Administration of Tyrosyl Radical–Oxidized HDL Inhibits the Development of Atherosclerosis in Apolipoprotein E–Deficient Mice
Objective— Tyrosyl radical–oxidized HDL (tyrHDL) increases the ability of cells to donate cholesterol to apolipoprotein (apo) A-I for HDL particle formation. We tested whether treatment with tyrHDL raises endogenous HDL cholesterol levels and decreases atherosclerosis development in apoE-deficient mice.
Methods and Results— Tyrosyl radical oxidation of mouse HDL induced formation of apoAI-AII heterodimers and enhanced the ability of mouse HDL to deplete cultured fibroblasts of their regulatory pool of cholesterol. 125I-labeled HDL and tyrHDL delivered intraperitoneally were cleared at similar rates from plasma of chow-fed apoE-deficient mice. ApoE-deficient mice injected intraperitoneally twice weekly with 150 μg tyrHDL from age 10 to 18 weeks showed a maximum 2.3-fold increase in endogenous HDL cholesterol levels, which fell toward the end of the treatment period. tyrHDL treatment resulted in 37% less aortic lesion development than in control HDL-treated mice (P<0.001) and 67% less than in saline-injected animals (P<0.001).
Conclusions— Administration of tyrHDL for 8 weeks resulted in significantly less atherosclerosis development in apoE-deficient mice than injection of HDL or saline. Molecules increasing mobilization of cellular cholesterol to apoAI for HDL particle formation would be expected to decrease atherosclerosis without necessarily causing sustained increases in circulating HDL cholesterol levels.
Despite extensive evidence that HDL cholesterol (HDL-C) levels are as strong or stronger predictors of risk for coronary heart disease than LDL levels,1–3⇓⇓ the development of therapies that specifically raise HDL has lagged far behind those that lower LDL. HDL is felt to exert its major protection by stimulating the return of cholesterol to the liver for excretion, but until recently there was a lack of clear understanding of the major mechanisms of HDL formation and therefore better ways to increase this clinically. The realization that the ATP-binding cassette transporter AI (ABCA1) is an absolute requirement for effective HDL particle formation has confirmed that active rather than passive mobilization of cellular lipids to apolipoprotein (apo) AI and small HDL particles is the rate-limiting step in this process.4–7⇓⇓⇓
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The antiatherosclerotic effects of injecting HDL or apoAI and of raising HDL levels by transgenic expression of human apoAI or ABCA1 have been shown in numerous animal studies.8–15⇓⇓⇓⇓⇓⇓⇓ In humans, agents that increase the availability of cellular lipids for apo-mediated HDL particle formation would be expected to increase total reverse cholesterol transport and decrease the development of atherosclerosis.16 We previously showed that tyrosyl radical oxidized or tyrosylated HDL (tyrHDL) markedly enhances depletion of the regulatory pool of cell cholesterol available for esterification by acyl-CoA:cholesterol acyltransferase (ACAT) compared with HDL in cultured human fibroblasts, mouse peritoneal macrophages, and human arterial smooth muscle cells.17,18⇓ This pool of cell cholesterol becomes available for HDL particle formation, as shown by a marked increase in efflux to apoAI after incubation of fibroblasts with low levels of tyrHDL.19 The effect of tyrHDL is an active process and is not attributable to tyrHDL being a better passive acceptor of cell cholesterol than HDL.19 ApoAI and AII heterodimers formed in HDL as a result of tyrosyl radical oxidation are responsible for this enhanced ability to mobilize cholesterol for removal from cells.20
The in vitro actions of tyrHDL suggested that administration of this modified lipoprotein might enhance the formation of HDL particles in vivo, raising endogenous HDL levels and decreasing the development of atherosclerosis. To test this hypothesis, apoE-deficient mice were treated twice weekly for 8 weeks with control mouse HDL or tyrosylated mouse HDL. We found that mice treated with tyrHDL had significantly increased maximal endogenous HDL-C levels during the 8-week treatment period and inhibition of atherosclerotic lesion development compared with animals injected with control HDL or the saline-treated reference animals. These results support the hypothesis that molecules enhancing the availability of cellular cholesterol for lipidation of apoAI increase HDL particle formation and decrease the development of atherosclerosis.
