Rabbit Aortic Endothelial Cells Research Articles

Effects of syndecan-4 silencing on the extracellular matrix remodeling in anoikis-resistant endothelial cells.

Anoikis is a process of programmed cell death induced by the loss of cell/matrix interactions. In previous work, we have shown that the acquisition of anoikis resistance upregulates syndecan-4 (SDC4) expression in endothelial cells. In addition, SDC4 gene silencing by microRNA interference reverses the transformed phenotype of anoikis-resistant endothelial cells. Due to this role of SDC4 in regulating the behavior of anoikis-resistant endothelial cells, we have evaluated that the functional consequences of SDC4 silencing in the extracellular matrix (ECM) remodeling in anoikis-resistant rabbit aortic endothelial cells submitted to SDC4 gene silencing (miR-Syn4-Adh-1-EC). For this, we evaluated the expression of adhesive proteins, ECM receptors, nonreceptor protein-tyrosine kinases, and ECM-degrading enzymes and their inhibitors. Altered cell behavior was monitored by adhesion, migration, and tube formation assays. We found that SDC4 silencing led to a decrease in migration and angiogenic capacity of anoikis-resistant endothelial cells; this was accompanied by an increase in adhesion to fibronectin. Furthermore, after SDC4 silencing, we observed an increase in the expression of fibronectin, collagen IV, and vitronectin, and a decrease in the expression of integrin α5β1 and αvβ3, besides that, silenced cells show an increase in Src and FAK expression. Quantitative polymerase chain reactionand Western blot analysis demonstrated that SDC4 silencing leads to altered gene and protein expression of MMP2, MMP9, and HSPE. Compared withparental cells, SDC4 silenced cells showed a decrease in nitric oxide production and eNOS expression. In conclusion, these data demonstrate that SDC4 plays an important role in ECM remodeling. In addition, our findings represent an important step towardunderstanding the mechanism by which SDC4 can reverse the transformed phenotype of anoikis-resistant endothelial cells.

Best Practices for Authentication of Cell Lines to Ensure Data Reproducibility and Integrity.

Cell line misidentification and contamination are major contributors to the reproducibility crisis in academic research. Authentication of cell lines provides assurances of the data generated; however, commercially available cells are often not subjected to rigorous identification testing. In this study, commercially available cell lines underwent testing to confirm cell identity and purity. The methods reported here outline the best practices for cell line authentication. Briefly, a commercially available primary rabbit aortic endothelial cell line was purchased for the intent of producing target proteins necessary for generating species-specific recombinant antibodies. These rabbit-specific antibodies would then be utilized for the development of in-house enzyme-linked immunosorbent assays (ELISA) to evaluate blood-based biomarkers of vascular injury after total-body irradiation. To authenticate the cell line, cell identity and purity were determined by single tandem repeat (STR) testing, flow cytometry, polymerase chain reaction (PCR), and cytochrome c oxidase subunit 1 (CO1) DNA Barcoding in-house and/or through commercial vendors. Fresh cells obtained from a New Zealand White rabbit (Charles River, Wilmington, DE) were used as a positive control. The results of STR and flow cytometry analyses indicated the cells were not contaminated with human or mouse cells, and that the cells were not of endothelial origin. PCR demonstrated that cells were also not of rabbit origin, which was further confirmed by a third-party vendor. An unopened vial of cells was submitted to another vendor for CO1 DNA Barcoding analysis, which identified the cells as being purely of bovine origin. Results revealed that despite purchase through a commercial vendor, the cell line marketed as primary rabbit aortic endothelial cells were of bovine origin. Purity analysis found cells were misidentified rather than contaminated. Further investigation to determine the cell type was not performed. The most cost-effective and efficient methodology for confirming cell line identity was found to be CO1 DNA Barcoding performed by a commercial vendor.

Low-Intensity Focused Ultrasound-Responsive Ferrite-Encapsulated Nanoparticles for Atherosclerotic Plaque Neovascularization Theranostics.

