© 1999 American Heart Association, Inc.
Original Contributions |
Inhibition of Arterial Thrombus Formation by ApoA1 Milano
From the Departments of Medicine (D.L., S.W., B.Y., W.W.N., J.L.M.) and Pathology (D.S.Z., S.K.), University of Florida College of Medicine; the VA Medical Center (D.L., S.W., B.Y., D.S.Z., W.W.N., J.L.M.), Gainesville, Florida; and the Department of Forensic Medicine (T.S.), University of Uppsala, Uppsala, Sweden.
Correspondence to J.L. Mehta, MD, PhD, Department of Medicine, University of Florida, College of Medicine, Box 100277, JHMHC, Gainesville, Florida 32610. E-mail mehta{at}medmac.ufl.edu
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Abstract |
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Abstract—The mutant form of human apoA1, known as apoA1 Milano, is formed as a result of arginine 173 to cysteine substitution and inhibits experimental atherosclerosis in cholesterol-fed animals. This study was designed to determine if apoA1 Milano would modify arterial thrombogenesis. Sprague Dawley rats were intravenously administered the carrier alone (n=8) or apoA1 Milano (20 mg · kg-1 · d-1 for 4 to 10 days, n=17). The abdominal cavity was opened, and the abdominal aorta was isolated. Whatman paper impregnated with 35% FeCl3 was wrapped around the surface of the aorta, and aortic flow was recorded continuously. In carrier-treated rats, an occlusive platelet-fibrin-rich thrombus was formed in 21.2±4.1 (mean±SD) minutes. Treatment of rats with apoA1 Milano markedly delayed time to thrombus formation (38.8±11.9 versus 21.2±4.1 minutes, P<0.01), inhibited platelet aggregation (25±7% versus 50±11%, P<0.01), and reduced weight of the thrombus (18.5±1.8 versus 23.7±2.3 mg/cm, P<0.01). Total cholesterol and HDL levels remained similar in both groups of rats, but plasma apoA1 Milano levels were elevated in apoA1 Milano–treated rats. In in vitro studies, incubation of platelets with apoA1 Milano reduced ADP-induced platelet aggregation by about 50%, but apoA1 Milano had no direct effect on vasoreactivity. This study provides further evidence for critical role of platelets in thrombosis. Use of apoA1 Milano offers a novel approach to inhibit arterial thrombosis.
Key Words: apoA1 • apolipoproteins • lipoproteins, HDL • thrombosis
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Introduction |
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Thrombus formation in the atherosclerotic coronary artery is the immediate cause of acute myocardial ischemia. Coronary artery thrombosis often is initiated by abrupt disruption of the atherosclerotic plaque and deposition and activation of platelets on the subendothelial layers in the disrupted plaque.1 2 Experimental and clinical studies have shown reduction in atherosclerosis and cardiac events with aggressive lowering of serum total and LDL cholesterol levels.3 4 5 Epidemiological studies also show an inverse relationship between the plasma levels of HDL cholesterol and atherosclerosis.6 7 Plasma levels of apoA1, the major protein component of HDL cholesterol, are reduced in patients with premature coronary artery disease.8 Whether increased HDL cholesterol or apoA1 is a marker for protection from atherosclerosis or has a direct vasoprotective effect is not known; however, studies in transgenic animal models that overexpress apoA1 suggest that elevated levels of apoA1 are antiatherogenic.9
In 1980, Franceschini et al10 described a family from
Limone sur Garda in Northern Italy with a lipoprotein disorder
characterized by a striking absence of atherosclerosis
despite very low plasma levels of HDL cholesterol.
Subsequent studies by this group11 showed that the absence
of atherosclerosis in this family was associated with
the presence of a mutant form of apoA1 with a single amino acid
substitution, arginine 173 to cysteine, which favors the
formation of dimers. This mutant form of apoA1, thereafter termed apoA1
Milano, constitutes a large component of the apolipoprotein content in
the affected family. All subjects in this family were found to be
heterozygous for the mutant allele and had very low levels of HDL
cholesterol, high triglyceride levels, and yet
no atherosclerosis. The apoA1 Milano has a shortened
residence time and causes rapid catabolism of apoA1 in these
subjects.12 The substitution of cysteine for arginine
appears to alter the amphipathic nature of the 
-helical fragment of
apoA1, increasing exposure of its hydrophobic residues.13
This structural modification is associated with high affinity of apoA1
Milano for lipids in the lipid-protein complexes and their easy
removal. The gene for apoA1 Milano has been cloned by Pharmacia,
and the genetically engineered version of the mutant protein has been
used in experimental studies. In a study by Ameli et al,14
administration of the genetically engineered apoA1 Milano caused a
marked reduction in the magnitude of intimal lesions and regression of
preexisting lesions in cholesterol-fed rabbits.
