Showing papers by "Garret A. FitzGerald published in 1998"
TL;DR: The demonstration that platelet MP may modulate important aspects of endothelial and monocyte function provides a novel mechanism by which platelets may interact with such cells in human atherosclerosis and inflammation.
Abstract: Platelets, activated by various agonists, produce microparticles (MP) from the plasma membrane, which are released into the extracellular space. Although the mechanism of MP formation has been clarified, their biological importance remains ill defined. We have recently shown that platelet-derived MP influence platelet and endothelial cell function. In this study, we have further examined the mechanism of cellular activation by platelet MP. To address the possibility that they may influence monocyte-endothelial interactions, we used an in vitro assay to examine their effects on the adhesion of monocytes to human umbilical vein endothelial cells (HUVEC). Platelet MP increased the adhesion of monocytes to HUVEC in a time- and dose-dependent manner. Maximal adhesion of monocytes to resting HUVEC was observed after 24 h of stimulation with MP. Similar kinetics were observed with U-937 (human promonocytic leukemia) cells, used as a model for the blood-borne monocyte. Maximal adhesion of resting monocytes to MP-stimulated HUVEC was observed after 5 h of stimulation with MP. The EC50s for MP-induced increases in HUVEC, monocyte, and U-937 cell adhesion is 8.74, 43.41, and 10.83 microg/ml of MP protein, respectively. The induction of monocyte-endothelial adhesion was mimicked by arachidonic acid isolated from MP. The observed increased cellular adhesiveness correlated with MP-induced upregulation of cell adhesion molecules. MP-stimulated HUVEC increased intracellular cell adhesion molecule-1 (ICAM-1) but not vascular cell adhesion molecule-1 (VCAM-1), P-, or E-selectin expression. Monocyte and U-937 lymphocyte function-associated antigen-1 (CD11a/CD18) and macrophage antigen-1 (CD11b/ CD18, alpham/beta2) were both upregulated upon MP stimulation, but an increase in p150,95 (CD11c/CD18), very late antigen-1, or ICAM-1 expression was not observed. The functional importance of these changes was demonstrated with blocking antibodies. MP also induced the chemotaxis of U-937 cells in a dose-dependent manner with an EC50 of 4.40 microg/ml of MP protein. Similarly, arachidonic acid isolated from MP mimicked the chemotactic response. A role for PKC was implicated in both adhesion and chemotaxis. GF 109203X, a specific inhibitor of PKC, significantly reduced monocyte-endothelial adhesion, as well as U-937 chemotaxis. The demonstration that platelet MP may modulate important aspects of endothelial and monocyte function provides a novel mechanism by which platelets may interact with such cells in human atherosclerosis and inflammation.
535 citations
TL;DR: Results indicate that oxidative stress is increased in the apoE–/– mouse, is of functional importance in the evolution of atherosclerosis and can be suppressed by oral administration of vitamin E.
Abstract: Oxidative modification of low density lipoprotein (LDL) has been implicated in atherogenesis. Evidence consistent with this hypothesis includes the presence of oxidized lipids in atherosclerotic lesions, the newly discovered biological properties conferred on LDL by oxidation and the acceleration of atherogenesis by in vivo delivery of the gene for 15-lipoxygenase, an oxidizing enzyme present in atherosclerotic lesions. However, it is still unknown whether oxidative stress actually coincides with the evolution of the disease or whether it is of functional relevance to atherogenesis in vivo. Isoprostanes are products of arachidonic acid catalyzed by free radicals, which reflect oxidative stress and lipid peroxidation in vivo. Elevation of tissue and urinary isoprostanes is characteristic of human atherosclerosis. Here, deficiency in apolipoprotein E in the mouse (apoE-/-) resulted in atherogenesis and an increase in iPF2alpha-VI, an F2-isoprostane, in urine, plasma and vascular tissue. Supplementation with vitamin E significantly reduced isoprostane generation, but had no effect on plasma cholesterol levels in apoE-/- mice. Aortic lesion areas and iPF2alpha-VI levels in the arterial wall were also reduced significantly by vitamin E. Our results indicate that oxidative stress is increased in the apoE-/- mouse, is of functional importance in the evolution of atherosclerosis and can be suppressed by oral administration of vitamin E.
