Bovine Aortic Endothelial Cells Research Articles

Abstract MP26: Activating P2Y 1 Receptors Improves Function In Arteries With Repressed Autophagy

Objective: The importance of endothelial cell (EC) autophagy to vascular homeostasis is evolving. Earlier we reported that purinergic 2Y 1 receptor (P2Y 1 -R) activation rejuvenates shear-stress induced nitric oxide (NO) generation in bovine aortic endothelial cells that is otherwise compromised after pharmacological and genetic autophagy repression. Here we determined the translational and functional relevance of these findings. Approach and Results: First we assessed translational relevance using older humans and mice that exhibit blunted EC autophagy at rest together with impaired arterial function vs. appropriate controls. Rhythmic handgrip exercise elevated radial artery shear rate similarly in adult and older males for 60-min. Compared to baseline, autophagy initiation, p-eNOS S1177 activation, and NO generation, occurred in radial artery ECs from adult but not older subjects.Regarding mice, indexes of autophagy and p-eNOS S1177 activation were robust in ECs from adult but not older mice in response to 60-min treadmill-running. Next we questioned whether an inability to initiate EC autophagy precipitates arterial dysfunction. Age-associated reductions in intraluminal flow-mediated vasodilation observed in older vs. adult mice were recapitulated in arteries from adult mice by : (i) NO synthase inhibition; (ii) autophagy impairment using 3-methyladenine (3-MA); (iii) EC Atg3 depletion (iecAtg3KO mice); (iv) P2Y 1 -R blockade; and (v) germline depletion of P2Y 1 -Rs. Importantly, P2Y 1 -R activation using 2-methylthio-ADP (2-Me-ADP) improved vasodilatory capacity in arteries from : (i) adult mice treated with 3-MA; (ii) adult iecAtg3KO mice; and (iii) older animals with repressed EC autophagy. Conclusions: Arterial dysfunction concurrent with pharmacological, genetic, and age-associated EC autophagy compromise is improved by activating P2Y 1 -Rs.

Anti-inflammatory HDL effects are impaired in atrial fibrillation

High-density lipoprotein (HDL), best known for cholesterol transport, also has anti-inflammatory effects. Previous studies suggest involvement of myeloperoxidase (MPO) in modification of HDL. HDL bound Sphingosine-1-phosphate (S1P) has been implied to be an essential protein regarding beneficial HDL effects. In this study, we analyzed anti-inflammatory HDL properties in patients with atrial fibrillation (AF), a disease involving atrial inflammation, compared to non-AF controls and whether anti-inflammatory properties improve upon catheter ablation. Additionally, association with serum concentrations of MPO and S1P were assessed. We isolated HDL from 25 AF patients, 13 non-AF individuals and 14 AF patients at follow-up (FU) after catheter ablation. S1P was measured in a cohort of 141 AF and 21 FU patients. Following preincubation with HDL from either group, bovine aortic endothelial cells were stimulated using tumor necrosis factor α and expression of pro-inflammatory genes intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), E-selectin (SELE) and P-selectin (SELP) was assessed using qPCR. Concentrations of circulating protein of these genes as well as MPO and S1P were measured in serum samples. Compared to non-AF individuals HDL from AF patients suppressed gene expression of the pro-inflammatory adhesion molecules ICAM1, VCAM1, SELE and SELP 27%, 18%, 21% and 57% less, respectively (p < 0.05 for all except SELE p = 0.06). In FU patients, the anti-inflammatory HDL activity was improved (suppression of ICAM1 + 22%, VCAM1 + 10%, SELE + 38% and SELP + 75%, p < 0.05 for all except VCAM1 p = 0.08). AF patients using angiotensin converting enzyme inhibitors or angiotensin receptor blockers had better anti-inflammatory HDL properties than non-users (gene expression suppression at least 28% more, p < 0.05 for all except ICAM1 p = 0.051). Circulating protein concentrations were not correlated with in vitro gene-expression, but circulating P-selectin was generally elevated in AF and FU patients compared to non-AF patients. MPO plasma concentration was positively associated with gene-expression of ICAM1, VCAM1 and SELP (r2 > 0.4, p < 0.05). Serum concentrations of S1P were increased in FU patients {1.201 µM [1.077–1.543]} compared to AF patients {0.953 µM [0.807–1.135], p < 0.01} but not correlated with ICAM1, VCAM1 and SELP gene expression. We conclude that the anti-inflammatory activity of HDL is impaired in AF patients, which might promote AF progression and AF-associated complications.

