphenol epigallocatechin-3-gallate on high-fat diet-induced insulin resistance and endothelial dysfunction

Insulin resistance, a hallmark of metabolic disorders, is a risk factor for diabetes and cardiovascular disease. Impairment of insulin responsiveness in vascular endothelium contributes to insulin resistance. The reciprocal relationship between insulin resistance and endothelial dysfunction augments the pathophysiology of metabolism and cardiovascular functions. The most abundant green tea polyphenol, epigallocatechin-3-gallate (EGCG), has been shown to have vasodilator action in vessels by activation of endothelial nitric oxide synthase (eNOS). However, it is not known whether EGCG has a beneficial effect in high-fat diet (HFD)-induced endothelial dysfunction. Male C57BL/6J mice were fed either a normal chow diet (NCD) or HFD with or without EGCG supplement (50 mg·kg(-1)·day(-1)) for 10 wk. Mice fed a HFD with EGCG supplement gained less body weight and showed improved insulin sensitivity. In vehicle-treated HFD mice, endothelial function was impaired in response to insulin but not to acetylcholine, whereas the EGCG-treated HFD group showed improved insulin-stimulated vasodilation. Interestingly, EGCG intake reduced macrophage infiltration into aortic tissues in HFD mice. Treatment with EGCG restored the insulin-stimulated phosphorylation of eNOS, insulin receptor substrate-1 (IRS-1), and protein kinase B (Akt), which was inhibited by palmitate (200 μM, 5 h) in primary bovine aortic endothelial cells. From these results, we conclude that supplementation of EGCG improves glucose tolerance, insulin sensitivity, and endothelial function. The results suggest that EGCG may have beneficial health effects in glucose metabolism and endothelial function through modulating HFD-induced inflammatory response.

Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells: A POTENTIAL ROLE FOR REDUCING LIPID ACCUMULATION

Epigallocatechin gallate (EGCG) is a major polyphenol in green tea that has beneficial effects in the prevention of cardiovascular disease. Autophagy is a cellular process that protects cells from stressful conditions. To determine whether the beneficial effect of EGCG is mediated by a mechanism involving autophagy, the roles of the EGCG-stimulated autophagy in the context of ectopic lipid accumulation were investigated. Treatment with EGCG increased formation of LC3-II and autophagosomes in primary bovine aortic endothelial cells (BAEC). Activation of calmodulin-dependent protein kinase kinase β was required for EGCG-induced LC3-II formation, as evidenced by the fact that EGCG-induced LC3-II formation was significantly impaired by knockdown of calmodulin-dependent protein kinase kinase β. This effect is most likely due to cytosolic Ca(2+) load. To determine whether EGCG affects palmitate-induced lipid accumulation, the effects of EGCG on autophagic flux and co-localization of lipid droplets and autophagolysosomes were examined. EGCG normalized the palmitate-induced impairment of autophagic flux. Accumulation of lipid droplets by palmitate was markedly reduced by EGCG. Blocking autophagosomal degradation opposed the effect of EGCG in ectopic lipid accumulation, suggesting the action of EGCG is through autophagosomal degradation. The mechanism for this could be due to the increased co-localization of lipid droplets and autophagolysosomes. Co-localization of lipid droplets with LC3 and lysosome was dramatically increased when the cells were treated with EGCG and palmitate compared with the cells treated with palmitate alone. Collectively, these findings suggest that EGCG regulates ectopic lipid accumulation through a facilitated autophagic flux and further imply that EGCG may be a potential therapeutic reagent to prevent cardiovascular complications.

Abstract 141: Histone Deacetylase 1 Regulates Nitric Oxide Production and Nitric Oxide Synthase-3 Acetylation in Endothelial Cells

