Stimulatory Effects of Atorvastatin on Extracellular Nucleotide Degradation in Human Endothelial Cells

Endothelial degradation of extracellular nucleotides is known to be an important mechanism in regulation of thrombosis, inflammation and immune response. It is possible that this pathway is a target for pleiotropic drugs such as atorvastatin. We studied therefore the effect of atorvastatin on extracellular nucleotide degradation in human endothelial cells. Atorvastatin treatment of human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1) resulted in significant increase in ATP breakdown and adenosine formation both if analysed in intact cell studies and as enzyme activity in cell lysates. We conclude that one of the beneficial effects of atorvastatin may include acceleration of extracellular nucleotide breakdown. This will attenuate nucleotide mediated pro-inflammatory effect and stimulate protective mechanisms of adenosine.     

Stimulatory effects of atorvastatin on extracellular nucleotide degradation in human endothelial cells

Endothelial degradation of extracellular nucleotides is known to be an important mechanism in regulation of thrombosis, inflammation and immune response. It is possible that this pathway is a target for pleiotropic drugs such as atorvastatin. We studied therefore the effect of atorvastatin on extracellular nucleotide degradation in human endothelial cells. Atorvastatin treatment of human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1) resulted in significant increase in ATP breakdown and adenosine formation both if analysed in intact cell studies and as enzyme activity in cell lysates. We conclude that one of the beneficial effects of atorvastatin may include acceleration of extracellular nucleotide breakdown. This will attenuate nucleotide mediated pro-inflammatory effect and stimulate protective mechanisms of adenosine.     

Extracellular nucleotide catabolism in human B and T lymphocytes. The source of adenosine production

Extracellular nucleotide degradation was studied in intact human B and T lymphocyte subpopulations and in lymphoblastoid cell lines. Cells of B lymphocyte lineage showed high nucleotide degrading activity, whereas T lymphocytes were unable to degrade extracellular nucleotides. The external surface of B cells contained active sites of ecto-triphosphonucleotidase (ecto-ATPase), ecto-diphosphonucleotidase (ecto-ADPase), and ecto-monophosphonucleotidase (ecto-AMPase). The expression of all three ectoenzyme activities seemed closely associated with B cell development. ATPase and ADPase activities increase continuously during B cell maturation, ecto-AMPase activity, on the other hand, reaches maximal activity in late pre-B cells. These results combined with our previous studies of intracellular ATP catabolism (Barankiewicz, J., and Cohen, A. (1984) J. Biol. Chem. 259, 15178-15181) provide evidence that extracellular ATP catabolism may represent exclusive source for adenosine in lymphocytes. It is suggested that adenosine may serve as a means of communication between B and T cells in lymphoid organs, B lymphocytes being the sole producers of adenosine and T lymphocytes being the recipients of this signal.     

The effects of pro- and anti-atherosclerotic factors on intracellular nucleotide concentration in murine endothelial cells

Abstract

Endothelial cell activation and dysfunction could lead to endothelial injury that is an important factor in the development of vascular diseases. Vascular injury is strongly associated with disturbed endothelial cell energetics and pyridine nucleotide pool. This study aimed to evaluate the effects of inflammatory stimuli (IL-6, LPS), uric acid, hyperglycemia, fatty acids, flavonoids, statins and nonsteroidal anti-inflammatory drugs on cellular concentration of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and nicotinamide adenine dinucleotide (NAD⁺) in cultured endothelial cells. Murine-immortalized heart endothelial cells (H5V cells) were treated with different concentrations of pro- and anti-atherosclerotic factors and intracellular concentration of nucleotides were measured using high performance liquid chromatography. Intracellular ATP concentration in H5V cells was not changed by inflammatory stimuli (IL-6 and LPS), uric acid, glucose, atorvastatin, acetylsalicylic acid, monounsaturated and polyunsaturated fatty acids. Only high concentration of palmitic acid (1?mM) and kaempferol (>0.1?mM) decreased intracellular ATP concentration. The concentration of intracellular ADP has not been altered by any of tested compounds. In turn, intracellular NAD⁺ pool was modified only by polyunsaturated fatty acids and atorvastatin. Linoleic acid, docosahexaenoic acid and atorvastatin increased cellular NAD⁺ concentration. Tested compounds have a small influence on murine endothelial cell energetics, but polyunsaturated fatty acids and atorvastatin increased intracellular NAD⁺ concentration that could be an important protective mechanism against endothelial cell injury.     