Total HDL from pooled plasma of male C57BL/6 mice was isolated by density gradient ultracentrifugation (d=1.07 to 1.21 g/mL)21 in the presence of 1 mmol/L EDTA, 100 μmol/L DTPA, and 1 mmol/L PMSF. HDL apoE was removed by heparin-Sepharose chromatography.22 Tyrosyl radical–oxidized mouse HDL was prepared as described previously in detail17 and reisolated by density gradient ultracentrifugation (d=1.21) to remove horseradish peroxidase and other reaction components. HDL and tyrHDL were dialyzed against 150 mmol/L saline containing 1 mmol/L EDTA and filter sterilized (0.22 mm low-protein-binding filters, Millipore), and protein content was determined by the method of Bradford using BSA as standard.23 HDL and tyrHDL apoproteins were separated using 7% to 20% nonreducing SDS-PAGE and transferred to nitrocellulose for immunoblotting using rabbit anti-mouse apoAI and apoAII antibodies (Biodesign International).20 125I-HDL and tyr125I-HDL for residence time studies were prepared using the method of Bilheimer et al.24 Total cholesterol and HDL-C in mouse plasma were measured enzymatically using Sigma Diagnostics kits 352 and 352-3, respectively.
Normal human skin fibroblasts were cultured in DMEM supplemented with 10% FBS, loaded with nonlipoprotein cholesterol, and equilibrated before HDL incubation as previously described.20 After a 16-hour incubation with the indicated concentration of mouse HDL or tyrHDL, the remaining cell cholesterol available for esterification by ACAT was determined during a 1-hour incubation with [14C]oleate (55 mCi/mmol, Amersham Pharmacia Biotech).20
Residence Time Studies
Eight-week-old female apoE-deficient mice were from Jackson Laboratory (Bar Harbor, Maine), and male and female C57BL/6 mice were from Charles River Laboratories (Ste. Constant, Quebec). All animals were housed in viral antibody–free conditions and cared for according to the Canadian Council on Animal Care guidelines. The residence times of HDL and tyrHDL were determined in female animals of both strains of mice with lipoproteins delivered either intravenously (IV) or intraperitoneally (IP). Groups of 6 mice were injected IV or IP with 10 μg 125I-labeled lipoprotein in 0.1 mL 150 mmol/L saline, and 50-μL blood samples were drawn from the tail vein from alternating subgroups of 3 mice within each group at 5 minutes and 1-, 4-, 8-, 24-, 48-, 72-, and 96-hour time points and counted in a gamma counter. The subgroups were alternated at each time point to ensure each mouse was bled only 4 times during the course of the study. The 5-minute time point was taken as 100% of the injected dose.
Female apoE-deficient mice were fed a normal chow diet (0.02% cholesterol and 4% fat) ad libitum from 8 weeks of age to completion of the study. At 9 weeks of age, mice were divided into 3 treatment groups (saline, n=8; HDL, n=7; tyrHDL, n=7) having similar ranges and means of plasma total and HDL-C. From age 10 to 18 weeks, mice were injected IP twice weekly with 150 μg of HDL or tyrHDL or saline (0.1 mL injection volume). This amount (15 mg/kg per week) was chosen based on doses of HDL protein previously found to inhibit progression and induce regression of atherosclerosis in rabbits.8,9⇓ A 200-μL fasting blood sample was collected biweekly just before reinjection to measure total and HDL-C.