Previous reports showed that NOS3 is regulated by acetylation through transcriptional mechanisms via histone acetylation or through direct lysine acetylation. Histone deacetylase (HDAC) enzymes and histone acetyltransferases (HATs) modulate acetylation processes. Recent work by our lab, demonstrated increased expression of aortic HDAC1 and HDAC6 while HATs were unchanged in a mouse model of early life stress with endothelial dysfunction. These data suggest a negative correlation between endothelial dysfunction and HDAC expression. The purpose of this study was to test the hypothesis that HDAC1 and 6 regulate endothelial NO production and/or NOS3 acetylation. Initial immunoprecipitation studies with anti-acetyl lysine and anti-NOS3 antibodies demonstrated that NOS3 is basally acetylated in primary bovine aortic endothelial cells (BAECs). Treatment with the HDAC inhibitor, trichostatin A (500 nM) for 1 hr, significantly increased NOS3 acetylation. BAECs were transfected with HDAC1, HDAC6, vector expression plasmids, or untransfected, with nitrite production determined by HPLC and NOS3 acetylation and expression probed by immunoprecipitation and Western blotting. Untransfected and vector transfected control BAECs had similar NO production (357 ± 10 and 344 ± 30 pmol/mg pr/h, respectively, N=6) as well as NOS3 acetylation (7.8 ± 1.6 and 6.8 ±0.3 AU, N=3). HDAC6 transfected BAECs had similar NO production to the control BAECs (272 ± 93 pmol/mg pr/h, N=3) with an increase in NOS3 acetylation (17.4 ± 1.7 AU, N=3). In contrast, HDAC1 overexpression significantly decreased NO production (89 ± 50 pmol/mg pr/h, P< 0.05, N=3) and reduced NOS3 acetylation (3.8 ± 0.5 A.U, N=3), P <0.05). Control transfections, HDAC6, and HDAC1 transfected BAECS all had similar NOS3 expression (10.14 ± 1.8; 9.8 ±1.6; 8.9 ± 1.5; 10.6 ± 1.0 AU, respectively, N=3). Thus, we conclude that HDAC1 regulates NO production via direct lysine deacetylation of NOS3.

Abstract 9675: Ultrasound and Microbubble-Targeted Delivery of Small Interfering RNA into Primary Endothelial Cells Is More Effective than Delivery of Plasmid DNA

Introduction Ultrasound and microbubble-targeted delivery (UMTD) is a promising non-viral technique for genetic-based therapy. Recently, we demonstrated that with UMTD employing low-intensity ultrasound small dextran molecules enter endothelial cells through transient pores, while the entry of larger dextrans was dependent upon endocytosis. Extrapolating these findings to UMTD of therapeutic compounds for effective genetic-based therapy, we hypothesized that UMTD employing low-intensity ultrasound is more effective when delivering small interfering RNA (siRNA) than plasmid DNA (pDNA). Methods Red fluorescently-labeled siRNA against housekeeping gene GAPDH, pDNA encoding GAPDH, and red fluorescently-labeled pDNA encoding green fluorescent protein (GFP) were delivered into primary bovine aortic endothelial cells using ultrasound (1 MHz, 0.22 MPa) and SonoVue microbubbles. Cellular localization and GFP expression were determined using fluorescent microscopy. The effect of siRNA and pDNA delivery on GAPDH protein levels was determined by western blotting. Results UMTD of siRNA transfected 97.9±1.5% of the cells, with labeled siRNA localized homogenously in the cytoplasm directly after ultrasound exposure. Whereas UMTD of pDNA resulted in only 43.0±4.2% transfected cells, with localization mainly in vesicular structures, co-localizing with endocytosis markers clathrin and caveolin. Delivery of siRNA decreased GAPDH protein levels to 24.3±7.9% of non-treated controls (p<0.01, n=5). In contrast, 24h after delivery of pDNA no increase in GAPDH protein levels could be detected. Transfection efficiency, verified with red fluorescently-labeled pDNA encoding GFP, showed that of the transfected cells only 2.0=0.7% expressed the transgene. Conclusions The localization of small RNA and large DNA molecules after UMTD employing low-intensity ultrasound is an important determinant for the effectiveness of these two genetic-based technologies, as the target for siRNA is located in the cytosol, but for pDNA to be transcribed it needs be in the nucleus. In conclusion, this compartmentalization of therapeutic compounds should be taken into account when designing new genetic-based therapies with UMTD.

Hydrogen peroxide signaling mediates DHEA-induced vascular endothelial cell proliferation

Dehydroepiandrosterone (DHEA) activates a putative plasma membrane G i-protein coupled receptor to induce vascular endothelial proliferation. We now test the hypothesis that hydrogen peroxide (H 2O 2) signaling mediates this effect. Incubation of EA.hy926 cells, a human vascular endothelial cell line, with DHEA for 5 min produced a significant increase in H 2O 2 production, measured by oxidation of either p-hydroxyphenylacetate or dichlorodihydrofluorescein. The DHEA effect on H 2O 2 production was maximal at 1 nM DHEA, was evident within the first minute of incubation, and remained for 10 min. Similar results were present in primary bovine aortic endothelial cells. The induction of H 2O 2 in EA.hy926 cells was mimicked by a membrane-impermeable albumin-conjugated DHEA and was inhibited by either catalase or pertussis toxin. Incubation of endothelial cells with DHEA for 5 min resulted in a 2-fold increase of cyclin D1 mRNA and protein expression at 4 h. These effects were abolished by co-incubation with catalase. DHEA induced a 50 ± 7% increase in cell proliferation over 24 h, measured as cellular Ki-67 immunoreactivity. This proliferative effect was abolished by either catalase or pertussis toxin co-incubation, indicating an H 2O 2 and G i-protein-dependent effect. We conclude that H 2O 2 is a key signaling molecule mediating the proliferative effects of DHEA in vascular endothelial cells, possibly by up-regulating cell-cycle associated genes, such as cyclin D1.