Degradation of extracellular nucleotides and their analogs in HeLa and HUVEC cell cultures

The use of nucleotides and their analogs in the pharmacological studies of nucleotide receptors (P2 class) should be preceded by detailed studies on their degradation connected with ecto-enzymes of a given cell type. In the present studies we have analyzed stability of some phosphorothioate and phosphonate analogs of ATP and ADP in the HeLa epitheloid carcinoma and endothelial HUVEC cells cultures. Our studies have revealed that ecto-nucleotide pyrophosphatase (E-NPP) is one of the main enzymes involved in the extracellular degradation of ATP and other nucleotides in the HeLa cells. On the other hand, the ecto-ATPDase is responsible for the hydrolysis of extracellular nucleotides in human endothelial cell cultures, while the E-NPP-like enzymes of the HUVEC cells are not essential to this degradation. The concerted action of the aforementioned ecto-enzymes and nucleotide pyrophosphatase, 5'-nucleotidase and adenosine deaminase present in fetal bovine serum (FBS) supplied to the culture medium, results in partial or complete degradation of the phosphorothioate (ATPgammaS) and phosphonate analogs of adenosine nucleotides (alpha,beta-methylene-ATP and beta,gamma-methylene-ATP) in the cell cultures. Only ADPbetaS appears to be resistant to these enzymes. The influence of some nucleotides and their analogs on the proliferation of the HeLa cells in presence or absence of FBS is also discussed.     

MEK/MAPK as a signaling element in ATP control of endothelial myosin light chain

Phosphorylation of endothelial myosin light chains (MLC) is a key mechanism in control of endothelial contractile machinery. Extracellular ATP influences endothelial MLC phosphorylation by either activation of Ca²⁺-dependent MLC kinase or Ca²⁺-independent MLC phosphatase. Here, the role of the MEK/MAPK pathway in this signaling was investigated in porcine aortic endothelial cells. Phosphorylation of ERK2 and phosphorylation of MLC were analyzed in cultured aortic endothelial cells. ATP (10 µM) increased ERK2 phosphorylation from basal 17 ± 3 to 53 ± 4%, an effect suppressed in the presence of the MEK inhibitors PD-98059 (20 µM) or U0126 (10 µM). Phosphorylation of ERK2 was not dependent on the ATP-induced cytosolic Ca²⁺ rise, because it was unaltered when this was suppressed by the Ca²⁺ chelator BAPTA (10 µM) or xestospongin C (3 µM), an inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release mechanism of the endoplasmic reticulum. Phosphorylation of ERK2 was neither induced by the adenosine analog 5'-(N-ethylcarboxamido)adenosine (1 µM) nor inhibited in the presence of the adenosine receptor antagonist 8-phenyltheophylline (10 µM). ATP increased MLC kinase activity, and this was blocked in presence of PD-98059. ATP also increased MLC phosphatase activity, which was not inhibited by PD-98059. The MEK/MAPK pathway is a Ca²⁺-independent part of ATP signaling toward MLC kinase but not of ATP signaling toward MLC phosphatase. mitogen-activated protein kinase; contractile machinery; myosin light chain kinase; myosin light chain phosphatase     

Intracellular adenosine formation and release by freshly-isolated vascular endothelial cells from rat skeletal muscle: effects of hypoxia and/or acidosis