Quantitation of Atherosclerotic Lesions in the Aorta
At 18 weeks of age, the mice were euthanized, terminal bleeds were taken, and the heart with aorta intact down to the iliac bifurcation was removed into PBS with 1 mmol/L EDTA. The heart and aortic root were cut and preserved in 1% formalin. The remaining aorta was cleaned of adipose and adventitial tissue under a dissecting microscope, cut longitudinally, and pinned open on a black background for en face photography without lipid staining using a Pentax K-1000 camera. Scanned images of aortas were analyzed for total aortic lesion area using Scion image analysis software (Scion Corporation). Each image was assessed by 2 observers blinded to treatment groups, and the average of lesion areas for the 2 observers was determined. Formalin-fixed aortic root and hearts were processed and embedded in paraffin, and 4- to 6-μm sections were cut for mounting on glass slides and staining with H&E. Sections were photographed using a Nikon Zoom SMZ-2T stereoscopic microscope equipped with a microflex HFX-35DX photomicrographic system.
Statistical analyses were done using SPSS version 10.1 (SPSS Inc). The data for plasma HDL levels and lesion area were found to be normally distributed. The treatment groups were compared using one-way ANOVA and least-squares difference as the post hoc test, with P<0.05. Residence times of control and tyrosylated mouse HDL were calculated using SAAM II (SAAM Institute).
Analysis of Tyrosylated Mouse HDL
Because of potential immunogenicity of human tyrHDL, tyrosylated mouse HDL was prepared using apoE-free HDL from C57BL/6 mice to administer to the apoE-deficient mice. ApoAI-AII heterodimers, with apoAII present as either a monomer or dimer in the heterodimer, are responsible for the enhanced ability of human tyrHDL to mobilize stored cholesterol and prevent the accumulation of LDL-derived cholesterol in cultured human fibroblasts.20 Oxidation of apoE-free mouse HDL with peroxidase-generated tyrosyl radical also resulted in formation of higher molecular weight multimers of HDL apolipoproteins, including a prominent band of approximately 36-kDa molecular weight containing apoAI and apoAII, consistent with an apoAI-apoAIImonomer heterodimer (Figure 1). This heterodimer comprised 14.1% or approximately one seventh of the total protein in mouse tyrHDL by densitometry.
Mobilization of ACAT-Accessible Cholesterol by Tyrosylated Mouse HDL
To validate the activity of tyrosyl radical-oxidized mouse HDL, the ability of these particles to deplete intracellular cholesterol was assessed in vitro. Similar to human tyrHDL, mouse tyrHDL markedly enhanced the depletion of cholesterol-enriched human fibroblasts of their pool of ACAT-accessible cholesterol compared with control mouse HDL (Figure 2). The Km for half-maximal depletion of this substrate pool was similar for tyrHDL and HDL (4.0 versus 4.9 μg/mL, respectively); however, maximal depletion of ACAT-accessible cholesterol was much greater for tyrHDL, with approximately 80% of cholesterol available for esterification removed in 1 hour versus only 20% to 25% for HDL. The previous demonstration of a direct correlation between depletion of the ACAT substrate pool and increase in cholesterol availability for removal by apoAI19,20⇓ suggested that, if active in vivo, tyrosylated mouse HDL would significantly increase the mobilization of cell cholesterol for removal by apoAI and thereby increase the formation of HDL in an animal model.
Residence Times of HDL and tyrHDL in ApoE-Deficient Mice
To examine the relative rates of clearance of HDL and tyrHDL and to establish an appropriate schedule for administering injections, we measured the residence times of iodinated HDL and tyrHDL injected intravenously and intraperitoneally in chow-fed apoE-deficient mice (Figure 3). The half-life of tyrHDL (3.2 hours) was shorter than HDL (6.3 hours) when given IV (P<0.05) but not significantly different from HDL when the particles were delivered IP (7.5 and 5.8 hours, respectively). At 1 hour, clearance of HDL given IP was slower than HDL given IV, after which the IP-administered particles were cleared more rapidly. tyrHDL delivered IP was cleared more slowly than when delivered IV at all time points. At 84 hours (3.5 days), less than 2% of exogenous HDL or tyrHDL remained in the circulation of apoE-deficient mice injected IV or IP. Endogenous HDL-C levels measured just before the next injection on a twice-weekly basis would therefore not be significantly affected by the small amount of residual injected tyrHDL.