510 citations
TL;DR: The data suggest that specific isoprostane analysis may reflect increased oxidative stress in AD, and increased F2‐isoprostanes in Alzheimer's disease: evidence for enhanced lipid peroxidation in vivo.
Abstract: Alzheimer's disease (AD) includes a group of dementing neurodegenerative disorders that have diverse etiologies but the same hallmark brain lesions. Since oxidative stress may play a role in the pathogenesis of AD and isoprostanes are chemically stable peroxidation products of arachidonic acid, we measured both iPF2alpha-III and iPF2alpha -VI using gas chromatography-mass spectrometry in AD and control brains. The levels of both isoprostanes, but not of 6-keto PGF1alpha, an index of prostaglandin production, were markedly elevated in both frontal and temporal poles of AD brains compared to the corresponding cerebella. Levels were also elevated compared to corresponding areas of brains from patients who had died with schizophrenia or Parkinson's disease or from nonneuropsychiatric disorders. iPF2alpha -IV, but not iPF2alpha-III, levels were higher in ventricular CSF of AD brains relative to the non-AD brains. These data suggest that specific isoprostane analysis may reflect increased oxidative stress in AD.
412 citations
TL;DR: Urinary excretion of the 2 F2 isoprostanes was significantly increased in hypercholesterolemic patients, whereas substrate AA in urine did not differ between the groups, and asymptomatic patients with moderate and severe hypercholesterol have evidence of oxidant stress in vivo.
Abstract: Background—F2 isoprostanes are stable, free radical–catalyzed products of arachidonic acid that reflect lipid peroxidation in vivo. Methods and Results—Specific assays were developed by use of mass spectrometry for the F2 isoprostanes iPF2α-III and iPF2α-VI and arachidonic acid (AA). Urinary excretion of the 2 F2 isoprostanes was significantly increased in hypercholesterolemic patients, whereas substrate AA in urine did not differ between the groups. iPF2α-III (pmol/mmol creatinine) was elevated (P<0.0005) in homozygous familial hypercholesterolemic (HFH) patients (85±5.5; n=38) compared with age- and sex-matched normocholesterolemic control subjects (58±4.2; n=38), as were levels of iPF2α-VI (281±22 versus 175±13; P<0.0005). Serum cholesterol correlated with urinary iPF2α-III (r=0.41; P<0.02) and iPF2α-VI (r=0.39; P<0.03) in HFH patients. Urinary excretion of iPF2α-III (81±10 versus 59±4; P<0.05) and iPF2α-VI (195±18 versus 149±20; P<0.05) was also increased in moderately hypercholesterolemic subjects (n...
284 citations
TL;DR: Increased urinary iPF2alpha-III is consistent with the hypothesis that oxidative stress occurs in COPD and provides a basis for dose finding and evaluation of antioxidant therapy in the treatment of this disease.
Abstract: Oxidative stress has been suggested as a potential mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). It has been difficult to address this hypothesis because of the limitations of conventional indices of lipid peroxidation in vivo. F2-isoprostanes (iPs) are prostaglandin isomers formed by free radical dependent peroxidation of arachidonic acid. Urinary iPF2alpha-III is a relatively abundant iPs produced in humans. In the present study, we investigated whether COPD is associated with enhanced oxidative stress by measuring urinary levels of this compound. Urinary excretion of iPF2alpha-III was determined in 38 patients with COPD and 30 sex- and age-matched healthy control subjects. Levels of iPF2alpha-III were significantly higher in patients with COPD (median, 84 pmol/ mmol creatinine; range, 38 to 321) than in healthy controls (median, 35.5 pmol/mmol creatinine; range, 15 to 65) (p < 0.0001). This elevation was independent of age, sex, smoking history, or duration of the disease. An inverse relationship was observed with the level of PaO2 (r = -0.38, p = 0. 019). Aspirin treatment failed to decrease urinary levels of iPF2alpha-III (102 +/- 8 versus 99.2 +/- 7.3 pmol/ mmol creatinine), whereas 11-dehydro TxB2 was significantly reduced (695 +/- 74 versus 95 +/- 10 pmol/mmol creatinine) (p < 0.0001). Elevated levels of iPF2alpha-III (median, 125 pmol/mmol creatinine; range, 110 to 170) in five patients with COPD declined (median, 90 pmol/mmol creatinine; range, 70 to 110) (p < 0.001) as an acute exacerbation in their clinical condition resolved. Increased urinary iPF2alpha-III is consistent with the hypothesis that oxidative stress occurs in COPD. This provides a basis for dose finding and evaluation of antioxidant therapy in the treatment of this disease.
