Cellular hypomethylation is associated with impaired nitric oxide production by cultured human endothelial cells.

Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease, but the underlying mechanisms remain incompletely defined. Reduced bioavailability of nitric oxide (NO) is a principal manifestation of underlying endothelial dysfunction, which is an initial event in vascular disease. Inhibition of cellular methylation reactions by S-adenosylhomocysteine (AdoHcy), which accumulates during HHcy, has been suggested to contribute to vascular dysfunction. However, thus far, the effect of intracellular AdoHcy accumulation on NO bioavailability has not yet been fully substantiated by experimental evidence. The present study was carried out to evaluate whether disturbances in cellular methylation status affect NO production by cultured human endothelial cells. Here, we show that a hypomethylating environment, induced by the accumulation of AdoHcy, impairs NO production. Consistent with this finding, we observed decreased eNOS expression and activity, but, by contrast, enhanced NOS3 transcription. Taken together, our data support the existence of regulatory post-transcriptional mechanisms modulated by cellular methylation potential leading to impaired NO production by cultured human endothelial cells. As such, our conclusions may have implications for the HHcy-mediated reductions in NO bioavailability and endothelial dysfunction.

A study of aspirin and clopidogrel in idiopathic pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is characterised by in situ thrombosis and increased thromboxane (Tx) A2 synthesis; however, there are no studies of antiplatelet therapy in IPAH. The aim of the current study was to determine the biochemical effects of aspirin (ASA) and clopidogrel on platelet function and eicosanoid metabolism in patients with IPAH. A randomised, double-blind, placebo-controlled crossover study of ASA 81 mg once daily and clopidogrel 75 mg once daily was performed. Plasma P-selectin levels and aggregometry were measured after exposure to adenosine diphosphate, arachidonic acid and collagen. Serum levels of TxB2 and urinary metabolites of TxA2 and prostaglandin I2 (Tx-M and PGI-M, respectively) were assessed. A total of 19 IPAH patients were enrolled, of whom nine were being treated with continuous intravenous epoprostenol. ASA and clopidogrel significantly reduced platelet aggregation to arachidonic acid and adenosine diphosphate, respectively. ASA significantly decreased serum TxB2, urinary Tx-M levels and the Tx-M/PGI-M ratio, whereas clopidogrel had no effect on eicosanoid levels. Neither drug significantly lowered plasma P-selectin levels. Epoprostenol use did not affect the results. In conclusion, aspirin and clopidogrel inhibited platelet aggregation, and aspirin reduced thromboxane metabolite production without affecting prostaglandin I2 metabolite synthesis. Further clinical trials of aspirin in patients with idiopathic pulmonary arterial hypertension should be performed.

Factor XIII (FXIII) and angiogenesis.

Factor XIII is a plasma transglutaminase that participates in the final stage of the coagulation cascade. Thrombin-activated FXIII (FXIIIa) catalyzes the formation of covalent cross-links between gamma-glutamyl and epsilon-lysyl residues on adjacent fibrin chains in polymerized fibrin to yield the mature clot. In addition to its role in hemostasis, FXIII is known to participate in wound healing and embryo implantation, which are processes involving angiogenesis. In this review, we discuss the role of FXIII in angiogenesis and the molecular mechanisms underlying its proangiogenic effects. The FXIII role in tissue repair and remodeling may at least in part be attributed to its pro-angiogenic activity.

L-arginine increases plasma homocysteine in apoE-/-/iNOS-/- double knockout mice.