Impact of diet-derived trimethylamine N-oxide (TMAO) on endothelial dysfunction: An in vitro study

Background and Aims: Trimethylamine N-oxide (TMAO) is an organic compound directly absorbed from food products or endogenously synthesized from its precursors, choline and L-carnitine, that are metabolized in trimethylamine by the gut microbiota and then oxidized in TMAO by hepatic flavin monooxygenases. At physiological concentrations TMAO acts as an osmolyte, a protein stabilizer and an electron acceptor. Emerging studies show an association between high TMAO circulating levels and atherosclerotic damage. Purpose of the study is to evaluate the direct effect of TMAO on bovine aortic endothelial cells (BAEC) key parameters related to vascular dysfunction.

Studying the anticancer activity in vitro of doxorubicin-encapsulated pH-sensitive redox nanoparticles

Doxorubicin (DOX) is known as a chemotherapeutic agent for treating a variety of cancers. However, due to non-specific distribution, DOX causes severe side effects, leading to the limitation of its clinical application. In this study, pH-sensitive redox nanoparticles containing DOX were studied to form drug-coated nanoparticles with the size of about 40-60 nm that increases the concentration of drugs accumulated at the tumor site in response to low pH conditions. The optimal properties of the nanoparticles were investigated by analysing the antioxidant activity, sensitivity to pH, drug delivery in a low pH environment, and toxicity on cancer cells, thereby observing the possibility of inhibiting the migration of cancer cells. Results showed that the combination of DOX and the pH-sensitive redox nanoparticles not only increases anticancer in colorectal cancer (C-26), breast cancer (MCF-7) but also reduces bovine aorta endothelial cells (BAEC) and inhibits C-26 cell migration in vitro.Thepaper results reveal the application potential of the pH-sensitive redox nanoparticles in improving the effectiveness of cancer treatment.

Revisiting Ehrlichia ruminantium Replication Cycle Using Proteomics: The Host and the Bacterium Perspectives.

The Rickettsiales Ehrlichia ruminantium, the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.

Red light stimulates vasodilation through extracellular vesicle trafficking.

Red light (670nm) promotes ex vivo dilation of blood vessels in a nitric oxide (NO) dependent, but eNOS independent manner by secreting a quasi-stable and transferable vasoactive substance with the characteristics of S-nitrosothiols (RSNO) from the endothelium. In the present work we establish that 670nm light mediated vasodilation occurs in vivo and is physiologically stable. Light exposure depletes intracellular S-nitroso protein while concomitantly increasing extracellular RNSO, suggesting vesicular pathways are involved. Furthermore, we demonstrate this RSNO vasodilator is embedded in extracellular vesicles (EV). The action of red light on vesicular trafficking appears to increase expression of endosome associated membrane protein CD63 in bovine aortic endothelial cells, enhance endosome localization in the endothelium, and induce exit of RSNO containing EVs from murine facialis arteries. We suggest a mechanism by which the concerted actions of 670nm light initiate formation of RSNO containing EVs which exit the endothelium and trigger relaxation of smooth muscle cells.

Syndecan-1 downregulates syndecan-4 expression by suppressing the ERK1/2 and p38 MAPK signaling pathways in cultured vascular endothelial cells

Syndecan-1 and syndecan-4 are members of the syndecan family of transmembrane heparan sulfate proteoglycans. Vascular endothelial cells synthesize both species of proteoglycans and use them to regulate the blood coagulation-fibrinolytic system and their proliferation via their heparin-like activity and FGF-2 binding activity, respectively. However, little is known about the crosstalk between the expressions of the proteoglycan species. Previously, we reported that biglycan, a small leucine-rich dermatan sulfate proteoglycan, intensifies ALK5–Smad2/3 signaling by TGF-β1 and downregulates syndecan-4 expression in vascular endothelial cells. In the present study, we investigated the crosstalk between the expressions of syndecan-1 and other proteoglycan species (syndecan-4, perlecan, glypican-1, and biglycan) in bovine aortic endothelial cells in a culture system. These data suggested that syndecan-1 downregulated syndecan-4 expression by suppressing the endogenous FGF-2-dependent ERK1/2 pathway and FGF-2-independent p38 MAPK pathway in the cells. Moreover, this crosstalk was a one-way communication from syndecan-1 to syndecan-4, suggesting that syndecan-4 compensated for the reduced activity in the regulation of vascular endothelial cell functions caused by the decreased expression of syndecan-1 under certain conditions.