265 citations
TL;DR: Urinary IPF2α-I is a novel index of lipid peroxidation in vivo, which can be measured with precision and sensitivity and is highly correlated with urinary 8-iso-PGF2 Alpha, an isomer of the PGG/H synthase (cyclooxygenase or COX) enzyme product.
Abstract: Isoprostanes are prostaglandin isomers produced from arachidonic acid by a free radical-catalyzed mechanism. Urinary excretion of 8-iso-prostaglandin F2α, an isomer of the PGG/H synthase (cyclooxygenase or COX) enzyme product, prostaglandin F2α (PGF2α), has exhibited promise as an index of oxidant stress in vivo. We have developed a quantitative method to measure isoprostane F2α-I, (IPF2α-I) a class I isomer (8-iso-PGF2α is class IV), using gas chromatography/mass spectrometry. IPF2α-I is severalfold as abundant in human urine as 8-iso-PGF2α, with mean values of 737 ± 20.6 pg/mg creatinine. Both isoprostanes are formed in a free radical-dependent manner in low density lipoprotein oxidized by copper in vitro. However, IPF2α-I, unlike 8-iso-PGF2α, is not formed in a COX-dependent manner by platelets activated by thrombin or collagen in vitro. Similarly, COX inhibition in vivo has no effect on IPF2α-I. Neither serum IPF2α-I, an index of cellular capacity to generate the isoprostane, nor urinary excretion of IPF2α-I, an index of actual generation in vivo, is depressed by aspirin or indomethacin. In contrast, both serum thromboxane B2 and urinary excretion of its 11-dehydro metabolite are depressed by the COX inhibitors. Although serum 8-iso-PGF2α formation is substantially depressed by COX inhibitors, urinary excretion of the compound is unaffected. Urinary IPF2α-I is elevated in cigarette smokers compared with controls (1525 ± 180 versus 740 ± 40 pg/mg creatinine; P < 0.01) and is highly correlated with urinary 8-iso-PGF2α (r = 0.9; P < 0.001). Urinary IPF2α-I is a novel index of lipid peroxidation in vivo, which can be measured with precision and sensitivity. It is an abundant F2-isoprostane formed in a free radical- but not COX-dependent manner. Although 8-iso-PGF2α may be formed as a minor product of COX, this pathway contributes trivially, if at all, to levels in urine. Urinary excretion of both isoprostanes is elevated in cigarette smokers.
214 citations
TL;DR: Heparin, administered orally in combination with the delivery agent SNAC, produces significant elevations in 4 indexes of anticoagulant effect in healthy human volunteers, establishing the feasibility of oral delivery of anticonvulsant doses of heparin in humans and may have broader implications for the absorption of macromolecules.
