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(Circulation. 2005;111:1543-1550.)
© 2005 American Heart Association, Inc.

Vascular Medicine

Reconstituted High-Density Lipoproteins Inhibit the Acute Pro-Oxidant and Proinflammatory Vascular Changes Induced by a Periarterial Collar in Normocholesterolemic Rabbits

Stephen J. Nicholls, MBBS, PhD, FRACP; Gregory J. Dusting, PhD; Belinda Cutri, BMedSc (Hons); Shisan Bao, MBBS, PhD; Grant R. Drummond, PhD; Kerry-Anne Rye, PhD; Philip J. Barter, MBBS, PhD, FRACP

From the Heart Research Institute, Sydney (S.J.N., B.C., K.-A.R., P.J.B., S.B.); the Department of Medicine, University of Adelaide, Adelaide (S.J.N.); the Howard Florey Institute, University of Melbourne, Melbourne (G.J.D., G.R.D.); the Department of Medicine, University of Sydney, Sydney (P.J.B.); and the Department of Pathology, University of Sydney, Sydney (S.B.), Australia.

Correspondence to Professor Philip Barter, The Heart Research Institute, 145 Missenden Rd, Camperdown, NSW 2050, Australia. E-mail p.barter{at}hri.org.au

Received August 12, 2004; revision received November 15, 2004; accepted December 13, 2004.

	Abstract

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Background— HDLs have antiinflammatory and antioxidant properties in vitro. This study investigates these properties in vivo.

Methods and Results— Chow-fed, normocholesterolemic New Zealand White rabbits received a daily infusion of (1) saline, (2) reconstituted HDL (rHDL) containing 25 mg apolipoprotein (apo) A-I and 50 mg of either 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) or 1,2-dipalmitoyl phosphatidylcholine (DPPC), (3) 25 mg lipid-free apoA-I, or (4) 50 mg of either PLPC-small unilamellar vesicles (SUVs) or DPPC-SUVs on each of 3 consecutive days. Nonocclusive carotid periarterial collars were implanted after the second dose of treatment. Forty-eight hours after insertion of the collars, the arteries were removed and analyzed for the presence of reactive oxygen species, the infiltration of neutrophils, and the expression of adhesion proteins and chemokines. Insertion of the periarterial collar induced a 4.1-fold increase in presence of vascular wall reactive oxygen species. This effect was completely abolished in the animals infused with rHDL. The periarterial collar was associated with a dense infiltration of the arterial wall by polymorphonuclear leukocytes. This infiltration was inhibited by 73% to 94% in the animals infused with rHDL, by 75% in the animals infused with lipid-free apoA-I, and by 51% to 65% in animals infused with SUVs. There were no significant differences between the effects of PLPC and DPPC in either the rHDL or SUVs. Endothelial expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemoattractant protein-1 was also increased by the collar insertion and inhibited by rHDL, lipid-free apoA-I, and, to a lesser extent, also by the SUVs.

Conclusions— Infusion of rHDL, apoA-I, and phospholipid-SUVs inhibits the early pro-oxidant and proinflammatory changes induced by a periarterial collar in normocholesterolemic rabbits.

Key Words: lipoproteins • inflammation • cholesterol • antioxidants • endothelium

	Introduction

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High-density lipoproteins (HDLs) have several functions that may contribute to their ability to protect against atherosclerosis. The best known of these relates to their role in promoting the efflux of cholesterol from macrophages in the artery wall. However, HDLs also have antioxidant, antithrombotic, and antiinflammatory functions that may contribute to their protective properties. The present study is concerned with the antiinflammatory effects of HDL in vivo.