Previous studies suggested indirectly that vascular endothelial cells (VECs) might be able to release intracellularly-formed adenosine. We isolated VECs from the rat soleus muscle using collagenase digestion and magnetic-activated cell sorting (MACS). The VEC preparation had >90% purity based on cell morphology, fluorescence immunostaining, and RT-PCR of endothelial markers. The kinetic properties of endothelial cytosolic 5′-nucleotidase suggested it was the AMP-preferring N-I isoform: its catalytic activity was 4 times higher than ecto-5′nucleotidase. Adenosine kinase had 50 times greater catalytic activity than adenosine deaminase, suggesting that adenosine removal in VECs is mainly through incorporation into adenine nucleotides. The maximal activities of cytosolic 5′-nucleotidase and adenosine kinase were similar. Adenosine and ATP accumulated in the medium surrounding VECs in primary culture. Hypoxia doubled the adenosine, but ATP was unchanged; AOPCP did not alter medium adenosine, suggesting that hypoxic VECs had released intracellularly-formed adenosine. Acidosis increased medium ATP, but extracellular conversion of ATP to AMP was inhibited, and adenosine remained unchanged. Acidosis in the buffer-perfused rat gracilis muscle elevated AMP and adenosine in the venous effluent, but AOPCP abolished the increase in adenosine, suggesting that adenosine is formed extracellularly by non-endothelial tissues during acidosis in vivo. Hypoxia plus acidosis increased medium ATP by a similar amount to acidosis alone and adenosine 6-fold; AOPCP returned the medium adenosine to the level seen with hypoxia alone. These data suggest that VECs release intracellularly formed adenosine in hypoxia, ATP during acidosis, and both under simulated ischaemic conditions, with further extracellular conversion of ATP to adenosine.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions.

Cell surface ecto-nucleotidases are considered the major effector system for inactivation of extracellular adenine nucleotides, whereas the alternative possibility of ATP synthesis has received little attention. Using a TLC assay, we investigated the main exchange activities of 3H-labeled adenine nucleotides on the cultured human umbilical vein endothelial cells. Stepwise nucleotide degradation to adenosine occurred when a particular nucleotide was present alone, whereas combined cell treatment with ATP and either [3H]AMP or [3H]ADP caused unexpected phosphorylation of 3H-nucleotides via the backward reactions AMP --> ADP --> ATP. The following two groups of nucleotide-converting ecto-enzymes were identified based on inhibition and substrate specificity studies: 1) ecto-nucleotidases, ATP-diphosphohydrolase, and 5'-nucleotidase; 2) ecto-nucleotide kinases, adenylate kinase, and nucleoside diphosphate kinase. Ecto-nucleoside diphosphate kinase possessed the highest activity, as revealed by comparative kinetic analysis, and was capable of using both adenine and nonadenine nucleotides as phosphate donors and acceptors. The transphosphorylation mechanism was confirmed by direct transfer of the gamma-phosphate from [gamma-32P]ATP to AMP or nucleoside diphosphates and by measurement of extracellular ATP synthesis using luciferin-luciferase luminometry. The data demonstrate the coexistence of opposite, ATP-consuming and ATP-generating, pathways on the cell surface and provide a novel mechanism for regulating the duration and magnitude of purinergic signaling in the vasculature.     

Extracellular ATP reduces tumor sphere growth and cancer stem cell population in glioblastoma cells

Glioblastoma is the most aggressive tumor in the CNS and is characterized by having a cancer stem cell (CSC) subpopulation essential for tumor survival. The purinergic system plays an important role in glioma growth, since adenosine triphosphate (ATP) can induce proliferation of glioma cells, and alteration in extracellular ATP degradation by the use of exogenous nucleotidases dramatically alters the size of gliomas in rats. The aim of this work was to characterize the effect of the purinergic system on glioma CSCs. Human U87 glioma cultures presented tumor spheres that express the markers of glioma cancer stem cells CD133, Oct-4, and Nanog. Messenger RNA of several purinergic receptors were differently expressed in spheres when compared to a cell monolayer not containing spheres. Treatment of human gliomas U87 or U343 as well as rat C6 gliomas with 100 μM of ATP reduced the number of tumor spheres when grown in neural stem cell medium supplemented with epidermal growth factor and basic fibroblast growth factor. Moreover, ATP caused a decline in the number of spheres observed in culture in a dose-dependent manner. ATP also reduces the expression of Nanog, as determined by flow cytometry, as well as CD133 and Oct-4, as analyzed by flow cytometry and RT-PCR in U87 cells. The differential expression of purinergic receptor in tumor spheres when compared to adherent cells and the effect of ATP in reducing tumor spheres suggest that the purinergic system affects CSC biology and that ATP may be a potential agonist for differentiation therapy.     

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.     

Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase

Nucleoside di- and triphosphates and adenosine regulate several components of the mucocilairy clearance process (MCC) that protects the lung against infections, via activation of epithelial purinergic receptors. However, assessing the contribution of individual nucleotides to MCC functions remains difficult due to the complexity of the mechanisms of nucleotide release and metabolism. Enzymatic activities involved in the metabolism of extracellular nucleotides include ecto-ATPases and secreted nucleoside diphosphokinase (NDPK) and adenyl kinase, but potent and selective inhibitors of these activities are sparse. In the present study, we discovered that ebselen markedly reduced NDPK activity while having negligible effect on ecto-ATPase and adenyl kinase activities. Addition of radiotracer [γ ³²P]ATP to human bronchial epithelial (HBE) cells resulted in rapid and robust accumulation of [³²P]-inorganic phosphate (³²Pi). Inclusion of UDP in the incubation medium resulted in conversion of [γ ³²P]ATP to [³²P]UTP, while inclusion of AMP resulted in conversion of [γ ³²P]ATP to [³²P]ADP. Ebselen markedly reduced [³²P]UTP formation but displayed negligible effect on ³²Pi or [³²P]ADP accumulations. Incubation of HBE cells with unlabeled UTP and ADP resulted in robust ebselen-sensitive formation of ATP (IC₅₀ = 6.9 ± 2 μM). This NDPK activity was largely recovered in HBE cell secretions and supernatants from lung epithelial A549 cells. Kinetic analysis of NDPK activity indicated that ebselen reduced the V _max of the reaction (K _i = 7.6 ± 3 μM), having negligible effect on K _M values. Our study demonstrates that ebselen is a potent non-competitive inhibitor of extracellular NDPK.     

Functional Analysis of Expression of Human Ecto-Nucleoside Triphosphate Diphosphohydrolase-1 and/or Ecto-5′-Nucleotidase in Pig Endothelial Cells

Adenine nucleosides and nucleotides are important signaling molecules involved in control of key mechanisms of xenotransplant rejection. Extracellular pathway that converts ATP and ADP to AMP, and AMP to adenosine mainly mediated by ecto-nucleoside triphosphate diphosphohydrolase 1, (ENTPD1 or CD39) and ecto-5′-nucleotidase (E5NT or CD73) respectively, is considered as important target for xenograft protection. To clarify feasibility of combined expression of human ENTPD1 and E5NT and to study its functional effect we transfected pig endothelial cell line (PIEC) with both genes together. To do this we have produced a dicistronic construct bearing F2A sequence in frame between human E5NT and human ENTPD1 coding sequences. PIEC cells were mock-transfected as transfection control or transfected with plasmids encoding human ENTPD1 or human E5NT. PIEC cells were exposed to 50 μM ATP or 50 μM ADP or 50 μM AMP. Conversion of extracellular substrates into products (ATP/ADP/AMP/adenosine) was measured by HPLC in the media collected at specific time intervals. Following addition of AMP, production of adenosine in the medium of E5NT/ENTPD1- and E5NT- transfected cells increased to 14.2 ± 1.1 and 24.5 ± 3.4 μM respectively while it remained below 1 μM in controls and in ENTPD1-transfected cells. A marked increase of adenosine formation from ADP or ATP was observed only in E5NT/ENTPD1-transfected cells (11.7 ± 0.1 and 5.7 ± 2.2 μM respectively) but not in any other condition studied. This study indicates feasibility and functionality of combined expression of human E5NT and ENTPD1 in pig endothelial cells using F2A sequence bearing construct.     

Identification and partial characterization of an adenosine(5'')tetraphospho(5'')adenosine hydrolase on intact bovine aortic endothelial cells.

The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A.     