Dihydroxyeicosatrienoic Acid, a Metabolite of Epoxyeicosatrienoic Acids Upregulates Endothelial Nitric Oxide Synthase Expression Through Transcription: Mechanism of Vascular Endothelial Function Protection.

The present study aimed to investigate the impacts and underlying mechanisms of 14,15-DHETs on eNOS and vascular endothelial functions. Bovine aortic endothelial cells (BAECs) were treated with various concentrations of 14, 15-DHET. The expressions of eNOS protein and mRNA were observed at different time points. The eNOS expression and phosphorylation were subsequently detected administered with 8,9-DHET, 11,12-DHET, and 14,15-DHET, respectively. Meanwhile, 14,15-DHET action on tube formation was observed in human umbilical vein endothelial cells (HUVECs). Finally, the aorta of male C57BL/6 mice was injected with 14,15-DHET via the tail vein. The impacts of 14,15-DHET (0.4 mg/kg body weight) on the expressions of eNOS protein and mRNA and endothelium-dependent vasodilation (EDV) were detected following 24 h. The expression of eNOS was greatly improved with the 14,15-DHET treatment compared with the BAECs, and eNOS phosphorylation sites at Ser1179, Ser635, and Thr497 were elevated. However, no statistically significant difference was revealed on total eNOS among the 8,9-DHET, 11,12-DHET, and 14,15-DHET treatment groups. Based on the upregulation of eNOS protein, eNOS mRNA levels were increased in BAECs and thoracic aorta of the male C57BL/6 mice treated with 14,15-DHET, suggesting that transcriptional activation was achieved in vascular eNOS. Moreover, 14,15-DHET enhanced tube formation abilities in HUVECs and acetylcholine(ACh)-induced EDV. These findings indicated that 14,15-DHET could improve the vascular endothelial diastolic functions of male C57BL/6 mice, and enhance the ability of tube formation, which might be related to the increase of eNOS expression.

TRPC channel-derived calcium fluxes differentially regulate ATP and flow-induced activation of eNOS

Endothelial dysfunction, characterised by impaired nitric oxide (NO) bioavailability, arises in response to a variety of cardiovascular risk factors and precedes atherosclerosis. NO is produced by tight regulation of endothelial nitric oxide synthase (eNOS) activity in response to vasodilatory stimuli. This regulation of eNOS is mediated in part by store-operated calcium entry (SOCE). We hypothesized that both ATP- and flow-induced eNOS activation are regulated by SOCE derived from Orai1 channels and members of the transient receptor potential canonical (TRPC) channel family. Bovine aortic endothelial cells (BAECs) were pre-treated with pharmacological inhibitors of TRPC channels and Orai1 to examine their effect on calcium signaling and eNOS activation in response to flow and ATP. The peak and sustained ATP-induced calcium signal and the resulting eNOS activation were attenuated by inhibition of TRPC3, which we found to be store operated. TRPC4 blockade reduced the transient peak in calcium concentration following ATP stimulation, but did not significantly reduce eNOS activity. Simultaneous TRPC3 & 4 inhibition reduced flow-induced NO production via alterations in phosphorylation-mediated eNOS activity. Inhibition of TRPC1/6 or Orai1 failed to lower ATP-induced calcium entry or eNOS activation. Our results suggest that TRPC3 is a store-operated channel in BAECs and is the key regulator of ATP-induced eNOS activation, whereas flow stimulation also recruits TRPC4 into the pathway for the synthesis of NO.