Because HDL cholesterol fraction per se decreases platelet aggregation and negates the stimulatory effect of oxidized LDL cholesterol on platelet aggregation,15 and apoA1 Milano in particular facilitates removal of LDL cholesterol, we hypothesized that administration of recombinant apoA1 Milano would inhibit platelet-dependent thrombus formation. This hypothesis was tested in a rat model of arterial thrombosis.
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Materials and Methods |
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Study Protocol
Twenty five Sprague Dawley (Harlan, Indianapolis, IN) rats weighing 250 to 350 g were fed standard rat chow. Seventeen rats received an intravenous injection of 20 mg/kg of apoA1 Milano in a carrier (or vehicle) of phosphatidylcholine-choline complex (dissolved in saline) in the tail vein every day for 8 to 10 days. Eight rats received an intravenous injection of the carrier of phosphatidylcholine-choline complex. Recombinant apoA1 Milano used in this study was supplied by Pharmacia and Upjohn.
An arterial thrombus model described by Kurz et al16 was used in this study. All animals were anesthetized with pentobarbital (30 mg/kg). Abdominal cavity was opened and approximately 1.2 cm length abdominal aorta was isolated. The aortic blood flow was recorded continuously by an ultrasonic Doppler flow probe (Crystal Biotech). The signal from the Doppler flow probe was calibrated against an electromagnetic flow probe, and the flow was expressed in mL/min as described earlier.17 Whatman paper soaked in 35% FeCl3 was wrapped around the external surface of the aorta. When thrombus was formed, thrombus along with the exposed aorta was taken out and weighed.
About 3 mL of blood was collected for platelet aggregation, measurement of plasma lipids, and apoA1 Milano levels. A segment of the thrombus along with aorta was saved in 2.5% glutaraldehyde, and another segment was saved in 10% neutral buffered formalin. ApoA1 Milano–treated rats were studied in parallel with carrier-treated rats.
Platelet Preparation and Aggregation
Blood was mixed thoroughly with 3.8% sodium citrate (9:1).
Blood was centrifuged at 1200 rpm for 10 minutes at room
temperature to obtain platelet-rich plasma (PRP) and
centrifuged again at 3000 rpm for 15 minutes to obtain
platelet-poor plasma (PPP). Platelet count in PRP was counted
and kept at about 2x108 to
3x108 cells/mL. ADP (final concentration
20 µmol/L) was used as stimulus for platelet aggregation as
described previously.18 All platelet aggregations were
performed in a 4-channel Chronolog aggregometer. Every time
platelet aggregation was performed in an apoA1 Milano–treated rat,
platelet aggregation was also done in a vehicle-treated rat. In
other in vitro studies, PRP was incubated with A1 Milano (0.6 mg/mL) or
the vehicle for 60 minutes at 37°C and platelet aggregation in
response to ADP determined.
Plasma HDL and Total Cholesterol Measurement
The blood samples were centrifuged at 1400g
for 10 minutes, and the supernatant was collected. Total serum
cholesterol was determined by enzymatic technique, and
serum HDL-cholesterol was measured after precipitation of
apoB-containing lipoproteins with phosphotungstic acid.
ApoA1 Milano Levels
Plasma levels of apoA1 Milano were determined by an ELISA
by Dr P.K. Shah of the University of California, Cedar-Sinai Medical
Center, Los Angeles, CA. The ELISA uses 2 different mouse monoclonal
antibodies (MAbs) developed against recombinant apoA1 Milano. The first
MAb was used to coat the microtiter wells. Standard recombinant apoA1
Milano and appropriate dilution of the serum samples were incubated.
Bound apoA1 Milano was detected with the other MAb, which was
biotinylated. The plate was developed using alkaline phosphatase, and
the absorbance was read at 405 nm.
