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In previous studies of the role of tissue plasminogen activator (tPA) in the lung inflammatory response, we observed that tPA expression was present exclusively in the small arteries and arterioles within the lung and absent from the capillaries, veins, and large pulmonary arteries. To define more completely the expression pattern of tPA, we evaluated the distribution of this protein during prenatal and postnatal development. tPA was first observed in the rat fetus at day 13 in the large arteries of both the thoracic and cranial cavities, including the dorsal aortas and pulmonary arteries in the former and the internal carotid and middle cerebral arteries in the latter. By day 15, tPA was no longer detectable in the aortas but appeared throughout the pulmonary, subclavian, vertebral, and basilar arteries. At day 17, tPA had disappeared from the subclavian artery and the proximal portion of the vertebral artery but was found in the smaller arterial branches of these 2 large vessels. By the end of gestation, tPA had also disappeared from the main pulmonary arteries but remained in the branches at the hilus of the lung. At birth, tPA was concentrated in the endothelia of arteries within the pia mater, the basilar and superficial cerebral arteries, and the lung arterial system. As the animals reached maturity, tPA disappeared from the larger cerebral arteries and their cortical branches but continued to be expressed in the vessels of the pia mater and lung. This study indicates that tPA expression is a dynamic process that responds to a changing arterial environment during vascular development.

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Anticardiolipin (anti-CL) antibodies, diagnostic for antiphospholipid antibody syndrome, are associated with increased risks of venous and arterial thrombosis. Because CL selectively enhances activated protein C/protein S-dependent anticoagulant activities in purified systems and because CL is not known to be a normal plasma component, we searched for CL in plasma. Plasma lipid extracts [chloroform/methanol (2:1, vol/vol)] were subjected to analyses by using TLC, analytical HPLC, and MS. A plasma lipid component was purified that was indistinguishable from reference CL (M:1448). When CL in 40 fasting plasma lipid extracts (20 males, 20 females) was quantitated by using HPLC, CL (mean +/- SD) was 14.9 +/- 3.7 microgram/ml (range 9.1 to 24.2) and CL was not correlated with phosphatidylserine (3.8 +/- 1.7 microgram/ml), phosphatidylethanolamine (64 +/- 20 microgram/ml), or choline-containing phospholipid (1,580 +/- 280 microgram/ml). Based on studies of fasting blood donors, CL (>/=94%) was recovered in very low density, low density, and high density lipoproteins (11 +/- 5.3%, 67 +/- 11.0%, and 17 +/- 10%, respectively), showing that the majority of plasma CL (67%) is in low density lipoprotein. Analysis of relative phospholipid contents of lipoproteins indicated that high density lipoprotein is selectively enriched in CL and phosphatidylethanolamine. These results shows that CL is a normal plasma component and suggest that the epitopes of antiphospholipid antibodies could include CL or oxidized CL in lipoproteins or in complexes with plasma proteins (e. g., beta(2)-glycoprotein I, prothrombin, protein C, or protein S) or with platelet or endothelial surface proteins.

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Estrogen replacement therapy has been shown to attenuate atherogenesis, although the mechanisms for this effect are incompletely defined. Previously, we showed that 17-beta estradiol (estradiol) attenuated oxidant stress-induced increases in vascular low density lipoprotein (LDL) accumulation. It was unclear whether estradiol's effect was imparted on the lipoprotein particle or the artery wall. To examine this, we chronically treated rats with the following sex hormones: low estradiol, high estradiol, progesterone, low estradiol + progesterone, placebo, or control. Carotid arteries (n = 8/group) were isolated and perfused with fluorescently labeled LDL. Rates of LDL accumulation were measured before and after treatment with 10 ng/ml tumor necrosis factor-alpha (TNF) using quantitative fluorescence microscopy. We observed a 50% decrease in basal LDL accumulation rates (P < 0.01) and a 25% decrease in endothelial layer permeability (P < 0.01) in arteries from estradiol-treated animals. There was no effect of hormone replacement on rate of TNF-induced LDL accumulation (P = 0.451), while incubation of LDL with 65 pg/ml estradiol attenuated the TNF effect (P < 0.01). These experiments suggest two independent mechanisms of anti-atherogenic protection by estradiol: 1) decreased endothelial layer permeability; and 2) incorporation of estradiol into the LDL particle and prevention of LDL binding to the artery wall.