MITOCHONDRIAL ROS MEDIATE ENDOTHELIAL DYSFUNCTION INDUCED BY CHRONIC PARATHYROID HORMONE EXPOSURE

Abstract Objective: Vitamin D deficiency contributes to cardiovascular risk (CVR), with hyperparathyroidism advocated as a putative mechanism. Indeed, mounting evidence supports the hypothesis that parathyroid hormone (PTH) impairs endothelial function, even though mechanisms are not fully elucidated. The present study was designed to verify in vitro the ability of sustained exposure to PTH to cause endothelial dysfunction, exploring the underlying mechanisms. Design and method: In bovine aortic endothelial cells (BAECs), we evaluated the effects of PTH exposure ([0,1 nM]-24 hours) on both endothelial response to vasodilators, such as bradykinin (Bk, [30 nM]) and acetylcholine (Ach [1 mM]), and angiogenic competence. Results: Pretreatment with PTH impaired endothelial response to Bk, but not to Ach, in terms of cytosolic calcium fluxes and NO production. In order to explore the underlying mechanisms, we assessed the production of total and mitochondrial ROS (respectively tROS and mROS) in response to PTH (at 1 and 3 hours). PTH increased ROS generation to an extent high enough to determine oxidation of Bk-receptor B2. Conversely, the oxidation levels oxidation of M1 and M3 Ach-receptors was not affected by PTH. A mROS selective scavenger (MitoTEMPO, [5 mM]) restored the endothelial responsiveness to Bk while the well known antioxidant properties of Vitamin D ([100 nM]) failed to counteract PTH-mediated oxidative stress. PTH determined mitochondrial calcium fluxes ([Ca2+]mt) and the inhibition of Mitochondrial Calcium Uniporter with inhibitor Ru360 [10 mM] reduced mROS production and prevented the following PTH-mediated endothelial dysfunction. Angiogenic competence was evaluated by tubular formations in Endothelial Matrigel Assay and showed a significant impairment in PTH-pretreated cells ([0,1 nM]-24 hours), despite the increase in VEGF transcriptional levels. VEGF-R2 oxidation occurred in response to PTH, suggesting that even the impairment of angiogenesis was due to ROS surge. Conclusions: These results indicate that PTH affects endothelial function through ROS production, driven by mitochondrial calcium overload. PTH-induced oxidative stress might act as signaling modifier, altering specific pathways (Bk and VEGF) and preserving others (Ach).

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.

Zearalenone Induces Endothelial Cell Apoptosis through Activation of a Cytosolic Ca2+/ERK1/2/p53/Caspase 3 Signaling Pathway.

Zearalenone (ZEN) is a mycotoxin that has been reported to damage various types of cells/tissues, yet its effects on endothelial cells (ECs) have never been investigated. Therefore, this study investigates the potential effects of ZEN using bovine aortic ECs (BAECs). In this study, we found that ZEN induced apoptosis of BAECs through increased cleavage of caspase 3 and poly ADP-ribose polymerase (PARP). ZEN also increased phosphorylation of ERK1/2 and p53, and treatment with the ERK1/2 or p53 inhibitor reversed ZEN-induced EC apoptosis. Transfection of BAECs with small interfering RNA against ERK1/2 or p53 revealed ERK1/2 as an upstream target of p53 in ZEN-stimulated apoptosis. ZEN increased the production of reactive oxygen species (ROS), yet treatment with the antioxidant did not prevent EC apoptosis. Similarly, blocking of estrogen receptors by specific inhibitors also did not prevent ZEN-induced apoptosis. Finally, chelation of cytosolic calcium (Ca2+) using BAPTA-AM or inhibition of endoplasmic reticulum (ER) Ca2+ channel using 2-APB reversed ZEN-induced EC apoptosis, but not by inhibiting ER stress using 4-PBA. Together, our findings demonstrate that ZEN induces EC apoptosis through an ERK1/2/p53/caspase 3 signaling pathway activated by Ca2+ release from the ER, and this pathway is independent of ROS production and estrogen receptor activation.

Epalrestat suppresses cadmium-induced cytotoxicity through Nrf2 in endothelial cells.

