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Biomedical subjects

J Loscalzo

Publications and source records attributed to J Loscalzo.

At least 91 records · Page 5Linked to original sources
PubMed1997

Changes in the amplitude of cyclic load biphasically modulate endothelial cell DNA synthesis and division.

Several physical factors, including shear stress and cyclic load, modulate the ability of endothelial cells to respond to injury. The objective of these experiments was to test the hypothesis that cyclic mechanical load stimulates endothelial cell DNA synthesis and division in vitro. Rabbit aortic endothelial cells were cultured on Flex I flexible-bottomed culture plates, and subjected to load amplitudes of increasing magnitude (0, 0.18, 0.24 and 0.27 load at 1 Hz) using a Flexercell strain unit. Cells were harvested enzymatically and cell numbers determined on days 1, 3 and 5 after initiating the load regimen. DNA synthesis was quantified after trichloroacetic acid precipitation of [3H]thymidine-labeled cells from: (1) whole culture wells and (2) areas of minimum and maximum strain in culture cells. Data were analyzed using analysis of variance and a Tukey's test (n = 6 observations/strain regimen per day in triplicate). Results from analysis of endothelial cells in whole, subconfluent cultures showed that cells subjected to strains of 0.18 had a decreased rate of cell division (76% of control) and DNA synthesis (63% of control), while cells subjected to strains of 0.24 and 0.27 had an increased rate of cell division (108 and 83% increase, respectively, compared with control; p < 0.001) and DNA synthesis (39 and 172% increase, respectively, compared with control; p < 0.001 for 0.27) on day 3 when compared with control cells. The results indicate that endothelial cells respond to various physiologic levels of cyclic load in a biphasic manner to initiate DNA synthesis and cell division. These data suggest that endothelial cell mitogenesis may be modulated by specific levels of cyclic load.

Animals↗
PubMed1997

New developments in nitrosovasodilator therapy.

Under basal conditions, nitric oxide (NO) modulates vascular tone, serves as an antithrombotic agent, and inhibits vascular smooth muscle cell proliferation. NO deficiency has been implicated in the pathophysiology of several vascular disorders, including hypertension, atherosclerosis, and restenosis, and provides a plausible biologic basis for the use of NO replacement therapy in these conditions. Treatment with conventional nitrate preparations is limited by a short therapeutic half-life, systemic absorption with potentially adverse hemodynamic effects, and drug tolerance. To overcome these limitations, novel delivery systems and novel NO donors have been developed that offer selective effects, a prolonged half-life, and a reduced incidence of tolerance.

Humans↗
PubMed1997

Endothelial dysfunction and atherothrombotic occlusive disease.

The endothelium plays a crucial role in the regulation of vascular function through the release of locally important molecular effectors such as endothelium-derived relaxing factor (EDRF) and prostacyclin. Endothelial cells also regulate vascular patency and tissue perfusion by inhibiting platelet aggregation and thrombosis, suppressing intimal proliferation, and maintaining vascular tone. Disturbances of the regulatory functions of the endothelium contribute to the pathophysiology of various disease states, including cardiovascular disease and stroke. Most studies focused on endothelial control of vasomotion and, in particular, on the action of EDRF; many studies have also emphasised that altered endothelial control of fibrinolysis and intimal growth influence the clinical expression of atherothrombotic disease. Importantly, understanding the pathophysiological role of endothelial dysfunction may lead to new therapeutic approaches for disease states caused by vascular disease.

Animals↗
PubMed1997

Nitric oxide and acetaminophen-mediated oxidative injury: modulation of interleukin-1-induced nitric oxide synthesis in cultured rat hepatocytes.

The role of endogenous hepatocyte synthesis of nitric oxide (NO) in states of oxidative stress is largely unknown. In a model of rat hepatocytes in primary culture, NO production was induced by exposure to interleukin-1beta (IL-1beta, 50 ng/ml). Acetaminophen-mediated oxidative injury was analyzed in unstimulated and stimulated hepatocytes in the presence and absence of N-methyl-L-arginine, a substrate inhibitor of NO synthesis (100 microM). Inhibition of NO synthesis was associated with exacerbation of acetaminophen-mediated oxidative injury. This effect was independent of guanylyl cyclase and cytochrome P450 activity. In addition, oxidative stress was associated with augmentation of interleukin-1beta-induced NO synthesis. Elevated NO synthesis occurred in parallel with increased inducible NO synthase (iNOS) enzyme activity and mass, steady-state levels of iNOS mRNA, increased transcription of the iNOS gene, and increased iNOS promoter activity. These effects were abrogated in the presence of antioxidants, suggesting that oxidative stress augments NO synthesis through a promoter-specific transcriptional regulatory mechanism. Thus, in conditions where oxidative injury may be a component of the overall proinflammatory state, induction of iNOS with subsequent elaboration of NO and augmentation of NO production may serve as an hepatoprotective mechanism against oxidative injury.