It has been shown previously in studies conducted in vitro that HDLs inhibit the cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in human umbilical vein endothelial cells (HUVECs) growing in tissue culture.¹ HDLs also inhibit binding of monocytes² and neutrophils³ to endothelial cells growing in culture. There is evidence that the antiinflammatory properties of HDL also operate in vivo, although in most cases this has been demonstrated in a setting of hypercholesterolemia and atherosclerosis. For example, intravenous infusion of reconstituted HDL (rHDL) reduces the vivo expression of endothelial adhesion molecules induced by insertion of carotid periarterial cuffs in cholesterol-fed, apoE-knockout mice.⁴ In another study of apoE-knockout mice, the increase in HDL concentration accompanying an overexpression of the human apoA-I gene reduced macrophage accumulation in the aortic root by more than 3-fold.⁵ This was associated with a reduced in vivo oxidation of ß-VLDL, lower ICAM-1 and VCAM-1 expression, and diminished ex vivo leukocyte adhesion.

However, there are also examples of in vivo antiinflammatory effects of HDL in the absence of hypercholesterolemia and atherosclerosis. For example, injection of rHDL inhibits the development of a local inflammatory infiltrate after the subcutaneous administration of interleukin-1 in a porcine model.⁶ Also, in studies of experimental stroke in rats, pretreatment with rHDL significantly and substantially reduced the brain necrotic area in a process possibly related to an rHDL-induced reduction in levels of reactive oxygen species (ROS).⁷ Furthermore, in a study of hemorrhagic shock in rats, the resulting multiple organ dysfunction syndrome was largely abolished by a single injection of human HDL given 90 minutes after the hemorrhage and 1 minute before resuscitation. In that model, injection of HDL prevented both the severe disruption of tissue architecture and the extensive cellular infiltration into the affected tissues.⁸

There is also circumstantial evidence that HDLs have acute antiinflammatory effects in vivo in humans. In one study, a single intravenous infusion of rHDL into hypercholesterolemic humans normalized endothelium-dependent vasodilation, possibly by increasing NO bioavailability.⁹ In a second human study, a single injection of rHDL corrected the endothelial dysfunction associated with low levels of HDL in ABCA1 heterozygotes.¹⁰

Thus, there is mounting evidence that HDLs have acute antiinflammatory effects that are extremely rapid and possibly unrelated to their cholesterol-transporting function. In the present study, we have used a model of acute arterial inflammation in normocholesterolemic, nonatherosclerotic rabbits to demonstrate in vivo for the first time that injection of rHDL markedly inhibits both the infiltration of neutrophils into the artery wall and the subsequent generation of ROS.

	Methods

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Animals
Male New Zealand White rabbits (Nanowie Small Animal Production Unit, Modewarre, Australia) weighing approximately 3 kg were maintained on a normal laboratory chow diet throughout the study. All procedures were approved by the Howard Florey Institute Animal Ethics Committee (protocol 02-009).

Isolation of Lipid-Free ApoA-I
HDLs were isolated from pooled samples of rabbit plasma (Quality Farms of Australia, Lara, Australia) by sequential ultracentrifugation in the 1.063- to 1.21-g/mL density range. The HDLs were lyophilized and delipidated.¹¹ ApoA-I was isolated by chromatography¹² on a Q-Sepharose Fast Flow column (Amersham Biosciences) attached to a fast performance liquid chromatography system (Amersham Biosciences). The purified apoA-I was lyophilized and stored at –20°C until used. Lyophilized apoA-I was reconstituted in 3 mol/L guanidine hydrochloride and dialyzed against endotoxin-free PBS (pH 7.4; Sigma) containing 0.2 g/L KH₂PO₄, 0.2 g/L KCl, 8 g/L NaCl, and 1.15 g/L Na₂PO₄ before being infused or used to prepare rHDL. The concentration of apoA-I was determined by use of an immunoturbidimetric assay.¹³

Preparation of Reconstituted HDL
Discoidal rHDL containing apoA-I complexed to either 1-palmitoyl-2-linoleoyl phosphatidylcholine (PC) (PLPC, Sigma) or 1,2-dipalmitoyl PC (DPPC, Sigma), in a molar ratio of PC to apoA-I of 200:1, were prepared by use of the cholate dialysis method.¹⁴ The resulting rHDLs were dialyzed extensively against endotoxin-free PBS before use. Protein¹⁵ and phospholipid¹⁶ concentrations were determined by enzymatic assay. The rHDLs were subjected to agarose gel electrophoresis¹⁷ and 3% to 40% nondenaturing gradient gel electrophoresis to determine their surface charge and Stokes’ diameter, respectively.