Cadmium (Cd) is an industrial and environmental pollutant that targets the vascular endothelium. The vascular system is critically affected by Cd toxicity. Recent studies have indicated an association between Cd and vascular diseases, although the mechanisms of Cd implications in vascular diseases are not clear. The purpose of the present study was to determine whether epalrestat (EPS), which is used for the treatment of diabetic neuropathy, protects against Cd-induced cytotoxicity in bovine aortic endothelial cells (BAECs). In the present study, the effects of EPS at near-plasma concentration were examined on Cd-induced cytotoxicity in BAECs. Cd-induced cytotoxicity was suppressed by pretreatment with EPS. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that serves a role in regulating the expression of glutamate cysteine ligase, the rate-limiting enzyme in glutathione (GSH) synthesis. In a previous study, EPS was demonstrated to increase GSH levels in BAECs in association with the Nrf2 pathway. In the present study, EPS increased GSH levels in BAECs exposed to Cd. The protective ability of EPS against the Cd-induced cytotoxicity disappeared following Nrf2 small interfering RNA transfection. In addition, EPS affected the intracellular levels of Cd, Cd transporter ZIP8 and metallothionein. To the best of our knowledge, the current study demonstrated, for the first time, that EPS suppresses Cd-induced cytotoxicity in BAECs. The upregulation of GSH may be associated with the suppression of Cd-induced cytotoxicity by EPS. From these findings, it may be proposed that the regulation of GSH, ZIP8 and metallothionein by EPS is a promising therapeutic approach to prevent Cd-induced toxicity.

Nuclear localization of endothelial nitric oxide synthase and nitric oxide production attenuates aphidicolin-induced endothelial cell death

Aphidicolin represses DNA replication by inhibiting DNA polymerase α and δ, which leads to cell cycle arrest and cell damage. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) plays an essential role in maintenance of endothelial integrity including endothelial cell (EC) survival. Previously, we reported that aphidicolin increases NO production in bovine aortic ECs (BAECs). However, the role of aphidicolin-induced NO on EC viability and its molecular mechanism remain to be elucidated. Treatment with 20 μM aphidicolin for 24 h reduced BAEC viability by ~40%, which was accompanied by increased NO production, phosphorylation of eNOS at Ser1179 (p-eNOS-Ser1179), and eNOS protein expression. The aphidicolin-increased eNOS expression and p-eNOS-Ser1179 were not altered by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a cell permeable and specific intracellular Ca2+ chelator. Co-treatment with 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), an NO scavenger, or Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor, exacerbated aphidicolin-stimulated BAEC death. Knockdown of eNOS gene expression using siRNA aggravated aphidicolin-induced BAEC death. However, exogenous NO donors including S-nitroso-l-glutathione (GSNO) or diethylenetriamine NONOate (DETA NO) had no effect on aphidicolin-decreased BAEC viability and aggravated BAEC viability at higher doses. Interestingly, aphidicolin accumulated eNOS protein in the active form, p-eNOS-Ser1179, in the nucleus. When cells were ectopically transfected with a wild-type (WT)-eNOS gene, aphidicolin induced significant localization of the protein product in the nucleus. Additionally, aphidicolin-elicited cell death was significantly reversed in WT-eNOS gene-transfected BAECs. Furthermore, overexpression of the eNOS gene containing nuclear localization signal (NLS) but not nuclear export signal (NES) significantly attenuated aphidicolin-induced BAEC death. When G2A-eNOS mutant lacking myristoylation at Gly2 was transfected, its intracellular distribution became diffuse and included the nucleus. Finally, expression of N-myristoyltransferase 2 (NMT2) but not NMT1 significantly decreased in aphidicolin-treated BAECs. Taken together, our results suggest that aphidicolin attenuates BAEC death in part by increasing nuclear eNOS localization and NO production.

Quantitative Examination of Changes in Fluidity and Cholesterol Content in Bovine Aortic Endothelial Cells using Phasor Analysis of Laurdan Fluorescence Lifetime

Cholesterol is important for maintaining cell membrane integrity and is known to have an effect on the function of ion channels in endothelial cells. Here, we use the properties of Laurdan to investigate how various cellular cholesterol manipulations, altering membrane fluidity and adding a cholesterol analog affect physical properties of bovine aortic endothelial cells (BAEC). Laurdan, a solvatochromic dye, has a spectral red shift in its emission when in a more liquid disordered (Ld) phase compared to a liquid ordered phase (LO). BAECs labeled with Laurdan were treated with methyl-ß-cyclodextrin (MßCD) to remove cholesterol, MßCD saturated with cholesterol to enrich cholesterol content, the cholesterol analog epicholesterol, or fluidizing alcohols benzyl alcohol (BA) or dodecanol and compared to BAECs treated with PBS+Ca2+ as a control. Two-photon excitation was used to excite Laurdan and lifetime measurements were compared at two emission channels (‘blue channel’ and ‘green channel’) in order to differentiate between changes in membrane fluidity and cholesterol content using two methods: traditional phasor analysis and a multiparametric distance analysis using phasor data. Treatment with MßCD, BA and dodecanol resulted in a shift to shorter lifetimes in the blue channel, indicative a more polar (Ld) environment, while enriching cells with cholesterol shifted phasors to longer lifetimes. In the green channel, the phasors of cholesterol enriched cells were shifted in the direction that corresponds to an increase in dipolar relaxation. Only treatment with a higher concentration of MßCD caused a shift in the opposite direction. Using Laurdan and phasor analysis to distinguish changes in fluidity from changes in cholesterol gives us the ability to manipulate these parameters to assist in determining the mechanisms by which cholesterol can impair cell signaling processes.