Erratum to: Raman spectroscopy of primary bovine aortic endothelial cells: a comparison of single cell and cell cluster analysis

There are many techniques that allow in vitro interactions among cells and their environment to be monitored, including molecular, biochemical and immunochemical techniques. Traditional techniques for the analysis of cells often require fixation or lysis from substrates; however, use of such destructive methods is not feasible where the expanded cell cultures are required to be used for clinical implantation. Several studies have previously highlighted the potential of Raman spectroscopy to provide useful information on key biochemical markers within cells. As such, we highlight the capability of Raman spectroscopy with different laser spot sizes for use as a non-invasive, rapid, and specific method to perform in situ analysis of primary bovine aortic endothelial cells (BAECs). Raman spectra were collected from both individual live cells cultured on fused silica substrates and on clusters of live cells placed on fused silica substrates, measured at 532 and 785 nm. The results obtained show notable spectral differences in DNA/RNA region indicative of the relative cytoplasm and nucleus contributions. Raman spectra of cell clusters show slight variations in the intensity of the phenylalanine peak (1004 cm) indicating variations in protein contribution. These spectra also highlight contributions from other cellular components such as, proteins, lipids, nucleic acids and carbohydrates, respectively.

Raman spectroscopy of primary bovine aortic endothelial cells: a comparison of single cell and cell cluster analysis

There are many techniques that allow in vitro interactions among cells and their environment to be monitored, including molecular, biochemical and immunochemical techniques. Traditional techniques for the analysis of cells often require fixation or lysis from substrates; however, use of such destructive methods is not feasible where the expanded cell cultures are required to be used for clinical implantation. Several studies have previously highlighted the potential of Raman spectroscopy to provide useful information on key biochemical markers within cells. As such, we highlight the capability of Raman spectroscopy with different laser spot sizes for use as a non-invasive, rapid, and specific method to perform in situ analysis of primary bovine aortic endothelial cells (BAECs). Raman spectra were collected from both individual live cells cultured on fused silica substrates and on clusters of live cells placed on fused silica substrates, measured at 532 and 785nm. The results obtained show notable spectral differences in DNA/RNA region indicative of the relative cytoplasm and nucleus contributions. Raman spectra of cell clusters show slight variations in the intensity of the phenylalanine peak (1004cm(-1)) indicating variations in protein contribution. These spectra also highlight contributions from other cellular components such as, proteins, lipids, nucleic acids and carbohydrates, respectively.

Poly(vinyl benzoate) nanoparticles for molecular delivery: Studies on their preparation and in vitro properties

The preparation and properties of poly(vinyl benzoate) nanoparticle suspensions as molecular carriers are described for the first time. These nanoparticles can be formed by nanoprecipitation of commercial poly(vinyl benzoate) in water using Pluronic F68 as surfactant, to create spherical nanostructures measuring 200–250 nm in diameter. These nanoparticles are stable in phosphate buffer and blood serum, and only slowly degrade in the presence of esterases. Pluronic F68 stabilizes the nanoparticle and also protects it from enzymatic degradation. Up to 1.6% by weight of a lipid-soluble molecule such as coumarin-6 can be introduced into the nanoparticle during nanoprecipitation, compared to a water-soluble compound (5(6)-carboxyfluorescein) which gave almost no loading. Kinetics experiments in phosphate buffer indicate that 78% of the coumarin-6 was encapsulated within the polymer matrix of the nanoparticle, and the residual 22% of coumarin-6 was surface-bound and quickly released. The nanoparticles are non-toxic in vitro towards human epithelial cells (IC 50> 1000 μg/mL) and primary bovine aortic endothelial cells (IC 50> 500 μg/mL), and non-bactericidal against a selection of representative test microbes (MIC > 250 μg/mL). These properties suggest that the poly(vinyl benzoate) nanoparticles may be suitable carriers for molecular delivery of lipophilic small molecules such as pharmaceutical and imaging agents.