Preparation of Phospholipid Small Unilamellar Vesicles
Small unilamellar vesicles (SUVs) containing either PLPC or DPPC and BHT (molar ratio of PC to BHT, 10:1) were prepared in PBS as described.¹⁸

Administration of Reconstituted HDL
Twenty-nine rabbits were randomly allocated to receive treatment with saline (n=6), PLPC-rHDL (n=4), DPPC-rHDL (n=4), lipid-free apoA-I (n=5), PLPC SUVs (n=5), or DPPC (n=5). Each treatment of rHDL and lipid-free apoA-I contained 25 mg of apoA-I (8 mg/kg). Each treatment of rHDL and SUVs contained 50 mg of PC (16 mg/kg). The treatments were administered via a marginal ear vein on each of 3 occasions: 24 hours before collar implantation, directly before the surgical procedure, and 24 hours after the collar implantation. The animals were euthanized 48 hours after the collar implantation.

Collar Implantation
The rabbits were anesthetized with intravenous propofol (5 mg/kg) followed by intramuscular ketamine/xylazine (50/10 mg/kg). Both carotid arteries were exposed surgically and cleared of connective tissue along a 30-mm length. Hollow, nonocclusive Silastic collars (length, 20 mm; internal diameter along bore, 4 mm; internal diameter at ends, 1 mm) were then placed around each artery and held in place with a nylon sleeve. The space inside the collar was filled with sterile saline (0.9%). Muscle, fat, and skin layers were sutured, the wound was dressed with antibiotic, and animals were allowed to recover for 48 hours before being euthanized.

Tissue Harvesting
Forty-eight hours after insertion of the collar, blood was sampled from a marginal ear vein. Animals were then heparinized (1000 U IV) before euthanasia with an overdose of sodium pentobarbitone (90 mg/kg IV). The collared segment of the carotid artery and approximately 10 mm of noncollared artery proximal to the collar were excised and placed in ice-cold Krebs-HEPES buffer (composition, in mmol/L: NaCl 99.0, KCl 4.7, KH₂PO₄ 1.0, MgSO₄ 1.2, CaCl₂ 2.5, NaHCO₃ 25.0, Na-HEPES 20.0, and glucose 11.0, pH 7.4). Collars were removed and arteries cleaned of fat and connective tissue. Three ring sections (3 mm) were cut both from the area enclosed by the collar and from the proximal noncollared segment of artery. One section from each segment was snap-frozen in Tissue-Tek OCT (Sakura) fixative for immunohistochemical analysis. The other sections were used to determine generation of ROS.

Detection of ROS
Levels of ROS in the carotid arteries were measured by 5 µmol/L lucigenin-enhanced chemiluminescence.¹⁹ The increased chemiluminescence signal, probably reflecting superoxide, is mediated by increased activity of NADPH oxidase.²⁰ To minimize the possibility that the lucigenin used in the assay may have contributed to the production of superoxide, we used a concentration of lucigenin that was not sufficient to participate in superoxide production by redox cycling.²¹ Ring segments were incubated for 45 minutes at 37°C in Krebs-HEPES buffer containing diethyldithiocarbamate (DETCA, 3 mmol/L) to irreversibly inactivate endogenous Cu²⁺/Zn²⁺ superoxide dismutase. The possibility that HDL may have interfered with the ability of DETCA to inactivate superoxide dismutase was excluded on the grounds that HDLs were not present in the wells during the incubation. Some rings were further treated with NADPH (10 µmol/L), the preferred substrate of NADPH oxidase, which is, in turn, the predominant source of superoxide in rabbit carotid arteries.²⁰ Each segment was then transferred to a separate well of a white, opaque 96-well plate containing 300 µL of 5-µmol/L lucigenin in Krebs-HEPES buffer as well as the appropriate drug treatment. DETCA was excluded from the lucigenin assay solution. The 96-well plate was loaded into a TopCount Single Photon Counter (Packard Bioscience), and photon emission per second was measured (6-second count time per cycle, 12 cycles, 1-minute delay between cycles). Ring segments were dried for 2 to 3 days in a 65°C oven, and the production of ROS was normalized to dry tissue weight (counts per second per milligram).