Morphologic Analysis of Thrombus
The method for scanning electron microscopy was similar to the
method described previously by us.17 Essentially, aortic
segments were fixed in 2.5% glutaraldehyde and then
placed in 1% osmium tetroxide in 1% cacodylate buffer (pH 7.2). After
several washes in cacodylate buffer, aortic segments were dehydrated in
graded alcohols and 1% acetone and then refrigerated overnight in amyl
acetate. Specimens were dried to the critical point and coated with
silver in a Hummer 5 sputter coating system (Anatech Ltd). Under a
dissecting microscope, tissues were cut with a razor blade for full
exposure of the intimal surface. Specimens were examined with a Hitachi
S450 scanning electron microscope (Hitachi Ltd).
For light microscopic examination, tissues from carrier and apo ApoA1 Milano–treated rats were fixed in 10% neutral buffered formalin for 3 days, processed routinely through alcohols and xylene, and then embedded in paraffin. Five micron thick sections were cut at 2 levels in the paraffin block and stained with standard hematoxylin-eosin stain. Additional sections were stained with a Prussian blue (iron) stain. Other sections were stained to illustrate fibrin and platelets using Carstairs' method19 20 ; in brief, 5 µm thick paraffin sections were hydrated to water, placed in 5% ferric alum for 5 minutes, rinsed in running tap water, stained in Mayer's hematoxylin for 5 minutes, and then rinsed again in running tap water. Slides were placed for 1 hour in picric acid-orange G solution (composition: 20 mL saturated aqueous picric acid, 80 mL saturated picric acid in isopropanol, and 0.2 g orange G) and then rinsed in distilled water. For 5 minutes, slides were placed in ponceau-fuchsin solution (composition: 0.5 g acid fuchsin, 0.5 g ponceau 2R, 1 mL acetic acid, and distilled water; final volume 100 mL) and then rinsed in distilled water. Slides were treated with 1% phosphotungstic acid until the muscle appeared red and the background pale pink and then rinsed in distilled water. Slides were stained with aniline blue solution (1 g aniline blue in 100 mL 1% acetic acid) for 30 minutes, rinsed in several changes of distilled water, dehydrated, cleared, and then mounted. With a fixation time >48 hours, Carstairs' method for fibrin and platelets produces differential staining of fibrin (bright red), platelets (gray-blue to navy), collagen (bright blue), muscle (red), and red blood cells (yellow).
ApoA1 Milano and Vascular Reactivity
To determine the direct effect of apoA1 Milano on vascular
reactivity, rat aortic rings (4 to 5 mm) were obtained from 6
different rats. Care was taken to avoid any unnecessary manipulation of
vessels. The rings were then mounted onto wire stirrups connected to
force transducers (Kistler Morse) and placed in custom-designed
tissue-organ baths filled with oxygen-saturated (95%
O2+5% CO2) Krebs-Ringer
buffer (composition in mmol/L: NaCl 118, KCl 4.7,
CaCl2 1.3,
KH2PO4 1.2,
MgCl2 1.2, NaHCO3 12.5,
Na-EDTA 0.01, and glucose 11.1, pH 7.4). The rings were then stretched
to and maintained at a preload tone of 2 g for approximately 60
minutes. During the period of equilibration, rings were incubated with
apoA1 Milano (0.6 mg/mL) or carrier for 1 hour at 37°C. Buffer was
changed every 30 minutes and continuously bubbled with 95%
O2+5% CO2. After
equilibration, rings were exposed to cumulative concentrations of
norepinephrine (NE,
10-9-10-6 M)
to determine the vasoconstrictor response. Other rings were contracted
with NE (10-7-10-6
M) to obtain 60% to 70% of maximal contraction, then
exposed to the endothelium-dependent receptor-mediated
vasorelaxant acetylcholine (Acetylcholine (ACh),
10-9-10-6 M) to
determine endothelium-dependent
vasorelaxation.21 22 23
Statistical Analysis
All data are given as mean±SD. ANOVA was used to compare the 2
experimental groups followed by unpaired t-test with
Bonferroni's correction. A P value of 
0.05 was considered
significant.
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Results |
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Blood Total Cholesterol, HDL, and ApoA1 Milano Levels
Both group of rats showed no significant change in body weight during the course of the study and did not differ significantly in their plasma levels of total cholesterol. The HDL cholesterol levels were slightly lower (P, NS) in the apoA1 Milano–treated rats. Plasma apoA1 Milano levels were identified only in the apoA1 Milano–treated rats (Table
).