Sodium trisulfide, a sulfane sulfur donor, stimulates bovine aortic endothelial cell proliferation in culture.

Reactive sulfur species (RSS) include biological persulfide molecules that protect cells against oxidative stress and heavy metal toxicity. Vascular endothelial cells regulate blood coagulation and fibrinolytic activity, and prevent vascular disorders such as atherosclerosis. We hypothesized that RSS protect vascular endothelial cells not only from nonspecific cell damage but also from specific functional damage through regulation of specific cell functions. In the present study, cultured bovine aortic endothelial cells were treated with sodium trisulfide, a sulfane sulfur donor, and both [3H]thymidine incorporation and effects on cell cycle were analyzed. These results suggest that RSS stimulate vascular endothelial cell proliferation. RSS may reduce the functional cytotoxicity of antiproliferative agents.

Impact of Maternal Vitamin D Receptor (VDR) Gene Polymorphisms on Spontaneous Preterm Birth (Egyptian Case-Control Study)

Diverse effects of Ba^(2+) and Co^(2+) on fura-2 fluorescence ratio signals (FR, F350/F380) were examined in cultured bovine aortic endothelial cells (BAECs). ATP (3 μM) induced FR oscillations in normal Ca^(2+)-containing solutions. Co^(2+), but not Ba^(2+), inhibited ATP-induced FR oscillations. In Ca^(2+)-free solutions, 3 μM ATP only elicited a single FR transient. When Ba^(2+) replaced Ca^(2+) in the perfusate, 3 μM ATP provoked a distinct FR transient. The maximal rate of rise of the signal was greater than seen without Ba^(2+) and the FR increased and maintained to a high steady level. When external Ca^(2+) was replaced by Co^(2+), ATP-induced FR transient oscillated with a long recovery time constant. In a survey of the single fluorescence at excitation wavelength 350 nm (F350), Co^(2+) decreased the intensity in the presence of ATP and Ca^(2+). Ba^(2+), however, increased F350 and FR as well, especially in Ca^(2+)-free solutions. The results suggest that ATP-induced Ca^(2+) fluctuation is balanced by Ca^(2+) release from internal stores and influx from external Ca^(2+). Ba2+ seems to be permeable through Ca^(2+) channels and competes with Ca^(2+) for the dye. Co^(2+) blocks Ca^(2+) channels but does not inhibit ATP-mediated Ca^(2+) release in cells.

L-Citrulline Supplementation Increases Plasma Nitric Oxide Levels and Reduces Arginase Activity in Patients With Type 2 Diabetes.

Type 2 diabetes mellitus (T2DM) is becoming a major contributor to cardiovascular disease. One of the early signs of T2DM associated cardiovascular events is the development of vascular dysfunction. This dysfunction has been implicated in increasing the morbidity and mortality of T2DM patients. One of the important characteristics of vascular dysfunction is the impaired ability of endothelial cells to produce nitric oxide (NO). Additionally, decreases in the availability of NO is also a major contributor of this pathology. NO is produced by the activity of endothelial NO synthase (eNOS) on its substrate, L-arginine. Reduced availability of L-arginine to eNOS has been implicated in vascular dysfunction in diabetes. Arginase, which metabolizes L-arginine to urea and ornithine, competes directly with NOS for L-arginine. Hence, increases in arginase activity can decrease arginine levels, reducing its availability to eNOS and decreasing NO production. Diabetes has been linked to elevated arginase and associated vascular endothelial dysfunction. We aimed to determine levels of plasma NO and arginase activity in (T2DM) patients and the effects of L-citrulline supplementation, a natural arginase inhibitor, on inhibiting arginase activity in these patients. Levels of arginase correlated with HbA1c levels in diabetic patients. Twenty-five patients received L-citrulline supplements (2000 mg/day) for 1 month. Arginase activity decreased by 21% in T2DM patients after taking L-citrulline supplements. Additionally, plasma NO levels increased by 38%. There was a modest improvement on H1Ac levels in these patients, though not statistically significant. The effect of L-citrulline on arginase activity was also studied in bovine aortic endothelial cells (BAECs) grown in high glucose (HG) conditions. HG (25 mM, 72 h) caused a 2-fold increase in arginase activity in BAECs and decreased NO production by 30%. L-citrulline (2.5 mM) completely prevented the increase in arginase activity and restored NO production levels. These data indicate that L-citrulline can have therapeutic benefits in diabetic patients through increasing NO levels and thus maintaining vascular function possibly through an arginase inhibition related pathway.