Immunohistochemistry
Frozen tissues were sectioned in 5-µm slices. A section from the middle of each of the collared and noncollared segments of both carotid arteries was then subjected to immunohistochemical staining. Sections were fixed with methanol/acetone (1:1, vol/vol) at room temperature for 5 minutes. Arterial wall infiltration by inflammatory cells was determined by use of mouse monoclonal antibodies against macrophages (RAM11, DakoCytomation), neutrophils (CD18, Serotag), and lymphocytes (CD43). In addition, the endothelial expression of adhesion proteins and chemokines was determined by use of murine monoclonal antibodies against rabbit VCAM-1 and ICAM-1 (gifts from Dr. M. Cybulsky, University of Toronto), monocyte chemotactic protein (MCP)-1 (a gift from Dr. A. Matsukowa, Kumamoto University), a goat polyclonal antibody against human E-selectin (R&D), endothelial nitric oxide synthase (eNOS) (BD Biosciences), and inducible nitric oxide synthase (iNOS) (BD Biosciences). These were applied and incubated overnight at room temperature. Endothelial integrity was determined by use of a mouse monoclonal antibody against human CD31 (DakoCytomation). Sections were incubated with biotinylated anti-mouse or anti-goat immunoglobulins for 30 minutes and then incubated with alkaline phosphatase-labeled streptavidin solution for 60 minutes. Slides were rinsed in PBS (pH 7.4) after each incubation. Peroxidase activity was revealed by diaminobenzamine. Slides were counterstained with hematoxylin and mounted. These slides were subsequently graded independently by 4 pathologists who were blinded to the treatment status of the animal. The degree of endothelial staining was graded^22,23 by use of a scale that incorporated both the strength of staining and the amount of endothelial surface involved (0=no staining, 1=weak staining of less than 50% of endothelium, 2=strong staining of less than 50% or weak staining of more than 50% of endothelium, 3=strong staining between 50% and 99% of endothelium, and 4=strong staining of 100% of endothelium). The collar-induced change in expression was calculated as the difference in mean score between noncollared and collared segments of each individual artery. Infiltration of the arterial wall by neutrophils, demonstrated by CD18 staining, was determined by quantitative immunohistochemistry. Digital micrographs were acquired with an Olympus BX40 microscope, and the percentage of total vessel wall area occupied by positive staining was determined by use of ImagePro Plus 4 (Cybernetics).²⁴

Plasma Analyses
Plasma collected at the commencement of the study and before euthanasia of the animal was stored at –80°C in EDTA until required for analysis. All chemical analyses were performed on a Roche Diagnostics/Hitachi 902 autoanalyzer (Roche Diagnostics GmbH). Triglyceride²⁵ was determined enzymatically. Total cholesterol was determined by use of a Roche Diagnostics kit. HDL cholesterol was determined by enzymatic assay after precipitation of apoB containing lipoproteins with polyethylene glycol.²⁶ ApoA-I concentrations were determined by an immunoturbidimetric assay using a sheep anti-rabbit apoA-I immunoglobulin.¹³

Data Analysis
All results are expressed as mean±SEM. Statistical comparisons were made by Student t tests and 1-way ANOVA using the statistical program in GraphPad Prism Version 4.0. A value of P<0.05 was considered significant.