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Platelet Aggregation in ApoA1 Milano–Treated Rats
Treatment with apoA1 Milano markedly inhibited platelet
aggregation in each of the treated rats (mean 25±7% versus 50±11%
in vehicle-treated rats, n=17 and n=8, respectively;
P<0.01). Representative examples of
platelet aggregation patterns in the 2 groups of rats are shown in
Figure 1. There was no difference in
platelet counts in the 2 groups of rats.
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Time to Thrombosis and Weight of Thrombus
Application of FeCl3 in vehicle-treated rats
resulted in oscillations in aortic blood flow for about 15
minutes. This was followed by rapid decrease in blood flow and
eventually total cessation, indicating occlusive thrombus formation.
Once the flow had totally ceased, there was no spontaneous return of
flow over 1 hour of observation in all vehicle-treated animals. A
typical pattern of thrombus formation in a vehicle-treated rat is shown
in Figure 1
.
Treatment of rats with apoA1 Milano markedly delayed time to thrombus
formation, and the mean value increased 86% to 38.8±11.9 minutes
(compared with 21.2±4.1 minutes in the vehicle-treated group) (Figure 2). Following cessation of blood flow,
the thrombus was often unstable as evident from return of flow
transiently in all apoA1 Milano–treated rats. A
representative example of markedly delayed time to
thrombus formation and unstable thrombus in an apoA1 Milano–treated
rat is shown in Figure 1. The weight of the thrombus in all
apoA1 Milano–treated rats was much less than in the vehicle-treated
rats (Figure 2).
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Morphology of the Thrombus
Scanning electron microscopic examination of the aortic region
with thrombus revealed endothelial disruption,
deposition of platelets on the intimal surface, fibrin strands, and
red blood cells, especially at the base of the thrombus (Figure 3A). Cross-sectional view of the aortas
of vehicle-treated animals showed extensive platelet-fibrin
deposition along the entire intimal surface. The main body of the
thrombus consisted of large number of red blood cells tethered with
fibrin but few platelets (Figure 3B). There was no
discernible difference in the morphology of the thrombus in the 2
groups of rats.
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Light microscopic examination of the thrombi (Figure 4) revealed irregular masses of
platelets with focal connections to the intimal surfaces, and
variable numbers of red blood cells, accounting for the majority of
the thrombus. Fibrin occupied spaces between the platelet
aggregates and the red blood cells, particularly adjacent to the
arterial wall. Subintimal deposits of crystalline material
were present and stained blue with the Prussian blue stain,
consistent with transvascular penetration of
FeCl3 (Figure 4). Again, there were no
discernible differences in the morphology of the thrombi in the
carrier-treated and apoA1 Milano–treated groups.
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Direct Effect of ApoA1 Milano on Platelet Aggregation
Incubation of PRP with apoA1 Milano markedly decreased ADP-induced
platelet aggregation in each of the 6 rats (mean platelet
aggregation at 5 minutes: 22.8±10.7% versus 54.5±20.9%;
P<0.01).
ApoA1 Milano and Vasoreactivity
Incubation of aortic rings with apoA1 Milano had no effect on
vasoconstrictor response to NE (EC50:
5.5±0.4x10-7 M versus
5.6±0.4x10-7 M; P,
NS). Likewise, incubation of aortic rings with apoA1 Milano had no
effect on relaxant response to ACh (IC50:
5.1±0.4x10-8 M versus
5.0±0.2x10-8 M;
P, NS).
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Discussion |
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Cholesterol and Thrombosis
It is generally presumed that high levels of LDL cholesterol lead to enhanced thrombus formation, probably a result of reduced vasomotion and enhanced platelet aggregability. This has been attributed in part to the loss of NO release and its activity in cholesterol-rich platelets and vascular tissues.15 24 Most episodes of acute myocardial infarction are a result of formation of occlusive platelet-rich thrombus in the atherosclerotic narrowed coronary arteries.1 2 There is also evidence that myocardial injury is greater in hypercholesterolemic animals subjected to coronary artery occlusion; this is related to diminished vasomotion of the coronary artery and enhanced platelet aggregation in the coronary arterioles.25 Sawa et al26 described a 2-fold increase in plasma levels of plasminogen activator inhibitor-1 (PAI-1) as well as PAI-1 mRNA in hypercholesterolemic rabbits with indwelling catheters. Abela et al27 showed that hypercholesterolemia causes markedly large thrombi in rabbits subjected to balloon injury. However, there are no data on the influence of alteration in lipid components on in situ platelet-fibrin rich thrombus formation that is akin to human arterial thrombus. Also, there are no data on the influence of modification of HDL cholesterol on thrombogenicity.