Pathological angiogenesis is a crucial factor that causes atherosclerotic plaque rupture. Sinoporphyrin sodium‐mediated sonodynamic therapy (DVDMS‐SDT) induces regression of plaque neovascularization in humans without causing obvious side effects. However, a clinical noninvasive theranostic strategy for atherosclerotic plaque neovascularization is urgently needed. A nanoplatform designed for multimodality imaging‐guided SDT in plaque angiogenesis theranostics, termed PFP–HMME@PLGA/MnFe2O4–ramucirumab nanoparticles (PHPMR NPs), is fabricated. It encapsulates manganese ferrite (MnFe2O4), hematoporphyrin monomethyl ether (HMME), and perfluoropentane (PFP) stabilized by polylactic acid‐glycolic acid (PLGA) shells and is conjugated to an anti‐VEGFR‐2 antibody. With excellent magnetic resonance imaging (MRI)/photoacoustic/ultrasound imaging ability, the distribution of PHPMR NPs in plaque can be observed in real time. Additionally, they actively accumulate in the mitochondria of rabbit aortic endothelial cells (RAECs), and the PHPMR NP‐mediated SDT promotes mitochondrial‐caspase apoptosis via the production of reactive oxygen species and inhibits the proliferation, migration, and tubulogenesis of RAECs. On day 3, PHPMR NP‐mediated SDT induces apoptosis in neovessel endothelial cells and improves hypoxia in the rabbit advanced plaque. On day 28, PHPMR NP‐mediated SDT reduces the density of neovessels, subsequently inhibiting intraplaque hemorrhage and inflammation and eventually stabilizing the plaque. Collectively, PHPMR NP‐mediated SDT presents a safe and effective theranostic strategy for inhibiting plaque angiogenesis.

Effect of angiotensin-converting enzyme inhibitor on DNA damage and inflammatory molecules expression in rabbit aortic endothelial cells cultured in vitro in hypercholesterolemic and hyperglycemic conditions

Effect of angiotensin-converting enzyme inhibitor on DNA damage and inflammatory molecules expression in rabbit aortic endothelial cells cultured in vitro in hypercholesterolemic and hyperglycemic conditions

Live endothelial cells on plasma-nitrided and oxidized titanium: An approach for evaluating biocompatibility.

We evaluated the effects of titanium plasma nitriding and oxidation on live endothelial cell viscoelasticity. For this, mechanically polished titanium surfaces and two surfaces treated by planar cathode discharge in nitriding (36N2 and 24H2) and oxidant (36O2 and 24H2). Surfaces were characterized regarding wettability, roughness and chemical composition. Rabbit aortic endothelial cells (RAECs) were cultured on the titanium surfaces. Cell morphology, viability and viscoelasticity were evaluated by scanning electron microscopy (SEM), methyl thiazolyl tetrazolium (MTT) assay and atomic force microscopy (AFM), respectively. Grazing Incidence X-ray Diffraction confirmed the presence of TiN0,26 on the surface (grazing angle theta 1°) of the nitrided samples, decreasing with depth. On the oxidized surface had the formation of TiO3 on the material surface (Theta 1°) and in the deeper layers was noted, with a marked presence of Ti (Theta 3°). Both plasma treatments increased surface roughness and they are hydrophilic (angle <90°). However, oxidation led to a more hydrophilic titanium surface (66.59°±3.65 vs. 76.88°±2.68; p=0.001) due to titanium oxide films in their stoichiometric varieties (Ti3O, TiO2, Ti6O), especially Ti3O. Despite focal adhesion on the surfaces, viability was different after 24h, as cell viability on the oxidized surface was higher than on the nitrided surface (9.1×103 vs. 4.5×103cells; p<0.05). This can be explained by analyzing the viscoelastic property of the cellular cytoskeleton (nuclear and peripheral) by AFM. Surface oxidation significantly increased RAECs viscoelasticity at cell periphery, in comparison to the nucleus (2.36±0.3 vs. 1.5±0.4; p<0.05), and to the RAECs periphery in contact with nitrided surfaces (1.36±0.7; p<0.05) and polished surfaces (1.55±0.6; p<0.05). Taken together, our results have shown that titanium plasma treatment directly increased cell viscoelasticity via surface oxidation, and this mechanobiological property subsequently increased biocompatibility.