Pathways of purine metabolism in human adipocytes. Further evidence against a role of adenosine as an endogenous regulator of human fat cell function

Previous results demonstrated that the adenosine that accumulates in human fat cell suspensions is derived from extracellular sources (Kather, H. (1988) J. Biol. Chem. 263, 8803-8809). To get insight into the mechanisms responsible for the lack of adenosine release, extracellular adenine nucleotide catabolism was minimized by 10 mmol/liter beta-glycerophosphate and 10 mumol/liter alpha,beta-methyleneadenosine 5'-diphosphate. Intracellular adenine nucleotide catabolism resulted in a release of inosine and hypoxanthine under these conditions that was increased markedly by isoproterenol. Experiments with inhibitors of adenosine deaminase and adenosine kinase indicated that the production of inosine and hypoxanthine proceeded via AMP deamination. Consistently, IMP levels were increased transiently in the presence of isoproterenol. In addition, the cells possessed a nucleotide phosphomonoesterase that was resistant to the inhibitory actions of ATP and alpha,beta-methyleneadenosine 5'-diphosphate and showed preference for IMP over AMP. Adenosine (approximately 1 nmol/10(6) cells/h) was also produced inside the cells. However, adenosine production was unrelated to ATP turnover via adenylate cyclase, and any adenosine formed was immediately reconverted to adenine nucleotides in the absence and presence of isoproterenol. It was concluded that adenosine is not released by intact human adipocytes, because the alternative routes of intracellular AMP catabolism are compartmentalized (at least in functional terms), and adenosine kinase is not saturated with substrate in the absence and presence of isoproterenol.     

NTPDase and 5′-nucleotidase Activities of Synaptosomes from Hippocampus of Rats Subjected to Hyperargininemia

ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system. Since the ecto-nucleotidase cascade that hydrolyzes ATP to adenosine is involved in the control of brain functions and previous studies realized in our laboratory have recently reported that acute administration of Arg decreases the NTPDase and 5′-nucleotidase activities of rat blood serum, in the present study we investigated the effect of arginine administration on NTPDase and 5′-nucleotidase activities by synaptosomes from hippocampus of rats. First, sixty-days-old rats were treated with a single or a triple intraperitoneal injection of arginine (0.8 g/Kg) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. Second, rats received an intracerebroventricular injection of 1.5 mM arginine solution or saline (5 μL) and were killed 1 h later. We also tested the in vitro effect of arginine (0.1–1.5 mM) on nucleotide hydrolysis in synaptosomes from rat hippocampus. Results showed that intraperitoneal arginine administration did not alter nucleotide hydrolysis. On the other hand, arginine administered intracerebroventricularly reduced ATP (32%), ADP (30%) and AMP (21%) hydrolysis, respectively. In addition, arginine added to the incubation medium, provoked a decrease on ATP (19%), ADP (17%) and AMP (23%) hydrolysis, respectively. Furthermore, kinetic studies showed that the inhibitory effect of arginine was uncompetitive in relation to ATP, ADP and AMP. In conclusion, according to our results it seems reasonable to postulate that arginine alters the cascade involved in the extracellular degradation of ATP to adenosine.     

Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma × glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC).2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ecto-phosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP.3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide- diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole.4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells. Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells.5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.     

Inhibition of Rho and Rac Geranylgeranylation by Atorvastatin Is Critical for Preservation of Endothelial Junction Integrity

Background

Small GTPases (guanosine triphosphate, GTP) are involved in many critical cellular processes, including inflammation, proliferation, and migration. GTP loading and isoprenylation are two important post-translational modifications of small GTPases, and are critical for their normal function. In this study, we investigated the role of post-translational modifications of small GTPases in regulating endothelial cell inflammatory responses and junctional integrity.

Methods and Results

Confluent human umbilical vein endothelial cell (HUVECs ) treated with atorvastatin demonstrated significantly decreased lipopolysaccharide (LPS)-mediated IL-6 and IL-8 generation. The inhibitory effect of atorvastatin (Atorva) was attenuated by co-treatment with 100 µM mevalonate (MVA) or 10 µM geranylgeranyl pyrophosphate (GGPP), but not by 10 µM farnesyl pyrophosphate (FPP). Atorvastatin treatment of HUVECs produced a time-dependent increase in GTP loading of all Rho GTPases, and induced the translocation of small Rho GTPases from the cellular membrane to the cytosol, which was reversed by 100 µM MVA and 10 µM GGPP, but not by 10 µM FPP. Atorvastatin significantly attenuated thrombin-induced HUVECs permeability, increased VE-cadherin targeting to cell junctions, and preserved junction integrity. These effects were partially reversed by GGPP but not by FPP, indicating that geranylgeranylation of small GTPases plays a major role in regulating endothelial junction integrity. Silencing of small GTPases showed that Rho and Rac, but not Cdc42, play central role in HUVECs junction integrity.