Previous studies have shown that L-arginine (L-Arg) administration to apoE-/-/iNOS-/- double knockout mice (dKO) on a Western diet paradoxically results in an increase in atherosclerotic lesion size. We hypothesized that the potential beneficial effects of L-Arg could be offset, in part, by the byproducts of L-Arg catabolism, especially the atherogenic risk factor, homocysteine. In the kidney, L-Arg is converted to L-ornithine and guanidinoacetate (GAA) by L-arginine-glycine amidinotransferase. The efficient transmethylation of GAA by an S-adenosyl-methionine (SAM)-dependent methyltransferase in liver yields creatine and S-adenosylhomocysteine (SAH), which is readily hydrolyzed to homocysteine and adenosine. We, therefore, measured total plasma homocysteine in the dKO mice and control mice. We found that L-Arg supplementation caused a 37% increase in total plasma homocysteine (tHcy) levels in dKO mice compared to controls not treated with L-Arg (5.2+/-2.2 vs 3.8+/-1.5 microM Hcy, p<0.04). In a liver cell line, HepG2, addition of 10 and 50 microM GAA in the presence of 50 microM L-methionine (L-Met) increased tHcy production by approximately 1.47 (p<0.0001) and 2.3-fold (p<0.0001), respectively. In the presence of additional 100 microM L-Met, baseline homocysteine production was elevated by 20% (p<0.005), and 10 and 50 microM GAA augmented homocysteine production by an additional 1.88- (p<0.0001) and 3.4-fold (p<0.001), respectively, compared with 50 microM L-Met. These data suggest that increased concentrations of a methyl acceptor, such as L-Arg-derived GAA, drives SAM-dependent-methylation and consequent homocysteine formation. Furthermore, L-Met levels can also influence homocysteine production likely by regulating the synthesis of the methyl donor SAM. Epidemiological studies have suggested that homocysteine is a graded risk factor. In animal models, modestelevations of homocysteine can cause endothelial dysfunction and augment atherosclerosis. Our data suggest that L-arginine supplementation may contribute to vascular injury and atherogenesis under some circumstances by elevating homocysteine levels.

Nitric oxide in vascular biology.

Nitric oxide is a highly versatile heterodiatomic molecule that effects a variety of actions in the vasculture. Originally identified as a principal determination of vascular tone, nitric oxide has since been recognized to exert anti thrombotic, antiproliferative, and anti-inflammatory effects in the vasculture. At higher concentrations and in the setting of other oxidants, nitric oxide can promote vascular pathology. In this review, we summarize the molecular mechanisms of nitric oxides actions in vascular biology and pathology.

Induction of platelet formation from megakaryocytoid cells by nitric oxide.

Although the growth factors that regulate megakaryocytopoiesis are well known, the molecular determinants of platelet formation from mature megakaryocytes remain poorly understood. Morphological changes in megakaryocytes associated with platelet formation and removal of senescent megakaryocytes are suggestive of an apoptotic process. Previously, we have established that nitric oxide (NO) can induce apoptosis in megakaryocytoid cell lines. To determine whether there is an association between NO-induced apoptosis and platelet production, we exposed Meg-01 cells to S-nitrosoglutathione (GSNO) with or without thrombopoeitin (TPO) pretreatment and used flow cytometry and electron microscopy to assess platelet-sized particle formation. Meg-01 cells treated with TPO alone produced few platelet-sized particles (<3% of total counts), whereas treatment with GSNO alone produced a significant percentage of platelet-sized particles (22 +/- 4% of total counts); when combined with TPO pretreatment, however, GSNO led to a marked increase in platelet-sized particle production (48 +/- 3% of total counts). Electron microscopy confirmed that Meg-01 cells treated with TPO and GSNO yielded platelet-sized particles with morphological features specific for platelet forms. The platelet-sized particle population appears to be functional, because addition of calcium, fibrinogen, and thrombin receptor-activating peptide led to aggregation. These results demonstrate that NO facilitates platelet production, thereby establishing the essential role of NO in megakaryocyte development and thrombopoiesis.

Overexpression of cellular glutathione peroxidase rescues homocyst(e)ine-induced endothelial dysfunction.