205 - The role of Sdc-4 proteoglycan in the production of NO in endothelial cells

Proteoglycans are transmembrane proteins that act as a signal transducer in communication between cells and with the extracellular matrix. In a recently published article (Lucena et al., 2018) we have shown that there is an increase in the production of reactive oxygen species and nitric oxide (NO) in mutant CHO cells for the expression of Xylosyl transferase, the enzyme responsible for the addition of glycosaminoglycans (GAGs) to the core protein. This production is associated with an increase in endothelial Nitric Oxide Synthase (NOS3) activity. In another recent publication (Cavalheiro et al., 2017), syndecan-4 (Sdc-4), a heparan sulfate proteoglycan (HSPG), was silenced in a rabbit aortic endothelial cell (RAEC). It was observed that Sdc-4 is a central mediator in focal adhesion complex and, when absent, it led to a disruption of the cytoskeleton. This feature was further explored in this present work because we were unable to cultivate the cells in shear stress conditions, most of them would always detach from the surfaces. The wild-type endothelial cells presented an activation of focal adhesion complex. We also measured an increased level of intracellular oxidation (GSSG :GSH ratio) in this present work, as it was observed in the CHO model. Another resemblance was the higher production NO in comparison to the wild-type cells, in this case, the production is maintained by the inducible NOS (NOS2). In conclusion, we account an important role for HSPG on REDOX status alterations within cells, but Sdc-4 has been shown to regulate this by specifically altering iNOS expression. (Financing agencies: FAPESP, CAPES and CNPq).

A Novel Tissue Culture Tray for the Study of Magnetically Induced Rotation and Translation of Iron Oxide Nanoparticles

Iron oxide nanoparticles (IONs) hold promise for enhancing drug delivery for cancer, stroke, and other diseases. Here, we demonstrate the use of a multiple-lane tissue culture tray and rotating permanent magnet to study magnetically induced rotation and translation (MIRT) of magnetic microbeads (MBs). MBs are nanoparticle aggregates with single-crystalline magnetite cores which can be moved by surface traction. The MIRT tray was produced by additive manufacturing (3-D printing) or subtractive manufacturing (milling) of acrylic. The velocity of MBs in the MIRT tray through phosphate-buffered saline, culture media with fetal bovine serum (FBS), and 100 FBS was measured from 7.5 to 30cm above the rotating magnet. A maximum velocity of greater than 5cms occurred at a distance of 22.5cm. Cultured rabbit aortic endothelial cells, grown in lanes of the sterilized MIRT tray, cause some reduction in MB velocity. We conclude that MBs are easily rotated and moved at physiologic distances by the rotating permanent magnet. Thus, MIRT can potentially be used to direct MBs in clinical drug delivery, and the MIRT tray provides a convenient model system for testing variables which can affect ION movement.

Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration

The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras – ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (RasN17) or with the non-nitrosatable mutant of Ras (RasC118S); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.

The NF-κB pathway: regulation of the instability of atherosclerotic plaques activated by Fg, Fb, and FDPs

Recently, the molecular mechanism responsible for the instability of atherosclerotic plaques has gradually become a hot topic among researchers and clinicians. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play an important role in the processes of formation and development of atherosclerosis. In this study, we established and employed the transwell co-culture system of rabbit aortic endothelial cells and smooth muscle cells to explore the relationship between fibrin (Fb), fibrinogen (Fg), and/or their degradation products (FDPs) in relation to the instability of atherosclerotic plaques; meanwhile, we observed the effects of Fg, Fb, and FDPs on the mRNA levels of MMPs and VEGF as well as on the activation of nuclear factor-kappa B (NF-κB). We concluded that Fb, Fg, and FDPs are involved in the progression of the instability of atherosclerotic plaques via increasing the expression of MMPs and VEGF. This effect might be mediated by the NF-кB pathway.

Angiotensin (1–7) ameliorates angiotensin II-induced inflammation by inhibiting LOX-1 expression

Endothelial dysfunction plays an important role in all stages of atherosclerosis and is characterized by an increased proinflammatory response. This study investigated the effect of angiotensin (1-7) on angiotensin II (Ang II)-mediated inflammation in endothelial cells (ECs) and uncovered its molecular mechanism. Real-time PCR and western blot analysis were used to determine lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression. Ang II treatment induced inflammation, as measured by the production of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, by activating nuclear factor-κB (NF-κB) in ECs. Ang II also induced LOX-1 expression in human ECs and rabbit aortic ECs. LOX-1 played an essential role in Ang II-mediated inflammation because Ang II antagonists or small interference RNA significantly decreased Ang II-induced VCAM-1 production. LOX-1 overexpression enhanced Ang II-mediated inflammation. LOX-1 mediated Ang II-induced inflammation by inducing NF-κB DNA-binding activity. Angiotensin (1-7) inhibited LOX-1 expression and diminished Ang II-mediated inflammation in ECs. Our findings suggest that angiotensin (1-7) prevents Ang II-induced inflammation by inhibiting LOX-1 mRNA and protein expression in ECs and may represent a novel pleiotropic effect of angiotensin (1-7).