Arginine recycling in endothelial cells is regulated BY HSP90 and the ubiquitin proteasome system

Despite the saturating concentrations of intracellular l-arginine, nitric oxide (NO) production in endothelial cells (EC) can be stimulated by exogenous arginine. This phenomenon, termed the “arginine paradox” led to the discovery of an arginine recycling pathway in which l-citrulline is recycled to l-arginine by utilizing two important urea cycle enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Prior work has shown that ASL is present in a NO synthetic complex containing hsp90 and endothelial NO synthase (eNOS). However, it is unclear whether hsp90 forms functional complexes with ASS and ASL and if it is involved regulating their activity. Thus, elucidating the role of hsp90 in the arginine recycling pathway was the goal of this study. Our data indicate that both ASS and ASL are chaperoned by hsp90. Inhibiting hsp90 activity with geldanamycin (GA), decreased the activity of both ASS and ASL and decreased cellular l-arginine levels in bovine aortic endothelial cells (BAEC). hsp90 inhibition led to a time-dependent decrease in ASS and ASL protein, despite no changes in mRNA levels. We further linked this protein loss to a proteasome dependent degradation of ASS and ASL via the E3 ubiquitin ligase, C-terminus of Hsc70-interacting protein (CHIP) and the heat shock protein, hsp70. Transient over-expression of CHIP was sufficient to stimulate ASS and ASL degradation while the over-expression of CHIP mutant proteins identified both TPR- and U-box-domain as essential for ASS and ASL degradation. This study provides a novel insight into the molecular regulation l-arginine recycling in EC and implicates the proteasome pathway as a possible therapeutic target to stimulate NO signaling.

Transcriptional activation of urokinase by the Krüppel-like factor Zf9/COPEB activates latent TGF-β1 in vascular endothelial cells

Understanding the regulation of genes controlling fibrinolysis and matrix homeostasis is essential for elucidating the basis of tissue repair. A recently described novel Krüppel-like factor, Zf9, is up-regulated in acute liver injury in activated hepatic stellate cells. Because Zf9 can be induced widely, its activity was examined in vascular endothelium, a key cell in vascular injury. Zf9 is induced as an immediate-early response gene in bovine aortic endothelial cells (BAECs) following treatment with serum or phorbol ester. Zf9 transcriptionally activates urokinase plasminogen activator (uPA). Recombinant Zf9-GST binds to wild-type but not mutated 'GC-box' motifs within the human uPA promoter (-63 to -32), with greatest affinity to the middle of 3 contiguous GC boxes. Transient transfection of Zf9 drives transactivation of a full-length uPA promoter- and GC box-construct, but not a uPA promoter-construct devoid of GC boxes. Transactivation of uPA by Zf9 is also supported in Drosophila S2 cells. Most importantly, transiently transfected Zf9 up-regulates endogenous uPA messenger RNA and activity in BAECs, resulting in increased bioactive transforming growth factor-beta (TGF-beta) via enhancement of proteolytic activation of the latent molecule. Furthermore, concomitant expression of Zf9 and uPA proteins was observed in arterial endothelial cells after balloon injury in rats, suggesting a potential role of Zf9 in uPA expression not only in vitro but also in vivo. These findings suggest a role of Zf9 in the injury response by enhancing uPA synthesis and subsequent activation of latent TGF-beta. (Blood. 2000;95:1309-1316)