Observations in the Current Study
The major finding in this study was that administration of
recombinant apoA1 Milano produces a substantial inhibition of
platelet aggregation and delays formation of arterial
thrombus in rats. In vitro incubation of platelets with apoA1
Milano for a short period also decreased ADP-induced platelet
aggregation. However, there was no significant effect of apoA1 Milano
on vasoconstriction or endothelium-dependent
relaxation.
Model of Arterial Thrombosis
The model of arterial thrombosis used in these studies
has been used to study the effect of a variety of
thrombolytic effect of a variety of
agents.16 28 The in situ thrombus induced in the rat aorta
by external application of FeCl3 is akin to human
intracoronary thrombus in its cellular composition and fibrin
content.16 Light microscopy showed penetration of
FeCl3 across the vessel wall (Prussian blue
stain), adherence of large numbers of platelets to the
subendothelial layers at several points along the
circumference of the vessel wall, and the central mass of the thrombus
consisting predominantly of red blood cells with interspersed fibrin.
These are also characteristics of arterial thrombi in human
coronary arteries.16 The rapidity with which the
thrombus is formed in this model depends on the concentration of
FeCl3 applied on the external surface of the
blood vessel. The thrombus, once formed, is stable and not subject to
spontaneous dissolution. We chose the 35% FeCl3
concentration because this concentration causes occlusive thrombus
formation in 18 to 24 minutes in all rats weighing about 300 g.
Because of the relatively low cost of animals and the ability to study
formation of occlusive thrombus, vascular reactivity, and platelet
aggregation, this model may be considered useful in evaluation of
different pharmacologic agents that modulate platelet function,
vascular reactivity, and lipid profile.
Mechanism of Antithrombotic Effect of ApoA1 Milano
It has been suggested that apoA1 Milano provides relative
protection against vascular disease related to its ability to remove
cholesterol from tissues.13 14 The apoA1
mutant appears to substantially alter the amphipathic nature of the
-helical fragment of apoA1 , thus increasing the exposure of its
hydrophobic residues. This structural modification is associated with a
higher kinetic affinity of apoA1 Milano for lipids and an easier
dissociation from lipid-protein complexes, which could contribute to
its accelerated catabolism and increased efficiency for uptake of
tissue lipids.12 The protective effect of HDL against
atherosclerosis has accordingly been attributed in
large part to the ability of HDL to facilitate reverse
cholesterol transport from peripheral tissues
to the liver for removal or revitalization.29 Badimon et
al30 31 have shown that HDL cholesterol can
reverse atherosclerotic lesions in cholesterol-fed rabbits.
Burkey et al32 showed that elevated apoA1 fractions reduce
aortic smooth muscle cell proliferation, a key early feature of
atherosclerosis.
Recent studies from our laboratory have indicated that it is the oxidized LDL cholesterol fraction that enhances platelet aggregation and downregulates NO synthase expression.16 33 Studies from other laboratories have shown that HDL inhibits oxidation of LDL cholesterol.34 35 We have also shown that HDL cholesterol blocks the platelet stimulatory affect of LDL cholesterol,36 and this effect of HDL cholesterol appears in large part mediated by reversal of the suppressive effect of LDL cholesterol on NO synthase in platelets.33 Stimulation of NO synthesis in the endothelial cells and platelets has a powerful antithrombotic effect. This phenomenon has been adequately demonstrated in animal models of thrombosis, wherein NO donors37 were used. The current studies showed that administration of apoA1 Milano or in vitro incubation of PRP with apoA1 Milano markedly reduced platelet aggregation. Whether platelet NO production or its activity is enhanced by apoA1 Milano cannot be discerned from this study. However, vascular endothelium-dependent relaxation was not altered when aortic rings were incubated ex vivo with apoA1 Milano. It is, nonetheless, reasonable to postulate that platelet inhibition and/or modulation of vascular reactivity are operative in delaying thrombus formation in apoA1 Milano–treated animals.