Antiproliferative Activity of Fucan Nanogel

Sulfated fucans comprise families of polydisperse natural polysaccharides based on sulfated L-fucose. Our aim was to investigate whether fucan nanogel induces cell-specific responses. To that end, a non toxic fucan extracted from Spatoglossum schröederi was chemically modified by grafting hexadecylamine to the polymer hydrophilic backbone. The resulting modified material (SNFuc) formed nanosized particles. The degree of substitution with hydrophobic chains was close to 100%, as estimated by elemental analysis. SNFfuc in aqueous media had a mean diameter of 123 nm and zeta potential of −38.3 ± 0.74 mV, as measured by dynamic light scattering. Nanoparticles conserved their size for up to 70 days. SNFuc cytotoxicity was determined using the MTT assay after culturing different cell lines for 24 h. Tumor-cell (HepG2, 786, H-S5) proliferation was inhibited by 2.0%–43.7% at nanogel concentrations of 0.05–0.5 mg/mL and rabbit aorta endothelial cells (RAEC) non-tumor cell line proliferation displayed inhibition of 8.0%–22.0%. On the other hand, nanogel improved Chinese hamster ovary (CHO) and monocyte macrophage cell (RAW) non-tumor cell line proliferation in the same concentration range. The antiproliferative effect against tumor cells was also confirmed using the BrdU test. Flow cytometric analysis revealed that the fucan nanogel inhibited 786 cell proliferation through caspase and caspase-independent mechanisms. In addition, SNFuc blocks 786 cell passages in the S and G2-M phases of the cell cycle.

Effects of heparinoids from algae on hemostasis and their action on the cycle cell

Heparin was the first component used as an anticoagulant and antithrombotic agent; therefore, its use presents limitations due to side effects, limited source of material and occurrence of contamination. Marine sulfated polysaccharides provide a low level of contamination by prions and viruses as they are extracted exclusively from a marine source. Heparin-like compounds, sulfated galactans, extracted from red marine algae Hypnea musciformis and fractionated with 1 volume of acetone (F1.0), were evaluated for their anticoagulant activity by activated partial thromboplastin time (aPTT) and prothrombin time (PT) tests by examining their action on enzymatic activity of thrombin and FXa, and their stimulatory action on antithrombotic heparan sulfate synthesis using [35S]-sodium sulfate, and determining how they promote the inhibition of cell proliferation. The aPTT test displayed a low anticoagulant activity at 350μg of F1.0 (∼90s) P<0.001. F1.0 caused an inhibition of around 50% on the enzymatic activity of the coagulation factor thrombin. This compound also exhibited a strong stimulatory action on antithrombotic heparan sulfate synthesis (4.6 times) by rabbit aortic endothelial cells (RAEC). The analysis showed that F1.0 causes inhibition of proliferation of RAEC cells due to blocking the cycle cell progression once the cells accumulate at the S phase of the cycle; nevertheless, F1.0 is not cytotoxic for this cell lineage because it does not cause apoptosis or necrosis in RAEC cells. Thus, this polymer has potential against the occurrence of thrombosis once it provides antithrombotic action.

Dihydrotestosterone Inhibits Lectin-Like Oxidized-LDL Receptor-1 Expression in Aortic Endothelial Cells via a NF-κB/AP-1-Mediated Mechanism