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Low density lipoprotein (LDL) receptor-deficient (LDLR-/-) mice consuming a high fat diet were used to assess the effect of endogenous and exogenous estradiol (E2) on atherosclerosis. Sexually mature female mice were ovariectomized (OVX) and implanted with subcutaneous, slow-release pellets designed to release 6 microg/day of exogenous 17beta-estradiol (17beta-E2 ), 17alpha-estradiol (17alpha-E2 ), or placebo (E2- deficient). Sham-operated control female (endogenous E2 ) and male mice were studied as controls. Aortic atherosclerotic lesion area was reduced by physiologic amounts of both endogenous and exogenous E2 compared to E2-deficient female mice. Although plasma cholesterol levels were reduced by exogenous E2 despite the absence of the LDL receptor, endogenous E2 was not associated with any cholesterol changes. In contrast, only 17alpha-E2 was associated with decreased fasting triglyceride. In subgroup analyses matched for time-averaged plasma total cholesterol, aortic lesion area was reduced by the presence of estradiol (E2 ). E2 protected LDLR-/- female mice from atherosclerosis and this protection was independent of changes in plasma cholesterol levels.

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-Current research suggests that estrogen may have primary effects on the artery wall. To investigate the mechanisms of female sex hormone actions in the artery wall, we used an isolated, perfused, rat carotid artery model to examine the effects of estradiol on the rates of accumulation of normal (N-LDL) and minimally modified (MM-LDL) low density lipoprotein in ovariectomized rats. N-LDL, MM-LDL, and oxidized LDL (OX-LDL) were fluorescently labeled and perfused into individual arteries. The rate of LDL accumulation was measured by quantitative fluorescence microscopy before and after treatment with estradiol (1 nmol/L, 272 pg/mL). Estradiol had no effect on the rate of N-LDL accumulation (45+/-12 versus 48+/-15 ng cholesterol per cm2 per h). However, estradiol significantly decreased the rate of MM-LDL (240+/-48 versus 160+/-48 ng cholesterol per cm2 per h; P<0.05) and OX-LDL (191+/-53 versus 112+/-36 ng cholesterol per cm2 per h; P<0.05) accumulation. Further experiments showed that perfusion of unlabeled MM-LDL (100 microgram/mL) increased endothelial layer permeability when the rate of accumulation of a water-soluble, fluorescently labeled, reference molecule (64 000-molecular weight dextran) was determined before and after perfusion of MM-LDL (319+/-96 versus 510+/-191 ng per cm2 per h, n=6 arteries; P<0.05). Estradiol prevented the expected increase in the rate of dextran accumulation when perfused with MM-LDL (control, 415+/-49 ng per cm2 per h and MM-LDL+estradiol, 415+/-160 ng per cm2 per h). Our studies show that estradiol prevents compromise of the endothelial barrier mediated by MM-LDL and attenuates accumulation of MM-LDL in the artery wall.

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Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels are associated, respectively, with either increased risk or apparent protective effects for atherothrombosis. The ability of purified LDL and HDL to downregulate thrombin formation, a contributor to atherothrombotic processes, was assessed. Purified HDL, but not LDL, significantly enhanced inactivation of coagulation factor Va by activated protein C (APC) and protein S, and HDL stimulated protein S-dependent proteolytic inactivation of Va by APC, apparently due to cleavage at Arg306 in Va. In normal plasma, added HDL enhanced APC/protein S anticoagulant activity in modified prothrombin-time clotting assays. When the anticoagulant potency of HDL was compared with phospholipid (PL) vesicles of well-defined composition using this assay, HDL appeared qualitatively different from PL vesicles because HDL showed only good anticoagulant activity, whereas PL vesicles were rather procoagulant. When 20 normal plasmas were tested using this clotting assay, apoA-I levels correlated with anticoagulant response to APC/protein S (r = 0.47, P = 0.035), but not with activated partial thromboplastin time-based APC resistance ratios. Because HDL enhances the anticoagulant protein C pathway in vitro, we speculate that HDL may help downregulate thrombin generation in vivo and that this anticoagulant action is one of HDL's beneficial activities.