Lipoxygenase metabolites modulate vascular-derived platelet activating factor production following endotoxin challenge

Endotoxin promotes the production of potent pro-inflammatory lipid mediators, such as platelet activating factor (PAF) and eicosanoids, which contribute to the pathophysiology of endotoxic shock. Endothelial cells are both a target for and producers of these lipid mediators so it is vital to understand the pathways leading to their production in these cells. Previous research suggested a positive feedback loop between eicosanoids and PAF during endotoxemia. This study sought to determine if eicosanoids derived from the 15-lipoxygenase (15-LOX1) pathway can modulate the biosynthesis of PAF in cultured bovine aortic endothelial cells (BAEC) following endotoxin stimulation. Endotoxin stimulation increased the production of 15-LOX1-derived eicosanoids prior to PAF in primary BAEC. Exogenous addition of specific 15-LOX1 eicosanoids, as well as overexpression of 15-LOX1 in endotoxin-stimulated BAEC, further increased the endotoxin-induced production of PAF. Whereas increased expression of 15-LOX1 activity can further exacerbate endotoxin-induced PAF biosynthesis, inhibition of 15-LOX1 activity is not capable of abrogating the initial onset of endotoxin-induced PAF production. The results indicate that 15-LOX1 activity is not necessary for the initial induction of PAF following endotoxin stimulation. There may exist, however, a role for elevated 15-LOX1 activity in further escalating the extent of PAF biosynthesis in BAEC during endotoxic shock. Determining factors that can potentiate endotoxin-induced vascular dysfunction may lead to the development of novel therapeutic targets to diminish the pathophysiological effects of endotoxic shock.

The Natural Protective Mechanism Against Hyperglycemia in Vascular Endothelial Cells

OBJECTIVEVascular endothelial cells (VECs) downregulate their rate of glucose uptake in response to hyperglycemia by decreasing the expression of their typical glucose transporter GLUT-1. Hitherto, we discovered critical roles for the protein calreticulin and the arachidonic acid–metabolizing enzyme 12-lipoxygenase in this autoregulatory process. The hypothesis that 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), the peroxidation product of 12-lipoxygenase, mediates this downregulatory mechanism by activating peroxisome proliferator–activated receptor (PPAR) δ was investigated.RESEARCH DESIGN AND METHODSEffects of 4-HDDE and PPARδ on the glucose transport system and calreticulin expression in primary bovine aortic endothelial cells were evaluated by pharmacological and molecular interventions.RESULTSUsing GW501516 (PPARδ agonist) and GSK0660 (PPARδ antagonist), we discovered that high-glucose–induced downregulation of the glucose transport system in VECs is mediated by PPARδ. A PPAR-sensitive luciferase reporter assay in VECs revealed that high glucose markedly increased luciferase activity, while GSK0660 abolished it. High-performance liquid chromatography analysis showed that high-glucose incubation substantially elevated the generation of 4-HDDE in VECs. Treatment of VECs, exposed to normal glucose, with 4-HDDE mimicked high glucose and downregulated the glucose transport system and increased calreticulin expression. Like high glucose, 4-HDDE significantly activated PPARδ in cells overexpressing human PPAR (hPPAR)δ but not hPPARα, -γ1, or -γ2. Moreover, silencing of PPARδ prevented high-glucose–dependent alterations in GLUT-1 and calreticulin expression. Finally, specific binding of PPARδ to a PPAR response element in the promoter region of the calreticulin gene was identified by utilizing a specific chromatin immunoprecipitation assay.CONCLUSIONSCollectively, our data show that 4-HDDE plays a central role in the downregulation of glucose uptake in VECs by activating PPARδ.

Persistence of a Staphylococcus aureus small colony variants ( S. aureus SCV) within bovine mammary epithelial cells

Persistent bovine Staphylococcus aureus mastitis is attributable to the versatility of this pathogen within the mammary gland environment and to the formation of small colony variants (SCVs) that can survive within host cells. Previous studies had shown that S. aureus SCV Heba3231, isolated from a cow with chronic mastitis, had invaded and persisted in primary bovine aortic endothelial cells but caused minimal deleterious effects. The objective of this study was to investigate the interaction of SCV Heba3231 with bovine mammary epithelial cells (MAC-T cells) compared to its parent strain 3231 and to prototype strain Newbould 305. Monolayer cells were infected with each strain at various multiplicity of infections (MOIs) for 1 and 3.5 h, followed by 20 min incubation with lysostaphin. Recovery of the SCV was significantly higher ( P < 0.05) after 3.8 h with MOI of 100 compared to recovery of strains 3231 and Newbould 305. Upon further incubation, viable SCV were detected up to 96 h while 3231 were not isolated at 24 h or later. Transmission electron microscopy demonstrated SCV uptake by MAC-T cells following a series of events similar to those for strain 3231. At 24 h, multiple SCV were seen within enclosed vacuoles, while the 3231 parent strain was released extracellularly and the monolayer cells were damaged. The ability of SCV Heba3231 to survive inside vacuoles could be related to up-regulation of protective mechanisms. These findings highlight the potential role of bovine mammary epithelial cells and S. aureus SCV in persistent bovine mastitis.