Activation of endogenous fibrinolytic pathways by apoA1 fraction of HDL cholesterol has also been well characterized.38 A similar activation of fibrinolytic pathway in apoA1 Milano–treated rats may relate to unstable thrombus and its tendency to spontaneously dissolve.
The light and scanning electron microscopy findings showed in a semiquantitative fashion that the composition of the thrombus in carrier- and apoA1 Milano–treated rats was similar, which indicates that platelets are able to aggregate and form an occlusive thrombus in the presence of extensive arterial endothelial injury despite treatment with apoA1 Milano. This suggests that the inhibitory effects of these agents are quantitative rather than qualitative.
Limitations of the Study
There are some important limitations of the study. First, it would
have been ideal to conduct similar studies with wild-type apoA1 to
determine if prolongation of time to thrombosis and inhibition of
platelet aggregation are unique to the administration of apoA1
Milano. It is possible that wild-type apoA1 can decrease platelet
aggregation and hence prolong time to thrombosis. Second, the study
lacked information on apoA1 levels in rats treated with carrier or
apoA1 Milano. Because apoA1 Milano has been characterized to induce
catabolism of apoA1 as well as HDL
cholesterol,12 13 it is possible that the
apoA1 levels were low in apoA1 Milano–treated rats. This concept gains
support from the observation of lower HDL cholesterol
levels in apoA1 Milano–treated rats (41±8 versus 49±4 mg/dL in
carrier-treated rats). Similar observation was made by Ameli et
al14 in rabbits fed high cholesterol diet and
administered apoA1 Milano. Third, the apoA1 Milano levels were somewhat
lower in this study compared with another study wherein a similar dose
was given to rabbits.14 The differences may relate to
differences in species (rat versus rabbit), diet (regular diet versus
high cholesterol diet), and study end-point (thrombosis
versus atherosclerosis). In the original report on
apoA1 mutant, a wide range of apoA1 levels (35 to 116 mg/dL) were
identified in the index family.10 In the transgenic mice,
saturated fat diet increased apoA1 levels markedly from 38 to 58
mg/dL.39 Our studies were conducted in fasting rats fed
regular chow. In further studies, all these issues will need to
carefully evaluated.
Conclusion
This study confirms a critical role of platelet activation in
arterial thrombosis. The use of apoA1 Milano—or possibly
use of modalities that may increase apoA1 only—offers a novel approach
directed to the inhibition of platelet-mediated thrombosis.
However, the precise dose and duration of administration of apoA1
mutant needs to be determined and the consistency of these
findings across species assessed.
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Acknowledgments |
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Supported by a Merit Review grant from the VA Central Office (to J.L.M.), a grant-in-aid from the American Heart Association, Florida Affiliate, St. Petersburg, Florida (to B.Y.), and support from the Division of Sponsored Research, University of Florida, Gainesville, Florida (to J.L.M.), and Swedish Medical Research Council, Stockholm, Sweden (to T.S.). We thank Jerry Santiago, HTL, (ASCP) for his technical assistance.
Received April 17, 1998; accepted July 21, 1998.
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References |
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|---|
1. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med. 1992;326:310–318.[Medline] [Order article via Infotrieve]
2. Conti CR, Mehta JL. Acute myocardial ischemia: role of atherosclerosis, in situ thrombosis, platelet activation, coronary vasospasm, and altered arachidonic acid metabolism. Circulation. 1987;75(suppl V):V-84–V-95.
3. Zhu BQ, Sun YP, Sievers RE, Isenberg WM, Moorehead TJ, Parmley WW. Effects of etidronate and lovastatin on the regression of atherosclerosis in cholesterol-fed rabbits. Cardiology. 1994;85:370–377.[Medline] [Order article via Infotrieve]
4. Scandinavian Simvastatin Survival Study Group. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet. 1994;344:1383–1389.[Medline] [Order article via Infotrieve]
5.
Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford
JD, Cole TG, Brown L, Warnica JW, Arnold JM, Wun CC, Davis BR,
Braunwald E. The effect of pravastatin on coronary
events after myocardial infarction in patients with average
cholesterol levels: Cholesterol and Recurrent
Events Trial investigators. N Engl J Med. 1996;335:1001–1009.