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Serum amyloid A (SAA) is an acute phase reactant that can become the predominant apolipoprotein of high density lipoprotein (HDL) during severe inflammatory states. However, the function of SAA is unknown. To study the ability of SAA to form HDL in the absence of apolipoprotein A-I, we expressed the mouse SAA pI 6.15 (CE/J) isoform in apolipoprotein A-I knock-out (apoA-I (-/-)) mice using a recombinant adenovirus. As a control, apoA-I (-/-) mice were injected with an adenovirus expressing human apoA-I. High level expression of plasma SAA was obtained in the absence of any endogenous acute phase SAA production. SAA expression increased plasma HDL cholesterol levels about 2-fold, but to a lesser extent than the expression of apoA-I (about 10-fold). The HDL particles isolated by density ultracentrifugation from SAA-expressing mice were heterogeneous in size and composition and rich in free cholesterol as well as apoE and apoA-IV. Of the SAA expressed in the plasma, only a small fraction (4%) was associated with HDL particles in contrast to expressed apoA-I, of which 62% was associated with HDL. We conclude that SAA is unable to substitute for apoA-I in HDL particle formation.

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HDL protects LDL from oxidative damage known to contribute to the onset and progression of atherosclerosis. This antioxidant protection by HDL is mediated by intrinsic hydrolytic enzyme systems, by accelerated selective uptake of lipid peroxides from HDL and by antioxidant molecules. Oxidative damage suffered by HDL as a result of the transfer of reactive species from LDL may compromise HDL function in cholesterol transport.

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A direct approach is described for generating human apolipoprotein (apo) A-I-specific monoclonal antibodies with defined linear epitopes. The antibodies have comparable binding affinity for delipidated apoA-I and apoA-I on high density lipoproteins (HDL). The antibodies were obtained by immunizing mice with HDL, screening the fusions first for antibodies that bind native HDL and second, for antibodies that bind defined apoA-I synthetic peptides. ApoA-I antibody epitopes assigned on the basis of synthetic peptide binding were confirmed by solid phase and fluid phase antibody competition assays. These antibodies, which bind epitopes that encompass greater than 60% of apoA-I, should prove useful for identifying discrete functional domains of apoA-I on HDL.

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Normal high density lipoprotein (N-HDL) is remodeled during acute phase (AP) reactions by the association of serum amyloid A (SAA) and the depletion of apolipoprotein (apo) A-I. To determine the impact of this remodeling on HDL function, the capacities of N-HDL and AP-HDL to associate with and promote cholesterol efflux from human monocytic THP-1 cells were compared. THP-1 cells preferentially bound AP-HDL compared with N-HDL. Examination of the AP-HDL particles bound to THP-1 cells revealed a disproportionate association of an apoSAA-enriched, apoA-I-depleted subpopulation compared with the composition of the starting material. However, N-HDL and AP-HDL promoted cholesterol efflux from THP-1 cells equally efficiently and in a dose-dependent manner. When N-HDL was experimentally remodeled with apoSAA to achieve an apoprotein composition similar to that of the preferentially bound particles, cellular cholesterol efflux was reduced by 30%. The remodelling of HDL with apoSAA during the acute phase reaction alters cholesterol efflux only when apoSAA constitutes more than 50% of the HDL protein.

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Oxidized low-density lipoprotein (oxLDL) has been characterized as an atherogenic molecule responsible for the induction of a variety of gene products. One such gene, tissue factor (TF), the cellular initiator of the coagulation cascade, is not expressed in normal vascular tissue but is expressed by monocytes and foam cells in atherosclerotic lesions. Therefore, we examined the effect of oxLDL on TF expression in cultured human adherent monocytes. Endotoxin-free oxLDL alone did not induce TF expression in adherent monocytes. However, oxLDL significantly enhanced TF expression induced by the inflammatory mediator, bacterial lipopolysaccharide (LPS), in a time- and dose-dependent manner. In contrast, oxLDL did not alter LPS-mediated production of interleukin-8 and actually inhibited LPS-induced secretion of tumor necrosis factor-alpha, suggesting that some aspects of the signaling pathways for TF induction differ from those of other LPS-responsive monocyte/macrophage gene products. Thus, this study documents specific modulation of the expression of LPS-inducible genes in monocytic cells by oxLDL. Factors that enhance TF expression in monocyte/macrophage cells present in atheroma may contribute to the severity of thrombotic episodes and complications observed in atherosclerosis.

