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J Loscalzo

Publications and source records attributed to J Loscalzo.

At least 37 records · Page 2Linked to original sources

Oxidative stress and renal dysfunction in salt-sensitive hypertension.

Hypertension is a risk factor for the development of end-stage renal disease. The mechanisms underlying hypertensive nephropathy are poorly understood. There is evidence, however, that in hypertension there is an accumulation of partially reduced oxygen and its derivatives, known collectively as reactive oxygen species, which may contribute to progressive renal dysfunction. In the present study, we assess the contribution of oxidative stress in the development of salt-dependent hypertensive nephrosclerosis. Going beyond previous end point studies, which inferred renal function either indirectly or only qualitatively, we have determined oxidative stress concurrently with direct and quantitative measurements of renal function (via inulin and p-aminohippuric acid clearances). Moreover, in this time-dependent study, the measurements have been taken under low- as well as high-salt diets. As was expected from previous studies, in the Dahl salt-sensitive rat, a high-salt diet (8% NaCl) resulted in the development of hypertension, in a decreased glomerular filtration rate, and in a decreased renal plasma flow as compared with the normotensive control, the Dahl salt-resistant rat. In addition, however, we found clear evidence for the accumulation of reactive oxygen species in renal tissue homogenates of Dahl salt-sensitive rats on the high-salt diet. Our time-dependent protocol also indicated that renal oxidative stress follows, in time, the development of hypertension. We also found that after 2 weeks of increased salt loading, Dahl salt-sensitive rats excreted less cyclic guanosine monophosphate and NO(x) than Dahl salt-resistant rats on the same diet. It is known that urinary cyclic guanosine monophosphate and NO(x) represent the activity and stable derivatives of renal NO., respectively, and that they closely correlate with renal vascular resistance. Therefore, our results suggest that, in the Dahl salt-sensitive rat, increased oxidative stress is associated with salt-dependent hypertensive nephrosclerosis and that decreased NO. bioavailability may represent a common factor responsible for the vascular and glomerular dysfunction.

Animals↗

Heparin reacts with and inactivates nitric oxide.

Although heparin is a well-known anticoagulant, in some cases it promotes a prothrombotic state and does so through both antibody-dependent and antibody-independent platelet activation. In this study, heparin was found to reverse the antiplatelet effect of an NO donor. S-nitroso-glutathione (SNO-Glu), with an EC(50) of 1.8 U/mL. Ultraviolet/visible spectral analysis and the Griess assay showed that increasing heparin concentrations on a dose-dependent basis eliminated acidified NO(x) species. Since heparin is a heterogeneous mixture of glycosaminoglycans, the effects of six different heparin disaccharides were compared with various substitutions on the hexose rings to determine which functional group(s) of the polysaccharide interact with acidified NO(x). Among the six disaccharides tested, only types I-S and II-S had the effect, suggesting that the sulfamino-group at the C2 position of the glucosamine moiety was critical for the elimination of acidified NO(x) species. Mass spectrometry experiments gave results consistent with these observations, indicating that only the I-S and II-S heparin disaccharides were modified upon treatment with NaNO(2)/HCl. Negative-ion electro-spray ionization MS and tandem MS analyses of the native compounds and their deuterium-labeled analogs confirmed that the reaction products from nitrosation of these N-sulfated disaccharides had eliminated the C2-sulfamino-moiety and replaced it with methoxide derived from the solvent. Participation of the 6-sulfato-substituent appears to facilitate the elimination reaction. These data show that heparin can impair the antiplatelet properties of nitric oxide by interacting with the nitrosating species, and suggest that heparin-like glycosamino-glycans may interact with endothelium-derived nitric oxide in vivo to regulate the bioactivity of this important antiplatelet and vasorelaxant substance.

Anticoagulants↗

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.

Adenosine Triphosphate↗

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.

Antioxidants↗

Regulation of tissue factor expression in human microvascular endothelial cells by nitric oxide.