6. Gordon T, Castelli WP, Hjortland M, Kannel W, Dawber T. High density lipoprotein, a protective factor against coronary heart disease. Am J Med. 1977;62:707–714.[Medline] [Order article via Infotrieve]
7. Miller N, Forde O, Thelle D, Mjos O. The Tromoso Heart study: high density lipoprotein and coronary heart disease: a prospective case-control study. Lancet. 1977;622:965–968.
8. Puchois P, Kandoussi A, Fievet P, Fourrier JL, Bertrand M, Kore E, Fruchard JC. Apolipoprotein A-1 containing lipoproteins in coronary disease. Atherosclerosis. 1987;68:35–40.[Medline] [Order article via Infotrieve]
9. Rubin EM, Krauss RM, Spangler EA, Verstuyft JG, Clift SM. Inhibition of early atherogenesis in transgenic mice overexpressing apolipoprotein A-1. Nature. 1991;353:265–267.[Medline] [Order article via Infotrieve]
10. Franceschini G, Sirtori CR, Capurso A, Weisgraber KH, Mahley RW. A-1 Milano apoprotein: decreased high density lipoprotein cholesterol levels with significant lipoproteins modifications and without clinical atherosclerosis in an Italian family. J Clin Invest. 1980;66:892–900.
11. Weisraber KH, Bersot TP, Mahley RW, Franceschini G, Magani D, Sirtori CR. Isolation and characterization of a cysteine containing variant of the apoprotein A-1 from high density lipoprotein. J Clin Invest. 1980;66:901–907.
12. Roma P, Gregg RE, Meng MS, Ronan R, Zech LA, Franceschini G, Sirtori CR, Brewer HB Jr. In vivo metabolism of a mutant form of apolipoprotein A-1, apoA1 Milano, associated with familial hypoalphalipoproteinemia. J Clin Invest. 1993;91:1445–1452.
13.
Franceschini G, Vecchio G, Gianfranceschi G, Magani D,
Sirtori CR. Apolipoprotein A-1 Milano: accelerated binding and
dissociation from lipids of a human apolipoprotein variant.
J Biol Chem. 1985;260:16321–16325.
14.
Ameli S, Hultgardh-Nilsson A, Cercek B, Shah PK,
Forrester J, Ageland H, Nilsson J. Recombinant apolipoprotein A-1
Milano reduces intimal thickening after balloon injury in
hypercholesterolemic rabbits. Circulation. 1994;90:1935–1941.
15. Mehta JL, Chen LY. Reversal by high density lipoprotein of the effect of oxidized low density lipoprotein on nitric oxide synthase protein expression in human platelets. J Lab Clin Med. 1996;127:287–295.[Medline] [Order article via Infotrieve]
16. Kurz KD, Main BW, Sandusky GE. Rat model of arterial thrombosis induced by ferric chloride. Thromb Res. 1990;60:269–280.[Medline] [Order article via Infotrieve]
17. Nicolini FA, Nichols WW, Mehta JL, Saldeen TGP, Ross M, Player DW, Pohl G, Mattsson C. Sustained reflow in dogs with coronary thrombosis with K2P, a novel mutant of tissue plasminogen activator. J Am Coll Cardiol. 1992;20:228–235.[Abstract]
18. Wargovich TJ, Mehta JL, Nichols WW, Ward MB, Lawson DL, Franzini D, Conti CR. Reduction in myocardial neutrophil accumulation and infarct size following administration of thromboxane inhibitor U 63,557A. Am Heart J. 1987;114:1078–1085.[Medline] [Order article via Infotrieve]
19. Carstairs KC. The identification of platelets and platelets antigens in histological sections. J Pathol Bacteriol. 1965;90:225–231.[Medline] [Order article via Infotrieve]
20. Sheehan DC, Hrapchak BB. Theory and Practice of Histotechnology. 2nd ed. Columbus, OH: Battelle Press; 1987:281.
21. Yang BC, Lawson DL, Mehta JL. Role of eicosanoids in rat aortic ring response to agonists and acetylcholine with special reference to the biphasic effects of prostacyclin. Eicosanoids. 1992;5:135–139.[Medline] [Order article via Infotrieve]
22.
Yang BC, Mehta JL. Critical role of
endothelium in sustained arterial
contraction during prolonged hypoxia. Am J
Physiol. 1995;268:H1015–H1020.