	Results

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Plasma Lipid Concentrations
Plasma concentrations of triglyceride, total and free cholesterol, HDL cholesterol, and apoA-I at the commencement of the study and at the time of euthanasia of the animals are presented in the Table. No significant differences were found between the groups, nor were there significant differences in lipid levels resulting from the infusions of saline, rHDL, lipid-free apoA-I, or PC-SUVs.

View this table: [in this window] [in a new window]   Plasma Lipid Profiles From Animals Infused With Saline (n=6), PLPC-rHDL (n=4), DPPC-rHDL (n=4), Lipid-Free apoA-I (n=5), PLPC-SUV (n=5), and DPPC-SUV (n=5) at Baseline and Immediately Before Death

Generation of ROS
Application of the periarterial collar increased the production of ROS in the vessel wall both in the unstimulated state without NADPH (126.9±48.1 and 6.6±2.9 counts · s^–1 · mg^–1 in the collared and noncollared segments, respectively, P<0.005) and after stimulating by incubation with NADPH (540.3±169.1 and 130.3±42.1 counts · s^–1 · mg^–1 in the collared and noncollared segments, respectively, P<0.01) (Figure 1). The collar-induced increase in vascular ROS that was observed in both the unstimulated (Figure 1A) and the stimulated (Figure 1B) states was significantly reduced in the vessels isolated from animals infused with rHDL. Infusion of PLPC-rHDL inhibited the collar-induced increase in vascular ROS in the unstimulated state by 92% (10±4.1 and 126.9±48.1 counts · s^–1 · mg^–1 in the PLPC-rHDL and saline-infused animals, respectively, P<0.03) and in the stimulated state by 85% (82.3±34.4 and 540.3±169.1 counts · s^–1 · mg^–1 in the PLPC-rHDL and the saline-infused animals, respectively, P<0.001). The collar-induced increase in ROS in the stimulated state was also inhibited by 90% by infusion of DPPC-rHDL (56.7±29.8 and 540.3±169.1 counts · s^–1 · mg^–1 in the DPPC-rHDL- and saline-treated animals, respectively, P<0.001) (Figure 1B). The effects of infusing PLPC-rHDL and DPPC-rHDL were not significantly different.

View larger version (10K): [in this window] [in a new window]   Figure 1. Levels of ROS as determined by lucigenin-enhanced chemiluminescence. Collared and noncollared vascular segments from animals infused with saline (n=6), PLPC-rHDL (n=4), or DPPC-rHDL (n=4) were incubated with DETCA (3 mmol/L) in absence (A) or presence (B) of NADPH (10 µmol/L) at 37°C for 45 minutes. After washing, lucigenin (5 µmol/L) and DETCA (3 mmol/L) with or without NADPH (10 µmol/L) were added to segments. Detected chemiluminescent signal was expressed per dry weight of segment. Results are expressed as mean±SEM (*P<0.03, **P<0.001 for comparison with saline-infused animals).

Neutrophil Infiltration
Representative sections of collared and noncollared segments, stained for CD18 as a marker of neutrophils, are shown in Figure 2. Staining with an antibody directed against CD31 demonstrated that the endothelium was intact (results not shown). There was no accumulation of either macrophages or lymphocytes in the collared segments of any group of animals as determined by staining for RAM11 and CD43, respectively (results not shown). Application of the periarterial collar did, however, promote a dense infiltration of neutrophils into the vessel wall in the saline-infused animals (30±9% of total vessel wall area in the collared versus 0.7±0.2% in the noncollared segments, P<0.005) (Figure 3). The collar-induced neutrophil recruitment was inhibited 73% by infusion of PLPC-rHDL (from 30±9% of the total vessel wall area in the saline-infused rabbits to 8.2±3.7% in the animals infused with rHDL, P<0.03) and 94% by infusion of DPPC-rHDL (from 30±9% of the total vessel wall area in the saline-infused rabbits to 1.8±1.4% in the animals infused with rHDL, P<0.02). There was no significant difference in the degree of neutrophil infiltration in collared segments of animals infused with PLPC-rHDL and DPPC-rHDL.