Conclusions

In conclusion, our studies show that post-translational modification of small GTPases plays a vital role in regulating endothelial inflammatory response and endothelial junction integrity. Atorvastatin increased GTP loading and inhibited isoprenylation of small GTPases, accompanied by reduced inflammatory response and preserved cellular junction integrity.     

Atorvastatin prevents cell damage via modulation of oxidative stress,glutamate uptake and glutamine synthetase activity in hippocampal slices subjected to oxygen/glucose deprivation

Oxygen–glucose deprivation (OGD) in brain cells increases extracellular glutamate concentration leading to excitotoxicity. Glutamate uptake from the synaptic cleft is carried out by glutamate transporters, which are likely to be modulated by oxidative stress. Therefore, oxidative stress is associated with reduced activity of glutamate transporters and glutamine synthetase, thus increasing extracellular glutamate levels that may aggravate damage to brain cells. Atorvastatin, a cholesterol-lowering agent, has been shown to exert neuroprotective effects. The aim of this study was to investigate if in vivo atorvastatin treatment would have protective effects against hippocampal slices subjected to OGD, ex vivo. Atorvastatin pretreatment promoted increased cell viability after OGD and reoxygenation of hippocampal slices. Atorvastatin-induced neuroprotection may be related to diminished oxidative stress, since it prevented OGD-induced decrement of non-proteic thiols (NPSH) levels and increase in the production of reactive oxygen species (ROS). Atorvastatin pretreatment also prevented the OGD-induced decrease in glutamate uptake and glutamine synthetase activity, although it had no effect on OGD-induced excitatory aminoacids release. Addition of cholesterol before OGD and reoxygenation, abolished the protective effect of atorvastatin on cellular viability as well as on glutamate uptake and glutamine synthetase activity. Therefore, atorvastatin is capable of preventing OGD-induced cell death, an effect achieved due to modulation of glutamate uptake and glutamine synthetase activity, and associated with diminished oxidative stress. Additionally, atorvastatin effects were dependent on its action on cholesterol synthesis inhibition. Thus, atorvastatin might be a useful strategy in the prevention of glutamate exitotoxicity involved in brain injuries such as vascular disorders.     

Pharmacological dissection of the cellular mechanisms associated to the spontaneous and the mechanically stimulated ATP release by mesentery endothelial cells: roles of thrombin and TRPV

Endothelial cells participate in extracellular ATP release elicited by mechanosensors. To characterize the dynamic interactions between mechanical and chemical factors that modulate ATP secretion by the endothelium, we assessed and compared the mechanisms participating in the spontaneous (basal) and mechanically stimulated secretion using primary cultures of rat mesentery endothelial cells. ATP/metabolites were determined in the cell media prior to (basal) and after cell media displacement or a picospritzer buffer puff used as mechanical stimuli. Mechanical stimulation increased extracellular ATP that peaked within 1 min, and decayed to basal values in 10 min. Interruption of the vesicular transport route consistently blocked the spontaneous ATP secretion. Cells maintained in media lacking external Ca²⁺ elicited a spontaneous rise of extracellular ATP and adenosine, but failed to elicit a further extracellular ATP secretion following mechanical stimulation. 2-APB, a TRPV agonist, increased the spontaneous ATP secretion, but reduced the mechanical stimulation-induced nucleotide release. Pannexin1 or connexin blockers and gadolinium, a Piezo1 blocker, reduced the mechanically induced ATP release without altering spontaneous nucleotide levels. Moreover, thrombin or related agonists increased extracellular ATP secretion elicited by mechanical stimulation, without modifying spontaneous release. In sum, present results allow inferring that the spontaneous, extracellular nucleotide secretion is essentially mediated by ATP containing vesicles, while the mechanically induced secretion occurs essentially by connexin or pannexin1 hemichannel ATP transport, a finding fully supported by results from Panx1^?/? rodents. Only the latter component is modulated by thrombin and related receptor agonists, highlighting a novel endothelium-smooth muscle signaling role of this anticoagulant.