Acetaminophen↗
PubMed1996

alpha-tocopherol inhibits aggregation of human platelets by a protein kinase C-dependent mechanism.

BACKGROUND: Epidemiological studies indicate that vitamin E (alpha-tocopherol) exerts a beneficial effect on cardiovascular disease. The effect of vitamin E has generally been attributed to its antioxidant activity and the antioxidant protection of LDL. Distinct from its effect on LDL, vitamin E is also known to inhibit platelet aggregation and adhesion in vitro, but the mechanism(s) responsible for these observations are not known. METHODS AND RESULTS: Using gel-filtered platelets derived from platelet-rich plasma treated with alpha-tocopherol (500 mumol/L) or vehicle (0.5% ethanol), we found that inhibition of platelet aggregation by alpha-tocopherol was closely linked to its incorporation into platelets (r = -.78; P < .02). Platelet incorporation of alpha-tocopherol was associated with a significant reduction in platelet sensitivity to aggregation by adenosine 5'-diphosphate, arachidonic acid, and phorbol ester (PMA) by approximately, 0.15-, 2-, and 100-fold, respectively. In contrast, platelets treated similarly with butylated hydroxytoluene, another potent lipid-soluble antioxidant, did not demonstrate any change in sensitivity to these agents. Platelet incorporation of alpha-tocopherol inhibited PMA-induced stimulation of platelet protein kinase C (PKC) as determined by phosphorylation of the 47-kD PKC substrate. In 15 normal subjects, oral supplementation with alpha-tocopherol (400 to 1200 IU/d) resulted in an increase in platelet alpha-tocopherol content that correlated with marked inhibition of PMA-mediated platelet aggregation (r = .67; P < .01). Platelets derived from these subjects after supplementation also demonstrated apparent complete inhibition of PKC stimulation by PMA. CONCLUSIONS: These data indicate that platelet incorporation of alpha-tocopherol at levels attained with oral supplementation is associated with inhibition of platelet aggregation through a PKC-dependent mechanism. These observations may represent one potential mechanism for the observed beneficial effect of alpha-tocopherol in preventing the development of coronary artery disease.

Blood Platelets↗
PubMed1996

In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.

Animals↗
PubMed1996

Usefulness of fibrinogenolytic and procoagulant markers during thrombolytic therapy in predicting clinical outcomes in acute myocardial infarction. TIMI-5 Investigators. Thrombolysis in Myocardial Infarction.

Thrombin activity is increased in the setting of acute myocardial infarction (AMI) and has been shown to increase further after the administration of thrombolytic therapy for acute infarction. This increase in thrombin activity may play an important role in the 15% to 25% rate of failure to achieve initial reperfusion and in the 5% to 15% rate of early reocclusion after initially successful thrombolysis. To investigate potential mechanisms of thrombin formation in vivo, to understand better the balance of coagulation and fibrinolysis during treatment with recombinant tissue-type plasminogen activator (rt-PA), and to investigate the role of hemostatic markers as predictors of clinical events, we measured 3 markers of procoagulant activity: fibrinopeptide A (FPA), thrombin-antithrombin III complexes (TAT), and prothrombin fragment 1.2 (F1.2), and a marker of fibrinogenolytic activity (B beta 1-42) in patients enrolled in the Thrombolysis in Myocardial Infarction (TIMI)-5 study. This trial was a randomized, dose-ranging, pilot trial of hirudin versus heparin as adjunctive antithrombotic therapy with rt-PA administered to patients with AMI. Correlation of markers at 1 hour with clinical outcomes revealed that increased FPA and TAT levels were associated with increased mortality and TIMI grades 0, 1, or 2 flow at 90 minutes; increased F1.2 levels were associated with TIMI grade 0 or 1 flow at 90 minutes; and increased levels of all 3 procoagulant markers were associated with hemorrhagic events. Late (12 to 24 hours) increases in F1.2, TAT, and B beta 1-42 may be predictive of recurrent ischemia. These results suggest that selected markers of procoagulant and fibrinogenolytic activity may be useful in predicting clinical outcomes in patients treated with thrombolytic therapy for AMI.

Antithrombin III↗
PubMed1996

Nitrosation of tryptophan residue(s) in serum albumin and model dipeptides. Biochemical characterization and bioactivity.