View larger version (118K): [in this window] [in a new window]   Figure 2. Representative immunohistochemical staining (x40 magnification) for presence of neutrophils (CD18) in noncollared segments from saline-infused (n=6, A) and PLPC-rHDL-infused animals (n=4, B) and collared segments from animals infused with saline (C), PLPC-rHDL (D), DPPC-rHDL (n=4, E), lipid-free apoA-I (n=5, F), PLPC-SUV (n=5, G), and DPPC-SUV (n=5, H).

View larger version (18K): [in this window] [in a new window]   Figure 3. Percentage of total arterial wall area infiltrated by neutrophils, as determined by CD18 immunohistochemical staining in animals infused with saline (n=6), PLPC-rHDL (n=4), DPPC-rHDL (n=4), lipid-free apoA-I (n=5), PLPC-SUV (n=5), and DPPC-SUV (n=5). Results are expressed as mean±SEM (****P<0.005 for comparison with noncollared segments; P=NS, nonsignificant, *P<0.05, **P<0.03, and ***P<0.02 for comparisons with saline-infused animals).

The collar-induced neutrophil recruitment was also inhibited 73% by infusion of lipid-free apoA-I (30±9% of the total vessel wall area in the saline-infused rabbits reduced to 8±2.9% in the animals infused with lipid-free apoA-I, P<0.02). This effect of infusing lipid-free apoA-I was not significantly different from that of rHDL (Figure 3).

Infusion of SUVs also reduced the infiltration of neutrophils but appeared to be less effective than either rHDL or lipid-free apoA-I (Figure 3). The collar-induced neutrophil recruitment was 30±9% of the total vessel wall area in the saline-infused rabbits, 14.6±4.2% in the animals infused with the PLPC-SUVs (P=NS compared with saline) and 10.4% in the animals infused with the DPPC-SUVs (P<0.05 compared with saline) (Figure 3). The accumulation of neutrophils in the collared arteries of animals infused with PLPC-SUVs and DPPC-SUVs was not significantly different.

Vascular Adhesion Molecules and Chemokines
The ability of rHDL, lipid-free apoA-I, and phospholipid SUVs to inhibit the expression of endothelial adhesion proteins and chemokines was also investigated. The collar-induced changes in expression of endothelial VCAM-1, ICAM-1, E-selectin, and MCP-1 are presented in Figure 4. Forty-eight hours after implantation of a periarterial collar, there was an increase in the expression of endothelial VCAM-1 (3.43±0.13 versus 0.91±0.12 in the collared and noncollared segments, respectively, P<0.05), ICAM-1 (3.34±0.14 versus 2.27±0.13, P<0.05), and MCP-1 (1.63±0.16 versus 1.06±0.14, P<0.05). Endothelial expression of E-selectin had still not increased 48 hours after application of the collar (2±0.12 versus 1.83±0.1, P=NS). Endothelial expression of eNOS was not significantly changed 48 hours after insertion of the collar (0.78±0.49 versus 0.44±0.36, P=NS; results not shown). There was no detectable endothelial iNOS staining in either noncollared or collared segments in saline-infused animals (results not shown).

View larger version (29K): [in this window] [in a new window]   Figure 4. Collar-induced change in endothelial expression of VCAM-1, ICAM-1, MCP-1, and E-selectin as determined by difference in score between collared and noncollared segments of artery in animals infused with saline (n=6), PLPC-rHDL (n=4), DPPC-rHDL (n=4), lipid-free apoA-I (n=5), PLPC-SUV (n=5), and DPPC-SUV (n=5). Results are expressed as mean±SEM (*P<0.05, **P<0.01, ***P<0.005, and P=NS, nonsignificant, for comparisons with saline-infused animals).