The mechanisms involved in the antiatherosclerotic effects of androgens are unclear. Although lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in endothelial cells plays critical roles in atherosclerosis, the effects of androgens on endothelial LOX-1 expression has not been examined. Therefore, to investigate the effects of dihydrotestosterone (DHT) on LOX-1 expression in rabbit aortic endothelial cells and cultured human aortic endothelial cells (HAEC), pellets containing DHT or placebo were s.c. implanted into 26 male New Zealand white rabbits at the time of castration or sham operation. The rabbits were then fed a high-cholesterol diet (HCD) for 2 wk. Microscopic examination of the aortic arch revealed that DHT significantly reduced HCD-induced LOX-1 expression in endothelial cells compared with placebo. In cultured HAEC, DHT at concentrations above 10(-9) to 10(-7) mol/liter inhibited TNFα-induced LOX-1 mRNA and protein expression. Deletion and mutation analysis of human LOX-1 promoter-luciferase constructs transfected into HAEC with an androgen receptor (AR) expression plasmid revealed that the 12-O-tetradecanoylphorbol-13-acetate (TPA) response element (TRE; nucleotides -60/-53) contributed to the inhibitory effects of DHT on TNFα-induced LOX-1 expression. Chromatin immunoprecipitation (ChIP) and re-ChIP assays revealed that TNFα- and TPA-dependent enrichment of p65 and phosphorylated c-Jun in the TRE chromatin region was inhibited by DHT-AR. Consistent with these results, DHT also suppressed TPA-induced expression of LOX-1. In conclusion, DHT exerts antiatherosclerotic effects by suppressing endothelial LOX-1 expression. This effect is partly mediated by the suppression of nuclear factor-κB- and activator protein 1-dependent activation of the LOX-1 promoter.

The uraemic milieu, the ubiquitin-proteasome pathway and the endothelial cell: a pathway to dysfunction

Chronic kidney disease (CKD) is one of the most common chronic diseases and often leads to end-stage kidney disease (ESKD) requiring dialysis and transplantation. Cardiovascular disease (CVD) is the major cause of morbidity and mortality in CKD and ESKD (Collins et al. 2011). In fact, more CKD patients die from CVD than progress to ESKD. Much of the vascular disease seen in ESKD is caused by atherosclerosis and is a consequence of many interconnected risk factors. These may be traditional risk factors, such as hypertension, dyslipidaemia, diabetes and smoking, or non-traditional factors, including oxidative stress, inflammation and endothelial dysfunction, which lead to arterial stiffness and atherosclerosis. However, the exact mechanism whereby vascular damage occurs in CKD remains unclear. Oxidative stress and inflammation are now accepted pathophysiological processes seen in CVD and progression of CKD. However, uraemic toxins may specifically, as well as indirectly, contribute to endothelial dysfunction, arterial stiffness and atherosclerosis (Feng et al. 2011). Recent evidence from a study conducted in a population of CKD patients without comorbidities, such as diabetes and hypertension, suggests that in CKD, stages 4–5, with higher degrees of uraemia and close to dialysis, exhibit endothelial dysfunction, whereas the earlier stages (2–3) do not (Lilitkarntakul et al. 2011). This is in contrast to previous studies that included patients with comorbidities that showed the presence of endothelial dysfunction even in the earlier CKD stages (2–3). In this issue of Experimental Physiology, Feng et al. (2011) provide new information from in vitro experimental studies on endothelial cells exposed to uraemic serum. They explore potential mechanisms involving the ubiquitin–proteasome pathway (UPP), whereby uraemic toxins cause endothelial cell dysfunction. This pathway regulates cell homeostasis by clearing proteins and regulating signal transduction, proliferation and apoptosis. They had previously demonstrated that inflammation and atherosclerosis in the aortic wall were inhibited by the proteasome inhibitor, MG132, in uraemic rabbits (Feng et al. 2010). In the present experiments, they cultured rabbit aortic endothelial cells (RAECs) in the presence of 10% uraemic serum. The proliferative activity of RAECs was significantly increased when they were exposed to uraemic serum, which was attenuated by MG132. In addition, uraemic serum activated nuclear factor-κB in RAECs, which was inhibited by MG132. Also, tumour necrosis factor-α protein expression was increased in RAECs by uraemic serum, which was also inhibited by MG132. Finally, they showed that biomarkers of nitric oxide (NO) production were decreased after exposure to uraemic serum, which were again inhibited by MG132. Nitric oxide is considered to be a potent biomarker of endothelial function. This novel series of experiments clearly demonstrates uraemic activation of the UPP with resultant activation of inflammation. This provides at least one potential mechanism explaining uraemia-induced endothelial dysfunction assessed in vitro. Defining such mechanisms is important because it may lead to the development of new, targeted therapies, which may reduce the burden of vascular disease in patients with CKD. Therapies tested so far in uraemic patients on haemodialysis have endeavoured to reduce mortality and cardiovascular events. These have included large multicentre randomized controlled trials, which have all been unsuccessful. The only positive studies in this area used antioxidant therapies, such as N-acetyl cysteine and vitamin E. However, these trials were limited by study design and sample size, have not been translated into practice and hence require validation. Other new evidence from studies using a nephron reduction model in vivo have identified that bone marrow derived stromal cells (mesenchymal stem cells; MSCs), which may differentiate into endothelial cells, become functionally incompetent in the presence of uraemia (Noh et al. 2011). The investigators showed that there was decreased expression of vascular endothelial growth factor, vascular endothelial growth factor receptor and stromal cell derived factor-1α in uraemic animals. In addition, there was decreased angiogenesis in MSCs in the uraemic environment. In the present study reported in this issue of Experimental Physiology, Feng et al. (2011) demonstrated the role of the UPP on endothelial cells in uraemia but did not determine which molecules within the uraemic serum were responsible for activation of the UPP. One of the next challenges will be the identification of the individual uraemic toxins responsible for vascular damage. This would have the potential to lead to the design of specific targeted therapies, much needed in patients with uraemic kidney disease. In addition, the exact mechanism by which uraemic serum activates the UPP needs further elucidation. Future research should also investigate earlier stages of CKD with lower levels of uraemic toxins, using in vivo studies with animal CKD models, such as nephron reduction with five-sixth nephrectomy or the oral administration of adenine. This may determine the threshold where endothelial dysfunction is initiated and therapies need to be applied.