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The inverse correlation between plasma high density lipoprotein (HDL) levels and the risk for cardiovascular disease has been attributed in part to the role of HDL in facilitating the transport of cholesterol to the liver for catabolism. One component of this reverse cholesterol transport is removal of excess cholesterol from peripheral cells. An immunochemical approach was employed to evaluate the role of human apolipoprotein (apo) A-I in cellular cholesterol efflux and to test the hypothesis that discrete structural domains of the molecule mediate this function. Two apoA-I-specific monoclonal antibodies (AI-11 and AI-14) inhibited in vitro cellular cholesterol efflux from THP-1 monocytic cells to HDL or apoA-I proteoliposomes by approximately 50%. Six other antibodies had no effect although three of these bound significant proportions of the apoA-I proteoliposomes. Antibody AI-11 binds apoA-I amino acid residues 96-111 (Banka, C. L., Bonnet, D. J., Black, A. S., Smith, R. S., and Curtiss, L. K. (1991) J. Biol. Chem. 266, 23886-23892). The AI-14 epitope was localized to residues 74-105. Therefore, the two antibodies that inhibited HDL promotion of cellular cholesterol efflux bound overlapping but distinct regions of the apoA-I molecule.

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T lymphocytes, macrophages, and oxidized low-density lipoprotein (Ox-LDL) are collocalized in early atherosclerotic lesions. Using a low-endotoxin in vitro system, we observed that Ox-LDL but not native LDL induced the production, by both freshly adherent human peripheral blood monocytes and human monocytic THP-1 cells, of the alpha chemokine interleukin (IL)-8, a potent chemoattractant for T lymphocytes. Marked IL-8 induction by Ox-LDL did not require IL-1 beta generation in THP-1 cells. Ox-LDL-induced chemokine production was selective, as Ox-LDL did not stimulate the production by THP-1 cells of the T-lymphocyte chemotactic beta chemokine macrophage inflammatory protein (MIP)-1 alpha. IL-8 induction increased in proportion to the extent of oxidation of LDL as measured by the content of lipid oxidation end products. To identify potentially active components of Ox-LDL, we tested malondialdehyde, an arachidonate-derived lipid oxidation product, and 9-hydroxyoctadecadienoic acid, an oxidation product of linoleate, the major polyunsaturated fatty acid in LDL, and observed that they induced IL-8 generation in the absence of Ox-LDL. Furthermore, when most free lipid oxidation products were removed from Ox-LDL by dialysis, some IL-8-inducing activity was released into the dialysate. However, the major IL-8-inducing activity was not dialyzable. To address the nature of the LDL particle modification required to induce IL-8, acetylated or malondialdehyde-treated native LDL particles were monitored for activity. Neither procedure rendered LDL capable of inducing IL-8. However, phospholipase A2-treated LDL induced THP-1 cell expression of IL-8.(ABSTRACT TRUNCATED AT 250 WORDS)

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Apolipoprotein (apo) A-I, the major apoprotein of human high density lipoprotein, is a vital cofactor for lecithin-cholesterol acyltransferase (LCAT), the plasma enzyme responsible for esterification of free cholesterol associated with high density lipoprotein. This esterification is an important component of the reverse cholesterol transport process. An immunochemical approach was used to test the hypothesis that a discrete region of apoA-I was important for LCAT activation. Three human apoA-I-specific monoclonal antibodies were found to inhibit LCAT activation in vitro in a manner directly proportional to their ability to bind to apoA-I-proteoliposomes in fluid phase immunoassays. This relationship was not observed with another four apoA-I-specific antibodies that also were able to bind to the apoA-I proteoliposomes. The use of synthetic peptides representing short amino acid sequences of the apoA-I molecule facilitated the identification of discrete but overlapping apoA-I epitopes for those antibodies that interfered with LCAT-mediated cholesterol esterification. These epitopes spanned amino acid residues 95-121 of mature apoA-I. Therefore, this region is most likely involved in the activation of LCAT by apoA-I.