23. Yang BC, Nicolini FA, Nichols WW, Mehta JL. Decreased endothelium-dependent vascular relaxation following subtotal coronary artery occlusion in dogs. Free Radical Biol Med. 1993;14:295–302.[Medline] [Order article via Infotrieve]
24.
Matsuda Y, Hirata K, Inoue N, Suematsu M, Kawashima S,
Akita H. High density lipoprotein reverses inhibitory
effect of oxidized low density lipoprotein on
endothelium-dependent arterial relaxation.
Circ Res. 1993;72:1103–1109.
25.
Sakamoto S, Kashiki M, Imai N, Liang CS, Hood WB Jr.
Effects of short-term, diet-induced
hypercholesterolemia on systemic
hemodynamics, myocardial blood flow, and infarct size
in awake dogs with acute myocardial infarction. Circulation. 1991;84:378–386.
26. Sawa H, Sobel BE, Fujii S. Potentiation by hypercholesterolemia of the induction of aortic intramural synthesis of plasminogen activator type-I by endothelial injury. Circulation. 1993;73:671–680.
27.
Abela GS, Picon PD, Friedl SE, Gebara OC, Miyarnoto A,
Tofler GH, Muller JE. Triggering of plaque disruption and
arterial thrombosis in an atherosclerotic rabbit model.
Circulation. 1995;91:776–784.
28.
Farrehi PM, Ozaki CK, Carmeliet P, Fay WP. Regulation
of arterial thrombolysis by
plasminogen activator inhibitor-1
in mice. Circulation. 1998;97:1002–1008.
29. Franceschini G, Madema P, Sirtori CR. Reverse cholesterol transport: physiology and pharmacology. Atherosclerosis. 1991;88:99–107.[Medline] [Order article via Infotrieve]
30. Badimon JJ, Badimon L, Galvez A, Dische R, Fuster V. High density lipoprotein plasma fraction inhibits aortic fatty streaks in cholesterol-fed rabbits. Lab Invest. 1989;60:455–461.[Medline] [Order article via Infotrieve]
31. Badimon JJ, Badimon L, Fuster V. Regression of atherosclerotic lesions by high density lipoprotein plasma fraction in the cholesterol-fed rabbit. J Clin Invest. 1992;85:1234–1241.
32. Burkey BF, Vlasic N, France D, Hughes TE, Drelich M, Ma X, Stemerman MB, Paterniti JR. Elevated apoprotein A-I pools in human apoA1 transgenic rats decrease aortic smooth muscle cell proliferation following balloon angioplasty. Circulation. 1992;86(suppl I):I-472. Abstract.
33.
Chen LY, Mehta P, Mehta JL. Oxidized LDL decreases
L-arginine uptake and nitric oxide synthase protein expression in human
platelets: relevance of the effect of oxidized LDL on platelet
function. Circulation. 1996;93:1740–1746.
34. Parathasarathy S, Barnett J, Fong LG. High density lipoprotein inhibits the oxidative modification of low-density lipoprotein. Biochem J. 1993;294:829–834.
35. Mackness MI, Abbot C, Arrol S, Durrington PN. The role of high density lipoprotein and lipid-soluble antioxidant vitamins in inhibiting low-density lipoprotein oxidation. Biochem J. 1993;294:829–834.
36. Chen LY, Mehta JL. Inhibitory effect of high-density lipoprotein on platelet function is mediated by increase in nitric oxide synthase activity in platelets. Life Sci. 1994;55:1815–1821.[Medline] [Order article via Infotrieve]
37.
Folts JD, Stamler J, Loscalzo J.
Intravenous nitroglycerin infusion inhibits
cyclic blood flow responses caused by periodic platelet thrombus
formation in stenosed canine coronary arteries.
Circulation. 1991;83:2122–2127.
38. Saku K, Ahmad M, Glass-Greewalt P, Kashyap M. Activation of fibrinolysis by apolipoproteins of high density lipoproteins in man. Thromb Res. 1985;39:1–8.[Medline] [Order article via Infotrieve]
39.
Arzolan N, Odaka H, Breslow JL, Fisher EA. Dietary fat
elevates hepatic ApoA1 production by increasing apolipoprotein
A-1 mRNA in the translating pool. J Biol Chem. 1995;270:19833–19838.
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