Abstract: Background —Parenteral heparin is the anticoagulant of choice in hospitalized patients. Continued anticoagulation is achieved by subcutaneous administration of low-molecular-weight heparin or with an orally active anticoagulant such as warfarin. An oral heparin formulation would avoid the inconvenience of subcutaneous injection and the unfavorable drug interactions and adverse events associated with warfarin. A candidate delivery agent, sodium N -[8(-2-hydroxybenzoyl)amino]caprylate (SNAC), was evaluated with escalating oral heparin doses in a randomized, double-blind, controlled clinical study for safety, tolerability, and effects on indexes of anticoagulation. Methods and Results —Increases in activated partial thromboplastin time (aPTT), anti-factors IIa and Xa, and tissue factor pathway inhibitor (TFPI) concentrations were detected when normal volunteers were dosed with 10.5 g SNAC/20 000 IU heparin by gavage in some subjects. For the entire group, 30 000 IU SNAC and heparin elevated TFPI from 74.9±7.6 to 254.2±12.3 mg/mL ( P P P Conclusions —Heparin, administered orally in combination with the delivery agent SNAC, produces significant elevations in 4 indexes of anticoagulant effect in healthy human volunteers. These results establish the feasibility of oral delivery of anticoagulant doses of heparin in humans and may have broader implications for the absorption of macromolecules.
126 citations
TL;DR: Both PGF2α and 8,12-iso-iPF2α-III induce myocyte hypertrophy via discrete signaling pathways through JNK1 and c-Jun pathways and the hypertrophic effects of these two agonists on cardiomyocytes are additive.
120 citations
Journal Article•
TL;DR: It is demonstrated that lipid peroxidation is increased in vivo in patients with cirrhosis and suggests that oxidant stress might contribute to the deterioration of liver disease.
Abstract: BACKGROUND Lipid peroxidation is thought to play a role in the evolution of liver damage, based on evidence in experimental models. However, evidence that lipid peroxidation occurs in patients with liver disease remains to be provided. We addressed the hypothesis by measuring levels of 8-epi Prostaglandin F2 alpha a bioactive prostaglandin isomer produced by free radical catalyzed peroxidation of arachidonic acid, in patients with liver cirrhosis. METHODS In 42 patients with hepatic cirrhosis 8-epi Prostaglandin F2 alpha, factor VII activity, endotoxemia, carotenoids and alpha-tocopherol were measured. In 10 patients 8-epi Prostaglandin F2 alpha was also measured before and 30 days after 300 mg b.i.d. vitamin E administration. RESULTS Cirrhotic patients had significant higher 8-epi Prostaglandin F2 alpha, excretion than controls [median (range): 199.2 (60.0-812) vs 85.9 (55.6-160.0) pg/mg creatinine, p < 0.0001]. Patients with urinary 8-epi Prostaglandin F2 alpha above the range in controls were more likely to have moderate or severe than mild liver failure (p < 0.004). They also had lower factor VII activity (62 +/- 19 vs 74 +/- 15%, P < 0.02) than patients with normal levels of the isoprostane. Urinary excretion of 8-epi Prostaglandin F2 alpha correlated directly with endotoxemia (Rho = 0.56, p < 0.0002) and inversely with factor VII (Rho = -0.39, p < 0.02). Cirrhotic patients given vitamin E showed a significant decrease of urinary 8-epi Prostaglandin F 2 alpha [median (range): 342.5 (170 - 812) vs 292.5 (142-562) pg/mg creatinine, p < 0.04]. CONCLUSION This study demonstrated that lipid peroxidation is increased in vivo in patients with cirrhosis and suggests that oxidant stress might contribute to the deterioration of liver disease.
105 citations
TL;DR: The identification in human urine of two more isomers of F2-iP, 8,12-iso-iPF2α-VI and 5-epi-8,12 -iso- iPF2 α-VI, using high performance liquid chromatography/tandem mass spectrometry and gas Chromatography/mass Spectrometry.
85 citations
TL;DR: Results indicate that PKC-dependent phosphorylation is of critical importance to homologous regulation of hIP, and Ser-328 is a primary site for PKC phosphorylated of h IP.
TL;DR: Development of specific assays for these compounds in urine suggests their utility as noninvasive indices of oxidant stress in vivo, andCrystallization of the biosynthetic enzymes is likely to facilitate the development of highly specific inhibitors, as is the case already for COX-2.