Uraemic serum induces dysfunction of vascular endothelial cells: role of ubiquitin-proteasome pathway

The ubiquitin-proteasome pathway (UPP) has been indicated to contribute to dysfunction of endothelial cells (ECs). Nevertheless, the relationship between UPP and vascular complications of uraemia remains unknown. We aimed to determine whether the UPP is activated in vascular ECs when cultured with uraemic serum, and to examine the role of the UPP on dysfunction of ECs in uraemia. Rabbit aortic endothelial cells (RAECs) were cultured with normal serum or different concentrations of uraemic serum. The expression of the ubiquitin-activating enzyme (E1), an indicator of the UPP, was detected by real-time RT-PCR and Western blot; proteasome activity was determined by fluorescence spectrophotometry; and nuclear factor-κB (NF-κB) activity and expression, as well as tumour necrosis factor-α (TNF-α) expression, were also detected. We found that the expression of E1 and the activities of three kinds of proteasomes were increased significantly in RAECs after incubation with uraemic serum. Proliferation of RAECs was increased significantly by incubation with 3-15% uraemic serum but decreased markedly when incubated with uraemic serum above 15% (increased apoptosis). Incubation of RAECs with uraemic serum induced increased NF-B DNA-binding activity and nuclear translocation of NF-κB, decreased nitric oxide production and increased expression of TNF-α, which is the final effector of inflammatory activation of cells. All of these responses in RAECs were suppressed by the specific proteasome inhibitor, MG132. The inhibition of inflammatory responses by MG132 was further supported by a parallel experiment with pyrrolidine dithiocarbamate, a specific inhibitor of κNF-B. These findings suggest that the UPP was activated in RAECs by administration of uraemic serum, and played a pivotal role in the dysfunction of vascular ECs, such as inflammatory activation.

Uncovering the Vasorelaxant Effect Induced by Vale do São Francisco Red Wine: A Role for Nitric Oxide

The aim of this study was to investigate the mechanisms underlying the vasorelaxant effect induced by the polyphenolic compounds found in red wine from Vale do São Francisco. In phenylephrine (10 μM) precontracted mesenteric artery rings, the red wine caused a concentration-dependent relaxation (maximum response to phenylephrine 10 μM = 87.5% ± 6.5%, n = 10). After endothelium removal, the vasorelaxant effect elicited by red wine was attenuated (28.4% ± 4.9%, n = 10). In addition, the vasorelaxant effect induced by red wine in rings pretreated with 100 μM of N(w)-nitro-l-arginine methyl ester and 10 μM of 1H-[1,2,4] oxadiazolo-[4,3-a]-quinoxalin-1-one was attenuated (23.4% ± 5.1%, n = 7 and 11.8% ± 2.7%, n = 6, respectively). Pretreatment with atropine did not affect the vasorelaxant effect induced by red wine (81% ± 3.9%, n = 6). Furthermore, in rabbit aortic endothelial cell line, red wine 100 and 300 μg/mL caused concentration-dependent increases in nitric oxide levels (58 ± 1; 82 ± 7.9; Δ% of fluorescence, n = 5, respectively). In conclusion, we suggest that the alcohol free-lyophilized red wine induces an endothelium-dependent vasorelaxant effect due, at least in part, to a secondary increase in the concentration of nitric oxide and that this effect might be associated with phenolic compounds found in the red wine.