Compared with saline, infusion of rHDL inhibited the collar-induced increase in endothelial expression of VCAM-1 (by 53.9% and 54.4% with PLPC-rHDL and DPPC-rHDL, respectively, P<0.005), ICAM-1 (by 50% and 74.6% with PLPC-rHDL, P<0.01, and DPPC-rHDL, P<0.005, respectively), and MCP-1 (by 76.5% with PLPC-rHDL, P<0.01). Although DPPC-rHDL inhibited the collar-induced change in MCP-1 by 50% compared with saline, this did not reach statistical significance (P=0.09). The collar-induced change in MCP-1 did not differ significantly between the PLPC-rHDL and DPPC-rHDL groups. The infusion of rHDL, combining the PLPC and DPPC groups, significantly inhibited the collar-induced change in MCP-1 staining (by 63%, P<0.05). Infusion of rHDL had no measurable effect on the expression of either iNOS or eNOS (results not shown).

Infusion of lipid-free apoA-I had effects similar to those of the rHDL infusions. Compared with saline, infusion of lipid-free apoA-I inhibited the collar-induced increase in expression of VCAM-1 by 40.5% (P<0.01), ICAM-1 by 94.1% (P<0.0005), and MCP-1 by 76.5% (P<0.05).

Infusion of PLPC-SUVs inhibited the collar-induced increase in expression of VCAM-1 by 47.2% (P<0.01) and ICAM-1 by 79.7% (P<0.01), but the 55.6% reduction in MCP-1 expression was not statistically significant. The effects of infusing DPPC-SUVs on the expression of VCAM-1 (–17%), ICAM-1 (–23%), and MCP-1 (–64%) were not statistically significant.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This is the first study to demonstrate that HDLs act in vivo in inflamed, nonatherosclerotic arteries to reduce the acute infiltration of neutrophils and inhibit the generation of ROS. The antiinflammatory properties of HDL have been demonstrated previously both in vitro^1,2,27 and in vivo^4–6 in several, but not in all,^28,29 previous studies. In vivo studies have generally been conducted in animals with hypercholesterolemia and atherosclerosis, although in vivo antiinflammatory effects of rHDL have also been observed in the absence of atherosclerosis in a porcine model of acute inflammation induced by the intradermal injection of interleukin-1.⁶ In the porcine model, a single infusion of rHDL inhibited the expression of endothelial E-selectin in the intradermal vessels, but effects on neutrophil recruitment and generation of ROS were not reported. The present studies demonstrate a profound ability of rHDL to inhibit the acute inflammatory changes induced by insertion of a nonocclusive Silastic collar around carotid arteries of normocholesterolemic, nonatherosclerotic rabbits.

Periarterial collars have been used previously in rabbits,³⁰ in monkeys,³¹ and in both wild-type³¹ and hypercholesterolemic apoE-knockout⁴ mice to induce acute inflammatory changes in the artery wall. Early effects (within the first 3 days) include upregulation of cellular adhesion molecules and MCP-1, with an influx of neutrophils being apparent as early as 24 hours after the collar was inserted.³² Collar insertion has also been shown to increase iNOS and reduce eNOS in rabbit arteries, but only after about 5 days.³⁰ Monocytes first appear in the artery wall after 5 days, and by 7 to 21 days, there is demonstrable neointimal hyperplasia.^4,33 The present study was designed to assess the antiinflammatory effects of rHDL during the very early stages of the inflammatory response before there was observable infiltration of monocytes and before development of neointimal hyperplasia. The substantial HDL-mediated reduction in neutrophil infiltration was probably secondary to the known ability of HDL to inhibit endothelial cell ICAM-1,¹ a factor that promotes neutrophil infiltration into the artery wall.³⁴ The fact that neutrophils are an important component of many acute inflammatory conditions, including myocardial ischemia-reperfusion injury³⁵ and stroke,³⁶ raises the possibility that HDL may have an important protective role to play in these conditions.