Nitrosation of bovine serum albumin with acidified NaNO2 was compared to that of carboxymethyl-bovine serum albumin in which the thiol group is covalently blocked. Differential ultraviolet-visible (UV-Vis) spectroscopy and a modified Saville assay indicated that a non-cysteine residue(s) in carboxymethyl-bovine serum albumin was nitrosated. The nitrosated carboxymethyl-bovine serum albumin exhibited similar vasorelaxation activity as that observed with nitrosated bovine serum albumin. Identification of the nitrosated non-cysteine residue(s) was studied using 16 model dipeptides, each of which contained a glycyl residue and a variable residue. Using photolysis-chemiluminescence analysis, modified Saville assay, differential UV-Vis spectroscopy, and bioassays, L-glycyl-L-tryptophan (Gly-Trp) was found to be the only dipeptide that underwent significant nitrosation under these conditions. Liquid chromatography-UV-Vis spectroscopy-mass spectrometry showed that the NO group was attached to the indole nitrogen of tryptophan. Nitrosated Gly-Trp exhibited dose-dependent vasorelaxation and platelet inhibiting activity with apparent EC50 values of 1.1 +/- 0. 3 and 3.5 +/- 0.9 microM, respectively. Because N-nitroso-Gly-Trp does not release NO radical via spontaneous homolytic N-NO bond fission nor freely diffuse through cellular membranes, the ability of this compound to induce NO.-like biological effects suggests the existence of a (membrane-associated) transnitrosation system that facilitates delivery of -NO to its specific biologic target(s).

Animals↗
PubMed1996

Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis.

Highly reactive oxygen species rapidly inactivate nitric oxide (NO), and endothelial product which inhibits platelet activation. We studied platelet inhibition by NO in two brothers with a cerebral thrombotic disorder. Both children had hyperreactive platelets, as determined by whole blood platelet aggregometry and flow cytometric analysis of the platelet surface expression of P-selectin. Mixing experiments showed that the patients'platelets behaved normally in control plasma; however, control platelets suspended in patient plasma were not inhibited by NO. As determined by flow cytometry, in the presence of plasma from either patient there was normal inhibition of the thrombin-induced expression of platelet surface P-selectin by prostacyclin, but not NO. Using a scopoletin assay, we measured a 2.7-fold increase in plasma H2O2 generation in one patient and a 3.4-fold increase in the second patient, both compared woth control plasma. Glutathione peroxidase (GSH-Px) activity was decreased in the patients' plasmas compared with control plasma. The addition of exogenous GSH-Px led to restoration of platelet inhibition by NO. These data show that, in these patients' plasmas, impaired metabolism of reactive oxygen species reduces the bioavailability of NO and impairs normal platelet inhibitory mechanisms. These findings suggest that attenuated NO-mediated platelet inhibition produced by increased reactive oxygen species or impaired antioxidant defense may cause a thrombotic disorder in humans.

Cyclic GMP↗
PubMed1996

The macrophage and fibrinolysis.

The monocyte/macrophage plays a central role in fibrinolysis. Cell-surface of components of the plasminogen activator system leads to the elaboration of plasmin, which facilitates degradation of fibrin in the pericellular environment, as well as activation of matrixins, which promote degradation of matrix components. Fibrin degradation also occurs by way of a proteolytic system within the macrophage lysosome that does not involve plasmin. This alternate pathway involves first the binding of fibrin(ogen) to the surface integrin Mac-1 (CD11b/CD18) followed by internalization of the complex into the lysosome where the aspartyl protease cathepsin D degrades the protein. These molecular events underlie the many physiologic and pathophysiologic processes in which the monocyte/macrophage is involved, including adhesion, migration, matrix degradation and remodeling, wound healing, fibrinolysis, and atherosclerosis.

Animals↗
PubMed1996

Homocysteine, EDRF, and endothelial function.

Nitric oxide, a heterodiatomic molecule, serves as an endothelium-dependent vasodilator and an antithrombotic agent in the vasculature. Because of its reactivity under physiological conditions, accumulating data suggest that nitric oxide forms adducts with several classes of biologic compounds, one of which is the sulfhydryl functionality, to form thionitrites or S-nitrosothiols. In this overview, we will examine the effects of S-nitrosothiols in the vascular system as modulators of tone and inhibitors of platelet activation to understand better what role these compounds may play in physiological and pathological states. With this background, we will review the role nitric oxide (EDRF) plays in helping to ameliorate vascular injury by homocysteine.

Animals↗