Cardiovascular Effects Elicited by Milonine, a New 8,14‐Dihydromorphinandienone Alkaloid

The mechanisms underlying the cardiovascular responses evoked by milonine (i.v.), an alkaloid, were investigated in rats. In normotensive rats, milonine injections produced hypotension and tachycardia, which were attenuated after N(w) -nitro-L-arginine methyl esther (L-NAME; 20 mg/kg, i.v.). In phenylephrine (10 μM), pre-contracted mesenteric artery rings, milonine (10⁻¹⁰ M to 3 × 10⁻⁴ M) caused a concentration-dependent relaxation (EC₅₀ = 1.1 × 10⁻⁶ M, E(max) = 100 ± 0.0%) and this effect was rightward shifted after either removal of the vascular endothelium (EC₅₀ = 1.6 × 10⁻⁵, p < 0.001), or after L-NAME 100 μM (EC₅₀ = 6.2 × 10⁻⁵, p < 0.001), hydroxocobalamin 30 μM (EC₅₀ = 1.1 × 10⁻⁴, p < 0.001) or ODQ 10 μM (EC₅₀ = 1.9 × 10⁻⁴ p < 0.001). In addition, in rabbit aortic endothelial cells, milonine increased NO₃⁻ levels. The relaxant effect induced by milonine was attenuated in the presence of KCl (20 mM), a modulator efflux K(+) (EC₅₀ = 1.2 × 10⁻⁵, p < 0.001), or different potassium channel blockers such as glibenclamide (10 μM) (EC₅₀ = 6.3 × 10⁻⁵, p < 0.001), TEA (1 mM) (EC₅₀ = 2.3 × 10⁻⁵ M, n = 6) or Charybdotoxin (0.2 μM) plus apamin (0.2 μM) (EC₅₀ = 3.9 × 10⁻⁴ M, n = 7). In addition, pre-contraction with high extracellular potassium concentration prevented milonine-induced vasorelaxation (EC₅₀ = 1.0 × 10⁻⁴, p < 0.001). Milonine also reduced CaCl₂ -induced contraction in Ca²(+) -free solution containing KCl (60 mM). In conclusion, using combined functional and biochemical approaches, we demonstrated that the hypotensive and vasorelaxant effects produced by milonine are, at least in part, mediated by the endothelium, likely via nitric oxide release, activation of nitric oxide-cGMP pathway and opening of K(+) channels.

Potential effect of uremic medium on cell proliferation and apoptosis and the activation of nuclear factor-kappa B pathway of aortic endothelial cells in rabbit culture

To investigate the potential effect of uremic medium on cell proliferation and apoptosis of aortic endothelial cell (AEC), two key processes in the development of atherosclerosis, in rabbit culture. And to understand the effects of uremic medium on the activation of nuclear factor-kappa B (NF-κB) pathway and cytokines expression of AEC. Rabbit AEC were cultured with growth media supplemented with pooled sera from normal rabbits or those with chronic renal failure. The 80% confluent AEC were incubated for 24 h with media supplemented with pools of control or uremic sera. Cell proliferation was assessed by a MTT assay and cell cycle detected by flow cytometry. Hoechst33342 assay and flow cytometry were used to investigate the apoptotic effect of uremic medium in AEC. The expression of mRNA and protein levels for NF-κB, IκBα were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. NF-κB P65 nuclear translocation was analyzed by immunofluorescence. The activity of NF-κB was measured by electrophoretic mobility shift assay (EMSA). Concentrations of TNF-α and IL-6 in culture supernatants were evaluated by ELISA, and the expression of protein for TNF-α in cell lysates by Western blot. Uremic medium induced proliferation in the lower concentration range of 3%-10% while promoted apoptosis in the higher concentrations (> 10%). Uremic serum increased NF-κB mRNA (0.35 ± 0.05 vs 0.26 ± 0.02, P < 0.01) and protein (1.67 ± 0.15 vs 0.41 ± 0.05, P < 0.01) expression, decreased IκBα mRNA (0.13 ± 0.03 vs 0.24 ± 0.04, P < 0.01) and protein (0.29 ± 0.06 vs 0.65 ± 0.08, P < 0.01) expression. Uremic serum enabled NF-κB p65 nuclear translocation and increased NF-κB DNA binding activity. An increased secretion of cytokines IL-6 and TNF-α. in AEC was observed after a treatment of 10% uremic sera in a time dependent manner. The expression of TNF-α in AEC exposed to 10% uremic sera also increased significantly (0.37 ± 0.04 vs 0.14 ± 0.03, P < 0.01). Uremic medium induces the activation of AEC. A lower level of uremic medium accelerates the proliferation of AEC while a higher level induces the apoptosis of AEC. The increased proliferation may be related to a higher NF-κB activity and the expression of inflammation cytokines. Although the enhanced atherosclerosis can not be explained on the basis of an apoptotic process, the proliferative status can contribute to intimal proliferation, an earlier step in the development of atherosclerosis.

