Domenico Praticò

VITAMIN E SUPPRESSES ISOPROSTANE GENERATION IN VIVO AND REDUCES ATHEROSCLEROSIS IN APOE-DEFICIENT MICE

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 iPF2α-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 iPF2α-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.

Modulation of monocyte-endothelial cell interactions by platelet microparticles.

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.

Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles.

Microparticles are released during platelet activation in vitro and have been detected in vivo in syndromes of platelet activation. They have been reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. To address the mechanism(s) of cellular activation by platelet microparticles, we examined their effects on platelets and endothelial cells. Activation of human platelets by diverse stimuli (thrombin, 0.1 U/ml; collagen, 4 microg/ml; and the calcium ionophore A23187, 1 microM) results in shedding of microparticles. Pretreatment of these particles, but not membrane fractions from resting platelets, with (s)PLA2 evokes a dose-dependent increase in platelet aggregation, intracellular [Ca2+] movement, and inositol phosphate formation. These effects localize to the arachidonic acid fraction of the microparticles and are mimicked by arachidonic acid isolated from them. However, platelet activation requires prior metabolism of microparticle arachidonic acid to thromboxane A2. Thus, pretreatment of platelets with the cyclooxygenase (COX) inhibitor, indomethacin (20 microM), the thromboxane antagonist SQ29,548 (1 microM), or the protein kinase C inhibitor GF109203X (5 microM) prevents platelet activation by microparticles. However, platelet microparticles fail to evoke an inositol phosphate response directly, via either of the cloned thromboxane receptor isoforms stably expressed in human embryonic kidney (HEK) 293 cells. Prelabeling platelets with [2H(8)] arachidonate was used to demonstrate platelet metabolism of the microparticle-derived substrate to thromboxane. Platelet microparticles can also induce expression of COX-2 and prostacyclin (PGI2) production, but not expression of COX-1, in human endothelial cells. These effects are prevented by pretreatment with actinomycin D (12 microM) or cycloheximide (5 microg/ml). Expression of COX-2 is again induced by the microparticle arachidonate fraction, which it may then use to synthesize PGI2. Both PGE2 and iloprost, a stable PGI2 analog, evoke human umbilical vein endothelial cell COX-2 expression, albeit with kinetics that differ from the response to platelet microparticles. These studies indicate a novel mechanism of transcellular lipid metabolism whereby platelet activation may be amplified or modulated by concentrated delivery of arachidonic acid to adjacent platelets and endothelial cells.

Increased F2-isoprostanes in Alzheimer's disease: evidence for enhanced lipid peroxidation in vivo

Alzheimers 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 iPF2α‐III and iPF2α ‐VI using gas chromatography‐mass spectrometry in AD and control brains. The levels of both isoprostanes, but not of 6‐keto PGF1α, 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 Parkinsons disease or from nonneuropsychiatric disorders. iPF2α ‐ IV, but not iPF2α‐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.—Praticò, D., Lee, V. M.‐Y., Trojanowski, J. Q., Rokach, J., FitzGerald, G. A. Increased F2‐isoprostanes in Alzheimers disease: evidence for enhanced lipid peroxidation in vivo. FASEB J. 12, 1777–1783 (1998)

Localization of distinct F2-isoprostanes in human atherosclerotic lesions.

F2-Isoprostanes are prostaglandin (PG) isomers formed in situ in cell membranes by peroxidation of arachidonic acid. 8-epi PGF2alpha and IPF2alpha-I are F2-isoprostanes produced in humans which circulate in plasma and are excreted in urine. Measurement of F2-isoprostanes may offer a sensitive, specific, and noninvasive method for measuring oxidant stress in clinical settings where reactive oxygen species are putatively involved. We determined whether isoprostanes were present in human atherosclerotic lesions, where lipid peroxidation is thought to occur in vivo. 8-epi PGF2alpha ranged from 1.310-3.450 pmol/micromol phospholipid in atherectomy specimens compared with 0.045-0.115 pmol/micromol phospholipid (P < 0.001) in vascular tissue devoid of atherosclerosis. Corresponding values of IPF2alpha-I were 5.6-13.8 vs. 0.16-0.44 pmol/micromol phospholipid (P < 0.001). Levels of the two isoprostanes in vascular tissue were highly correlated (r = 0.80, P < 0.0001). Immunohistochemical studies confirmed that foam cells adjacent to the lipid necrotic core of the plaque were markedly positive for 8-epi PGF2alpha. These cells were also reactive with anti-CD68, an epitope specific for human monocyte/macrophages. 8-epi PGF2alpha immunoreactivity was also detected in cells positive for anti-alpha-smooth muscle actin antibody, which specifically recognizes vascular smooth muscle cells. Our results indicate that 8-epi PGF2alpha and IPF2alpha-I, two distinct F2-isoprostanes and markers of oxidative stress in vivo, are present in human atherosclerotic plaque. Quantitation of these chemically stable products of lipid peroxidation in target tissues, as well as in biological fluids, may aid in the rational development of antioxidant drugs in humans.

Oxidative injury in diseases of the central nervous system: focus on alzheimer’s disease

Alzheimers disease is one of the most challenging brain disorders and has profound medical and social consequences. It affects approximately 15 million persons worldwide, and many more family members and care givers are touched by the disease. The initiating molecular event(s) is not known, and its pathophysiology is highly complex. However, free radical injury appears to be a fundamental process contributing to the neuronal death seen in the disorder, and this hypothesis is supported by many (although not all) studies using surrogate markers of oxidative damage. In vitro and animal studies suggest that various compounds with antioxidant ability can attenuate the oxidative stress induced by beta-amyloid. Recently, clinical trials have demonstrated potential benefits from treatment with the antioxidants, vitamin E, selegiline, extract of Gingko biloba, and idebenone. Further studies are warranted to confirm these findings and explore the optimum timing and antioxidant combination of such treatments in this therapeutically frustrating disease.