Abstract: Arachidonic acid (AA) is an unsaturated fatty acid constituent of the phospholipid domain of cell membranes. It is subject to release via mobilization of phospholipases, particularly a cytoplasmic phospholipase A 2 . Thereafter, it may be metabolized by at least two cyclooxygenase (COX) isoforms to prostaglandins and related compounds, via lipoxygenases to leukotrienes and via p450-catalyzed metabolism to epoxyeicosatrienoic acids. Collectively, these bioactive lipids are termed eicosanoids. All of these lipids express potent bioactivity in vitro. Clinical studies have already demonstrated the importance of COX and lipoxygenase (LOX) products in human disease. The generation of models of COX, LOX and prostaglandin receptor gene inactivation is likely to broaden our insight into the importance of these compounds in vivo. Crystallization of the biosynthetic enzymes is likely to facilitate the development of highly specific inhibitors, as is the case already for COX-2. AA possesses intrinsic biological properties. It is also subject to free radical attack, generating isomeric eicosanoid species, the isoeicosanoids. These compounds may also express biological activity in vitro, although their importance in vivo is unclear. Development of specific assays for these compounds in urine suggests their utility as noninvasive indices of oxidant stress in vivo.
TL;DR: Two new mouse models closely mimic human atherosclerosis, providing exciting opportunities for elucidating the molecular mechanisms of this disease and for evaluating novel therapeutics.
Abstract: Two new mouse models closely mimic human atherosclerosis, providing exciting opportunities for elucidating the molecular mechanisms of this disease and for evaluating novel therapeutics (pages 934–938).
TL;DR: In this article, a new reliable index of oxidative stress based on iPF2α-VI (previously IPF 2α-I), a new Class VI isoprostane was proposed.
Journal Article•
TL;DR: It is suggested that this biologically active isoprostane does not directly contribute to the portal hypertension observed in hepatic cirrhosis and there is no correlation between iPF2 alpha-III portal levels and the portal pressure observed in these patients.
Abstract: BACKGROUND Isoprostane F2 alpha-III (iPF2 alpha-III), a recently described member of a family of prostaglandin F2 alpha isomers and a biologically active end-product of lipid peroxidation, has been reported to increase portal pressure in cirrhotic rats. We found that its urinary levels were elevated in cirrhotic patients. METHODS To investigate whether portal levels of iPF2 alpha-III were elevated in cirrhotic patients and whether there was a relationship between these levels and the portal pressure in the same patients, peripheral and portal plasma from cirrhotic patients (n = 18) undergoing elective transjugular intrahepatic portosystemic shunt and appropriate controls (n = 18) were assayed for iPF2 alpha-III levels by using a gas chromatography/mass spectrometry assay. Portal pressure was measured in all cirrhotic patients. RESULTS Cirrhotic patients had higher peripheral plasma levels of iPF2 alpha-III [78 (27-150) pg/mL] than controls [18(10-30)pg/mL] (P < 0.001). Portal iPF2 alpha-III levels were higher than plasma peripheral levels [129(50-375) pg/mL; P < 0.0001]. No correlation was found between peripheral and portal levels of iPF2 alpha-III (Rho = 0.17, P = 0.5). Portal levels of iPF2 alpha-III and portal pressure did not correlate (Rho = 0.17, P = 0.49). CONCLUSIONS This study shows that peripheral and portal levels of iPF2 alpha-III, marker of in vivo lipid peroxidation, are elevated in liver cirrhosis. There is no correlation between iPF2 alpha-III portal levels and the portal pressure observed in these patients. These findings suggest that this biologically active isoprostane does not directly contribute to the portal hypertension observed in hepatic cirrhosis.
TL;DR: A new and general synthetic methodology for the synthesis of isoprostanes, using the four key lactones which are constructed from D and L -glucose by a thionocarbonate-mediated radical cyclization reaction, is presented in this article.
Abstract: A new class of natural products called isoprostanes are formed in humans as a result of non-enzymatic free-radical-catalyzed lipid peroxidation. Isoprostanes possess biological activity and can be used as an index of free-radical lipid peroxidation and as a marker of oxidative stress. We report here a new and general synthetic methodology for the syntheses of isoprostanes, using the four key lactones which are constructed from D and L -glucose by a thionocarbonate-mediated radical cyclization reaction. These lactones possess the required stereochemistry and the right functional groups for the syntheses of isoprostanes.