Unravelling the cardiovascular effects induced by α‐terpineol: A role for the nitric oxide–cGMP pathway

Summary1. α‐Terpineol is a monoterpene found in the essential oils of several aromatic plant species. In the present study, we investigated the mechanisms underlying the cardiovascular changes induced by α‐terpineol in rats.2. In normotensive rats, administration of α‐terpineol (1, 5, 10, 20 and 30 mg/kg, i.v.) produced a dose‐dependent hypotension (−10 ± 3, −20 ± 8, −39 ± 16, −52 ± 21 and −57 ± 23 mmHg, respectively; n = 5) followed by tachycardia. The hypotensive responses to 1, 5, 10, 20 and 30 mg/kg, i.v., α‐terpineol were significantly attenuated following the administration of NG‐nitro‐l‐arginine methyl ester (l‐NAME; 20 mg/kg, i.v.; −2 ± 1, −5 ± 2, −7 ± 3, −22 ± 9 and −22 ± 10 mmHg, respectively; P &lt; 0.05; n = 5).3. In 10 μmol/L phenylephrine (PE)‐precontracted mesenteric artery rings, α‐terpineol (10−12 to 10−5 mol/L) caused a concentration‐dependent relaxation (maximum relaxation 61 ± 6%; n = 7). After removal of the endothelium, the vasorelaxation elicited by α‐terpineol was attenuated (maximum relaxation 20 ± 1%; P &lt; 0.05; n = 7). In addition, vasorelaxation induced by α‐terpineol in rings pretreated with 100 or 300 μmol/L l‐NAME, 30 μmol/L hydroxocobalamin or 10 μmol/L 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one was attenuated (maximum relaxation 18 ± 3, 23 ± 3, 24 ± 7 and 21 ± 1%, respectively; n = 6; P &lt; 0.05).4. Furthermore, in a rabbit aortic endothelial cell line, 10−6, 10−5 and 10−4 mol/L α‐terpineol induced concentration‐dependent increases in nitric oxide (NO) levels (12 ± 6, 18 ± 9 and 34 ± 12%Δ fluorescence, respectively; n = 3).5. In conclusion, using combined functional and biochemical approaches in the present study, we were able to demonstrate that α‐terpineol‐induced hypotension and vasorelaxation are mediated, at least in part, by the endothelium, most likely via NO release and activation of the NO–cGMP pathway.

Dose-dependent effects of propofol on expression ICAM-1 in rabbit aorta endothelial cells

The dose-dependent effects of propofol on expression intracellular adhesion molecule (ICAM-1) in rabbit aorta endothelial cells was examined. Twenty adult New Zeland albino rabbits were used in this study. One control and three experimantal groups were designed. In experimantal groups; for group I propofol was not applied, group II 0.5, group III 4.0 and group IV 8.0 mg/kg propofol were applied to rabbits by marginal ear vein. One hour after applying propofol, control and experimantal group rabbits were sacrificed and their aorta were removed. The sections were stained with APAAP immunohistochemical staining for evaluation using a light microscope. This marked ICAM-1 immunoreactivity was maintained in 8.0 mg/kg of propofol applied to rabbits of aorta endothelium, whereas mild stain was observed for other experimantal groups. The present study indicated that there is parallel relationship between endothelial cells ICAM-1 expression and different doses of propofol.