Increased 8,12‐iso‐iPF2α‐VI in Alzheimer's disease: Correlation of a noninvasive index of lipid peroxidation with disease severity

The isoprostane 8,12‐iso‐iPF2α‐VI is a sensitive and specific marker of in vivo lipid peroxidation. We found elevated levels in the urine, blood, and cerebrospinal fluid of patients with Alzheimers disease (AD) that correlated with measures of cognitive and functional impairment, established biomarkers of AD pathology (cerebrospinal fluid tau and amyloid) and the number of apolipoprotein E ε4 alleles. These results suggest that 8,12‐iso‐iPF2α‐VI is a useful biomarker of oxidative damage in AD. Ann Neurol 2000;48:809–812

Increased Formation of Distinct F2 Isoprostanes in Hypercholesterolemia

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 iPF2alpha-III and iPF2alpha-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. iPF2alpha-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 iPF2alpha-VI (281+/-22 versus 175+/-13; P<0.0005). Serum cholesterol correlated with urinary iPF2alpha-III (r=0.41; P<0.02) and iPF2alpha-VI (r=0. 39; P<0.03) in HFH patients. Urinary excretion of iPF2alpha-III (81+/-10 versus 59+/-4; P<0.05) and iPF2alpha-VI (195+/-18 versus 149+/-20; P<0.05) was also increased in moderately hypercholesterolemic subjects (n=24) compared with their controls. Urinary excretion of iPF2alpha-III and iPF2alpha-VI was correlated (r=0.57; P<0.0001; n=106). LDL iPF2alpha-III levels (ng/mg arachidonate) were elevated (P<0.01) in HFH patients (0.32+/-0.08) compared with controls (0.09+/-0.02). The concentrations of iPF2-III in LDL and urine were significantly correlated (r=0.42; P<0.05) in HFH patients. CONCLUSIONS Asymptomatic patients with moderate and severe hypercholesterolemia have evidence of oxidant stress in vivo.

8-Epi PGF2α Generation During Coronary Reperfusion

Background Myocardial reperfusion is believed to be associated with free radical injury. However, indexes of oxidative stress in vivo have been limited by their poor specificity and sensitivity. Isoprostanes are stable products of arachidonic acid formed in a nonenzymatic, free radical–catalyzed manner. We have developed a sensitive and specific assay for one of these compounds, 8-epi prostaglandin (PG) F2α. Methods and Results To address its utility as an index of oxidative stress during coronary reperfusion, we measured urinary levels by gas chromatography/mass spectrometry in a canine model of coronary thrombolysis, in patients with acute myocardial infarction treated with thrombolytic therapy, and in patients after elective coronary artery bypass surgery. Urinary 8-epi PGF2α was unchanged after circumflex artery occlusion in a canine model of coronary thrombolysis (n=13; 437.2±56.4 versus 432.7±55.2 pmol/mmol creatinine) but increased significantly ( P <.05) immediately after reperfusion (553.8±64.7 pmol/mmol). Urinary levels were increased ( P <.001) in patients (n=12) with acute myocardial infarction given lytic therapy (265.8±40.8 pmol/mmol) compared with age-matched control subjects (n=20; 91.5±11.8 pmol/mmol) and patients with stable coronary disease (n=20; 95.7±6.3 pmol/mmol). Preoperative levels rose from 113.2±11.8 to 248.2±86.3 pmol/mmol at 30 minutes into revascularization to 332.2±82.6 pmol/mmol by 15 minutes after global myocardial reperfusion ( P <.05) and dropped to 181.2±50.4 pmol/mmol at 30 minutes and 120.2±9.9 pmol/mmol at 24 hours after bypass surgery (n=5). Corresponding changes in spin adduct formation, found with electron paramagnetic resonance, were noted in 2 patients. Conclusions These data support the hypothesis that free radical generation occurs during myocardial reperfusion. Measurement of isoprostane production may serve as a noninvasive index of oxidative stress.

Platelet-derived microparticles stimulate proliferation, survival, adhesion, and chemotaxis of hematopoietic cells.

OBJECTIVE Peripheral blood platelet-derived microparticles (PMPs) circulate in blood and may interact directly with target cells affecting their various biological functions. METHODS To investigate the effect of human PMPs on hematopoiesis, we first phenotyped them for expression of various surface molecules and subsequently studied various biological responses of normal stem/progenitor (CD34(+)) and more differentiated precursor cells as well as several leukemic cell lines to PMPs. RESULTS We found that, in addition to platelet-endothelium attachment receptors (CD41, CD61 and CD62), PMPs express G-protein-coupled seven transmembrane-span receptors such as CXCR4 and PAR-1; cytokine receptors including TNF-RI, TNF-RII, and CD95; and ligands such as CD40L and PF-4. Moreover, we found that several of these receptors could be transferred by PMPs to the membranes of normal as well as malignant cells and observed that PMPs: 1) chemoattract these cells, 2) increase their adhesion, proliferation, and survival, and 3) activate in these cells various intracellular signaling cascades including MAPK p42/44, PI-3K-AKT, and STAT proteins. The biological effects of PMPs were only partly reduced by heat inactivation or trypsin digest, indicating that, in addition to the protein components of PMPs, lipid components are also responsible for their biological activity. CONCLUSIONS We conclude that PMPs modulate biological functions of hematopoietic cells and postulate that they play an important but as yet not fully understood role in intercellular cross-talk in hematopoiesis. Further studies, however, are needed to identify the PMP components that exert specific biological effects.