Homocyst(e)ine (Hcy) inhibits the expression of the antioxidant enzyme cellular glutathione peroxidase (GPx-1) in vitro and in vivo, which can lead to an increase in reactive oxygen species that inactivate NO and promote endothelial dysfunction. In this study, we tested the hypothesis that overexpression of GPx-1 can restore the normal endothelial phenotype in hyperhomocyst(e)inemic states. Heterozygous cystathionine beta-synthase-deficient (CBS((-/+))) mice and their wild-type littermates (CBS((+/+))) were crossbred with mice that overexpress GPx-1 [GPx-1((tg+)) mice]. GPx-1 activity was 28% lower in CBS((-/+))/GPx-1((tg-)) compared with CBS((+/+))/GPx-1((tg-)) mice (P < 0.05), and CBS((-/+)) and CBS((+/+)) mice overexpressing GPx-1 had 1.5-fold higher GPx-1 activity compared with GPx-1 nontransgenic mice (P < 0.05). Mesenteric arterioles of CBS((-/+))/GPx-1((tg-)) mice showed vasoconstriction to superfusion with beta-methacholine and bradykinin (P < 0.001 vs. all other groups), whereas nonhyperhomocyst(e)inemic mice [CBS((+/+))/GPx-1((tg-)) and CBS((+/+))/GPx-1((tg+)) mice] demonstrated dose-dependent vasodilation in response to both agonists. Overexpression of GPx-1 in hyperhomocyst(e)inemic mice restored the normal endothelium-dependent vasodilator response. Bovine aortic endothelial cells (BAEC) were transiently transfected with GPx-1 and incubated with dl-homocysteine (HcyH) or l-cysteine. HcyH incubation decreased GPx-1 activity in sham-transfected BAEC (P < 0.005) but not in GPx-1-transfected cells. Nitric oxide release from BAEC was significantly decreased by HcyH but not cysteine, and GPx-1 overexpression attenuated this decrease. These findings demonstrate that overexpression of GPx-1 can compensate for the adverse effects of Hcy on endothelial function and suggest that the adverse vascular effects of Hcy are at least partly mediated by oxidative inactivation of NO.

Anemia-induced increase in the bleeding time: implications for treatment of nonsurgical blood loss.

BACKGROUND: Preoperative bleeding time (BT) does not correlate with postoperative bleeding in patients subjected to surgical procedures. A significant positive correlation has been reported between the BT 2 hours after cardiopulmonary bypass surgery and the nonsurgical blood loss during the first 4 hours after bypass surgery. This study was done to investigate the effect of Hct and platelet count on the BT measurement in normal, healthy men and women. STUDY DESIGN AND METHODS: To assess the relative effect of RBCs and platelets on the BT, 22 healthy male and 7 healthy female volunteers were subjected to the removal of 2 units of RBCs (360 mL), followed by the return of the platelet-rich plasma (PRP) from both units and the infusion of 1000 mL of 0.9-percent NaCl. Four of the men and all seven women received their RBCs 1 hour after their removal. Shed blood levels of thromboxane B(2) (TXB(2)), 6-keto prostaglandin F(1 alpha), and peripheral venous Hct were measured. BTs were measured in 15 men and 13 women before and after a plateletpheresis procedure to collect 3.6 x 10(11) platelets per unit. RESULTS: The 2-unit RBC apheresis procedure produced a 60-percent increase in the BT associated with a 15-percent reduction in the peripheral venous Hct and a 9-percent reduction in the platelet count. The plateletpheresis procedure produced a 32-percent decrease in the platelet count, no change in peripheral venous Hct, and no change in the BT. After the removal of 2 units of RBCs, the shed blood TXB(2) level decreased significantly. Reinfusion of 2 units of RBCs restored the BT and restored the TXB(2) level to the baseline levels. CONCLUSION: The acute reduction in Hct produced a reversible platelet dysfunction manifested by an increase in BT and a decrease in the shed blood TXB(2) level at the template BT site. Return of the RBCs restored both the BT and the shed blood TXB(2) level to normal. The platelet dysfunction observed with the reduction in Hct was due in part to a reduction in shed blood TXB(2) and other, unknown mechanisms.

Detection of nitrosated proteins.

Three methods are provided in this unit to detect nitrosated proteins. The Saville assay is the most commonly used and has the advantages that it is less sensitive to interference than the quinine sulfate-based fluorescent assay, uses simple chemical reagents, and requires a spectrophotometer. The diaminonaphthalene assay (a fluorescence assay) and chemiluminescence assay are used when the experiment requires high sensitivity. Mass spectrometry is employed for precise structural determinations.

Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences.

5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP competed with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca(2+), which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO.

Cyclic strain modulates resistance to oxidant stress by increasing G6PDH expression in smooth muscle cells.

Vascular smooth muscle cells (VSMC) may be subjected to mechanical forces, such as cyclic strain, that promote the formation of reactive oxygen species (ROS). We hypothesized that VSMC modulate this adverse milieu by increasing the expression of glucose-6-phosphate dehydrogenase (G6PDH) to maintain or restore intracellular glutathione (GSH) levels. Cyclic strain increased superoxide formation, which resulted in diminished GSH because of an increase in oxidized glutathione formation; there was also an increase in glutathione peroxidase and glutathione reductase activities. G6PDH activity and protein expression were enhanced concomitant with decreases in GSH levels and remained elevated until intracellular GSH levels were restored. To confirm the role of G6PDH in repleting GSH stores, we inhibited G6PDH activity with DHEA or inhibited enzyme expression with an antisense oligodeoxynucleotide. Diminished G6PDH activity or expression was associated with persistently depleted GSH levels and inhibition of the cyclic strain-mediated increase in glutathione reductase activity. These observations demonstrate that cyclic strain promotes oxidant stress in VSMC, which, in turn, induces G6PDH expression. When G6PDH is inhibited, GSH levels are not restored because of impaired glutathione reductase activity. These data suggest that G6PDH is a critical determinant of the response to oxidant stress in VSMC.

Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia.

Homocysteine is a risk factor for the development of atherosclerosis and its thrombotic complications. We have employed an animal model to explore the hypothesis that an increase in reactive oxygen species and a subsequent loss of nitric oxide bioactivity contribute to endothelial dysfunction in mild hyperhomocysteinemia. We examined endothelial function and in vivo oxidant burden in mice heterozygous for a deletion in the cystathionine beta-synthase (CBS) gene, by studying isolated, precontracted aortic rings and mesenteric arterioles in situ. CBS(-/+) mice demonstrated impaired acetylcholine-induced aortic relaxation and a paradoxical vasoconstriction of mesenteric microvessels in response to superfusion of methacholine and bradykinin. Cyclic GMP accumulation following acetylcholine treatment was also impaired in isolated aortic segments from CBS(-/+) mice, but aortic relaxation and mesenteric arteriolar dilation in response to sodium nitroprusside were similar to wild-type. Plasma levels of 8-epi-PGF(2alpha) (8-IP) were somewhat increased in CBS(-/+) mice, but liver levels of 8-IP and phospholipid hydroperoxides, another marker of oxidative stress, were normal. Aortic tissue from CBS(-/+) mice also demonstrated greater superoxide production and greater immunostaining for 3-nitrotyrosine, particularly on the endothelial surface. Importantly, endothelial dysfunction appears early in CBS(-/+) mice in the absence of structural arterial abnormalities. Hence, mild hyperhomocysteinemia due to reduced CBS expression impairs endothelium-dependent vasodilation, likely due to impaired nitric oxide bioactivity, and increased oxidative stress apparently contributes to inactivating nitric oxide in chronic, mild hyperhomocysteinemia.

Nitric oxide induces apoptosis in megakaryocytic cell lines.

Cytokines that stimulate inducible nitric oxide (NO) synthase can suppress the growth and differentiation of normal human bone marrow cells, including megakaryocytes. Since NO promotes apoptosis in other cell systems, we chose to study the determinants of apoptosis in megakaryocytic cells. We show that both exogenous and endogenous sources of NO can induce apoptosis in megakaryocytoid cell lines. The megakaryocyte growth factor thrombopoietin suppresses NO-induced apoptosis, whereas treatment with peroxynitrite, a cytotoxic product formed when NO reacts with superoxide, promotes apoptosis. Superoxide inhibitors suppress NO-induced apoptosis, and pretreatment with megakaryocyte growth and maturation factors attenuates NO-induced apoptosis. These data show that NO modulates megakaryocyte apoptosis and suggest that this process may occur in the cytokine-rich marrow milieu to regulate megakaryocyte turnover.