Neutrophils are a major source of ROS.³⁷ In turn, ROS increase the expression of endothelial ICAM-1³⁸ and thus recruit additional neutrophils into the artery wall. The vicious circle that results may be broken at several points by HDL. For example, HDLs inhibit the expression of ICAM-1 in activated endothelial cells by a mechanism involving the inhibition of endothelial cell sphingosine kinase.³⁹ Such inhibition of ICAM-1 would reduce the infiltration of neutrophils into the artery wall and, as a consequence, reduce the generation of ROS, thus breaking the cycle. A reduction in the generation of ROS may also be achieved directly as a consequence of the antioxidant properties of HDL.^40,41

In the present study, the possibility was considered that a reduced availability of nitric oxide may have contributed to the acute inflammatory changes observed in collared segments of artery and that some of the beneficial effects of HDL may have been secondary to their ability to generate nitric oxide by upregulating eNOS.⁴² However, this was not supported by the observation that iNOS expression was undetectable in both the collared and noncollared artery segments, whether or not rHDLs were infused. In addition, neither collar insertion nor rHDL infusion had a statistically significant effect on the minimal constitutive expression of eNOS. These observations are consistent with a previous report showing that collar-induced effects on iNOS and eNOS become apparent only after 5 days,³⁰ and not at the 48-hour time point used in the present study. It is possible, however, that infusion of rHDL increased the bioavailability of nitric oxide by reducing collar-induced superoxide production (which would otherwise have inactivated nitric oxide). Such an effect would be independent of changes in NOS expression.

It was of interest to note that infusion of lipid-free apoA-I had an antiinflammatory effect comparable to that of rHDL. This contrasted with observations made in vitro²⁷ in which lipid-free apoA-I was found to have no antiinflammatory activity. It is possible, however, that the positive effects of lipid-free apoA-I in the present study reflected no more than a rapid in vivo lipidation of the lipid-free apoA-I to form apoA-I/PC complexes with a composition similar to that of the rHDLs that were infused. Another difference between the results of these in vivo studies and those previously obtained in vitro relates to the effects of PC composition. In the present in vivo studies, there were no differences in the antiinflammatory effects of PLPC and DPPC, whether administered as components of rHDL or SUVs, whereas in previous studies conducted in vitro, PLPC was found to be much more effective than DPPC in terms of inhibiting VCAM-1 expression in activated HUVECs.⁴³ The explanation for these differences is unclear.

The antiinflammatory effects of infusing rHDL and lipid-free apoA-I in the present study were achieved with remarkably small amounts of apoA-I. Each infusion contained only 25 mg of apoA-I. This amount, when given to a 3-kg rabbit (plasma volume approximately 120 mL), would have increased the plasma apoA-I concentration in the immediate postinfusion period by 0.2 mg/mL, an increase of only 40% over the preinfusion concentration of 0.5 mg/mL. Furthermore, with a fractional catabolic rate for rabbit plasma apoA-I of 0.8 pools/d,⁴⁴ it would be predicted (as observed) that the apoA-I concentration would have returned to preinfusion levels by the time of euthanasia 24 hours after the last infusion. Yet despite what was no more than a minor and transient increase in the concentration of plasma HDL, the infusions resulted in an almost complete inhibition of the collar-induced arterial inflammation. This suggests that the infused rHDL, apoA-I, and SUVs had profound antiinflammatory effects that extended well beyond those resulting from a simple increase in the concentration of plasma HDL.

The mechanism underlying these potent antiinflammatory effects of infused rHDL, lipid-free apoA-I, and SUVs is unknown. Despite this, the therapeutic implications of the present findings are substantial, providing strong support for a proposition that the infusion of rHDL (or indeed lipid-free apoA-I or PC SUVs) should be investigated as first-line therapy to minimize tissue damage in devastating acute inflammatory states such as acute coronary syndromes, stroke, and ischemia-reperfusion injury.

	Acknowledgments

 
This work was supported by a grant from the Pfizer International HDL Awards. Dr Nicholls was supported by a postgraduate research scholarship from the National Heart Foundation of Australia. Dr Drummond is supported by a Peter Doherty Fellowship from the National Health and Medical Research Council of Australia (007044). Dr Rye is a National Heart Foundation of Australia Principal Research Fellow.

	References

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