BACKGROUND: Tissue factor (TF) is a critical determinant of thrombin generation in normal hemostasis and in atherothrombotic disease. Nitric oxide has both antithrombotic and antiatherosclerotic actions in the vasculature, yet its role in the regulation of TF expression has not been examined. METHODS AND RESULTS: To study the effect of endogenous endothelium-derived nitric oxide on TF expression and activity, we induced TF in human microvascular endothelial cells with lipopolysaccharide or interleukin-1beta and observed a dose- and time-dependent increase in TF activity and expression by Northern and Western blotting. L-Arginine, the principal substrate for nitric oxide synthases, added to the media suppressed the induction of TF activity significantly (by 66% for lipopolysaccharide induction and by 59% for interleukin-1beta induction) at 24 hours. These changes in activity were accompanied by correlative changes in TF protein and steady-state mRNA. D-Arginine had no effect, and inhibition of endogenous nitric oxide production failed to increase TF expression. CONCLUSIONS: These data suggest that enhanced production of endothelium-derived nitric oxide reduces endotoxin- and cytokine-induced expression of TF and, thereby, the prothrombotic phenotype of the endothelial cell.

Arginine↗

Aspirin.

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Angina, Unstable↗

Roles of endothelial dysfunction in coronary artery disease.

Endothelial dysfunction is an early and persistent vascular abnormality in the evolution of atherothrombotic disease. Risk factors for atherosclerosis promote an inflammatory oxidative environment in the vasculature that induces pathologic changes in endothelial function, including the support of enhanced smooth muscle tone, thrombosis, and smooth muscle proliferation. This article provides an overview of the molecular basis of endothelial dysfunction and of its diagnosis and treatment.

Antioxidants↗

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.

Animals↗

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.

Acetylcholine↗

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.

Journal Article↗

S-nitrosoderivative of a recombinant fragment of von Willebrand factor (S-nitroso-AR545C) inhibits thrombus formation in guinea pig carotid artery thrombosis model.

Antiplatelet drugs are the mainstays of therapy for acute and chronic cardiovascular diseases. S-nitroso-AR545C - an S-nitrosoderivative of a recombinant von Willebrand factor fragment AR545C spanning Ala 444 to Asp 730 and containing an Arg 545 Cys mutation, was previously found to inhibit ristocetin- and ADP-induced platelet aggregation and the interaction of platelets with extracellular matrix (ECM). In the current study we tested the antithrombotic properties of S-nitroso-AR545C on guinea pig platelets and in a platelet-rich thrombosis model in the guinea pig. Preincubation of guinea pig platelets with 0.1 microM of S-nitroso-AR545C decreased ristocetin-induced agglutination by 40% (p = 0.009) and completely abolished ADP-induced aggregation (p <0.0001). At concentration of 1.0 microM, S-nitroso-AR545C completely inhibited platelet adhesion (represented by surface coverage - SC) and decreased aggregate formation (represented by average aggregate size - AS) by more than 50%. Treatment of guinea pigs with 1.0 mg/kg S-nitroso-AR545C resulted in a significantly delayed time to arterial occlusion (31.7+/-6.0 min vs. 13.9+/-3.2 min, p <0.02). Similarly, total patency time was longer in the group injected with S-nitroso-AR545C compared to the control group. However, the difference was not statistically significant (33.8+/-6.3 min vs. 20.2+/-3.3 min, p = 0.07). No change in platelet count, hematocrit and bleeding time was observed 60 min after injection compared to baseline. In contrast, a significant decrease in SC (p <0.0001) and AS (p <0.01) were observed 60 min after the injection of S-nitroso-AR545C, whereas no change in these parameters was observed in the control group. These observations indicate that S-nitroso-AR545C exhibits significant antiadhesive and antiaggregating effects in-vitro and inhibits clot formation in-vivo suggesting that this compound may have potential therapeutic advantages.

Animals↗