Oxidant stress as a critical determinant of endothelial function.

The endothelial dysfunction associated with nitric oxide (NO) depletion accompanies the development of atherothrombotic disease. Recent evidence suggests that oxidative reactions in the vasculature promote atherothrombosis. Several risk factors for atherothrombosis, such as diabetes mellitus, cigarette smoking, hypertension, hyperhomocyst(e)inemia and hypercholesterolemia, are associated with oxidative reactions in the vasculature and with endothelial dysfunction. Traditional and novel treatments for atherothrombosis increase bioavailable NO and may decrease oxidant stress; however, the effect of this treatment on improving the long-term prognosis of patients with risk factors for atherothrombosis has yet to be determined.

Endothelial cell nitric oxide production in acute chest syndrome.

Acute chest syndrome (ACS) is the most common form of acute pulmonary disease associated with sickle cell disease. To investigate the possibility that alterations in endothelial cell (EC) production and metabolism of nitric oxide (NO) products might be contributory, we measured NO products from cultured pulmonary EC exposed to red blood cells and/or plasma from sickle cell patients during crisis. Exposure to plasma from patients with ACS caused a 5- to 10-fold increase in S-nitrosothiol (RSNO) and a 7- to 14-fold increase in total nitrogen oxide (NO(x)) production by both pulmonary arterial and microvascular EC. Increases occurred within 2 h of exposure to plasma in a concentration-dependent manner and were associated with increases in endothelial nitric oxide synthase (eNOS) protein and eNOS enzymatic activity, but not with changes in nitric oxide synthase (NOS) III or NOS II transcripts, inducible NOS (iNOS) protein nor iNOS enzymatic activity. RSNO and NO(x) increased whether plasma was obtained from patients with ACS or other forms of vasoocclusive crisis. Furthermore, an oxidative state occurred and oxidative metabolites of NO, particularly peroxynitrite, were produced. These findings suggest that altered NO production and metabolism to damaging oxidative molecules contribute to the pathogenesis of ACS.

Unique antiplatelet effects of a novel S-nitrosoderivative of a recombinant fragment of von Willebrand factor, AR545C: in vitro and ex vivo inhibition of platelet function.

The recombinant fragment of von Willebrand factor (vWF) spanning Ala444 to Asp730 and containing an Arg545Cys mutation (denoted AR545C) has antithrombotic properties that are principally a consequence of its ability to inhibit platelet adhesion to subendothelial matrix. Endothelial-derived nitric oxide (NO) can also inhibit platelet function, both as a consequence of inhibiting adhesion as well as activation and aggregation. Nitric oxide can react with thiol functional groups in the presence of oxygen to form S-nitrosothiols, which are naturally occurring NO derivatives that prolong the biological actions of NO. Because AR545C has a single free cysteine (Cys545), we attempted to synthesize the S-nitroso-derivative of AR545C and to characterize its antiplatelet effects. We successfully synthesized S-nitroso-AR545C and found that it contained 0.96 mol S-NO per mole peptide. S-nitroso-AR545C was approximately 5-fold more potent at inhibiting platelet agglutination than was the unmodified peptide (IC(50) = 0.02 +/- 0. 006 micromol/L v 0.1 +/- 0.03 micromol/L, P =.001). In addition and by contrast, S-nitroso-AR545C was a powerful inhibitor of adenosine diphosphate-induced platelet aggregation (IC(50) = 0.018 +/- 0.002 micromol/L), while AR545C had no effect on aggregation. These effects were confirmed in studies of adhesion to and aggregation on extracellular matrix under conditions of shear stress in a cone-plate viscometer, where 1.5 micromol/L S-nitroso-AR545C inhibited platelet adhesion by 83% and essentially completely inhibited aggregate formation, while the same concentration of AR545C inhibited platelet adhesion by 74% and had significantly lesser effect on aggregate formation on matrix (P