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The human monocytic leukemia cell line, THP-1, shares many properties with human monocyte-derived macrophages and might be a useful model for studying foam cell formation in vitro. Therefore, we examined the ability of THP-1 cells to accumulate cholesteryl esters, the hallmark feature of foam cells, in response to culture with native low density lipoprotein (LDL), modified LDL, and platelets. THP-1 cells stored more cholesteryl esters than macrophages in response to 200 micrograms/ml of LDL. Down-regulation of LDL receptors occurred in macrophages at lower LDL concentrations than in THP-1 cells. Phorbol ester-treated THP-1 cells stored more cholesteryl esters than human macrophages in response to 25-200 micrograms/ml of acetylated LDL. Because we have previously demonstrated that activated platelets enhanced macrophage cholesteryl ester storage, we examined the ability of THP-1 cells to store cholesteryl esters in response to coculture with platelets. Compared with macrophages, dividing THP-1 cells and phorbol ester-treated THP-1 cells accumulated only 50% and 33% as much cholesteryl esters, respectively. Furthermore, although platelets induced a 90% reduction in cholesterol synthesis in macrophages by day 5, cholesterol synthesis in THP-1 cells and phorbol ester-treated THP-1 cells was inhibited less than 50% by platelets. Nevertheless, both THP-1 cells and macrophages responded to platelets by increasing their secretion of apolipoprotein E. Therefore, we conclude that dividing THP-1 cells and phorbol ester-treated THP-1 cells are capable of forming foam cells in response to physiologic doses of both LDL and acetylated LDL, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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The early fatty streak lesions of atherosclerosis are characterized by the presence of cholesteryl ester-loaded macrophages or "foam cells." Platelets are also present in the early lesions of atherosclerosis and are often found in close association with foam cells. We have investigated the hypothesis that platelets contribute to foam cell formation by inducing macrophage cholesteryl ester accumulation. Using an in vitro culture system of human monocyte-derived macrophages and autologous platelets, we have demonstrated a platelet-dependent stimulation of macrophage cholesterol esterification and cholesteryl ester accumulation. The response is specific to platelets and is dependent upon activation of the platelets. An active fraction can be isolated from the releasates of thrombin-stimulated platelets that contain large cholesterol-rich platelet membrane vesicles. The results suggest that platelet-derived free cholesterol is required for platelet-induced macrophage foam cell formation.

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The location of the medullary chemoreceptors is not conclusively established. The original experiments, which were believed to suggest a shallow surface location in the ventrolateral medulla, have been questioned because substances, particularly CO2, applied on the surface of the medulla could diffuse into small arterioles. Because the whole tissue blood flow is supplied by surface arterioles, they could transport substances from the surface into the tissue to the respiratory centers. We studied simple transport equations describing movement of CO2 in arterioles bathed by rapidly flowing cerebrospinal fluid (CSF) and arterioles in tissue perfused by capillaries. Substantial exchange of CO2 could occur across the arteriole wall for all expected sizes of vessels when the partial pressure of CO2 at the outside wall was determined by CSF. When an arteriole is surrounded by tissue, only vessels with inside diameters (ID) less than or equal to 50 micron will exchange substantial amounts of CO2 but the smallest arterioles may be nearly in equilibrium with the tissue. The CO2 gradient in tissue around the arteriole will extend approximately 1 mm. Our simple theoretical description of CO2 transport in arterioles predicts substantial exchange in precapillary vessels. CO2 picked up by the smallest surface arterioles when the medulla is perfused at a high rate with CSF will not stay in the blood past the putative depth of the chemoreceptors. In arterioles greater than 30 micron, however, the CO2 could be carried to the respiratory centers.

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GnRH has been shown to induce premature meiotic maturation in preantral follicles of the immature estrogen-primed hypophysectomized rat. As these animals are free of circulating gonadotropins and contain large numbers of full-grown oocytes in preantral follicles, we have investigated this model to determine its usefulness in studying meiotic maturation. We show that a maximum dose of the agonist D-Trp6,Pro9,Net-LRF (GnRH-a) induces approximately 25% of full grown oocytes to resume meiosis within a 12-h period. This response is dose dependent (ED50 = 0.24 microgram/rat) and specific for GnRH-a. GnRH-a stimulates germinal vesicle breakdown and first polar body formation within 2 and 8 h, respectively. More than 75% of those oocytes that initiate meiotic maturation reach metaphase II by 15 h. This effect of GnRH parallels the time course of physiological meiotic maturation triggered by LH as well as that of oocytes maturing spontaneously in vitro. Oocytes in primordial and primary follicles do not respond to GnRH. The majority of affected follicles are small tertiary follicles (200-400 micron in diameter) and show signs of atresia. This atresia is not caused by GnRH-a and does not, in itself, result in meiotic maturation, but appears to confer susceptibility to GnRH-a-induced meiotic maturation. Our studies indicate that this animal model will be useful to elucidate further the mechanisms and requirements for meiotic maturation. It will also facilitate investigation of the role of atresia in the GnRH response of tertiary follicles and the issue of follicle heterogeneity within these animals.

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