Quantitative Effects of tyrHDL Administration on Circulating HDL-C Levels
The presence of active apoAI-AII heterodimers in mouse tyrHDL and the increased ability of these particles to mobilize intracellular cholesterol in vitro suggested administration of these particles might increase HDL formation, raise HDL levels, and decrease atherosclerosis in vivo. The effects of twice-weekly IP injection of saline, HDL, or tyrHDL for 8 weeks on endogenous HDL-C levels are shown in Figure 4. Average HDL-C levels at baseline were similar in the saline-injected reference group and HDL and tyrHDL treatment groups (58.88±9.86, 63.38±7.76, and 64.86±8.17 mg/dL, respectively). Treatment with tyrHDL resulted in a 1.6-fold increase at 2 weeks and a maximal 2.3-fold increase in endogenous HDL-C levels at 4 weeks compared with baseline levels. Treatment with control HDL resulted in a maximal 1.7-fold increase over baseline in endogenous HDL-C levels at 6 weeks. Saline-injected reference animals had a nonsignificant increase in endogenous HDL-C levels at 8 weeks. Endogenous HDL-C levels in both tyrHDL- and HDL-treated animals tended to drop after reaching their maximum levels during the study and approached similar levels by 8 weeks. HDL-C levels were significantly higher in tyrHDL- and HDL-treated animals compared with saline-treated animals at 4, 6, and 8 weeks and were significantly higher in tyrHDL- compared with HDL-treated animals at 4 and 8 weeks. There were no significant changes in total plasma cholesterol levels over the duration of the study in the reference or treatment groups (saline group, 466±44 versus 413±35; HDL group, 484±61 versus 422±39; tyrHDL group, 470±90 versus 393±15 mg/dL, baseline versus terminal bleed [mg/dL, ±SEM], respectively).
Effects of Tyrosylated HDL Administration on Development of Aortic Atherosclerosis
Atherosclerotic lesion area in the entire aorta from aortic root to iliac bifurcation was determined by en face quantitation. As shown in Figure 5, average lesion area in tyrHDL-treated animals (1.13±0.48 mm2, average±SD) was 67% less than the saline-injected reference group (3.41±0.69 mm2) (P<0.001) and 37% less than the control HDL-treated animals (1.80±0.24) (P<0.001). HDL-treated animals showed an average 48% lower lesion area compared with the saline-injected reference group (P<0.001). In all animals most lesions were localized to the aortic root and aortic arch, with additional involvement of small artery branch points and the iliac bifurcation. A representative section of the aortic root for each group is shown in Figure 6. Sections from saline- and HDL-injected animals showed extensive intimal thickening, inflammatory infiltrate, macrophage foam cells, cell necrosis, and cholesterol clefts, with tyrHDL-treated animals showing lesser degrees of intimal thickening and earlier lesion stage in the aortic root. The percent of aortic root containing atherosclerotic lesions of any stage was 62.5±13%, 43.6±7%, and 27.1±14% (mean±SEM) for saline-injected, HDL-treated, and tyrHDL-treated mice, respectively.
The present studies show that tyrosyl radical oxidation of mouse HDL, like human HDL, induces the formation of apoAI-AII heterodimers and enhances the ability of mouse HDL to mobilize the regulatory pool of cholesterol available for esterification by ACAT from cultured fibroblasts. Intraperitoneal injection of apoE-deficient mice with tyrHDL resulted in significantly higher average endogenous HDL-cholesterol levels than in saline- or HDL-injected mice; however, this increase was diminishing at the end of the treatment period. Chow-fed apoE-deficient mice injected with tyrHDL developed significantly less aortic atherosclerosis by 18 weeks of age than the saline-injected reference and control HDL-treated animals. These results suggest that the enhanced effect of tyrHDL in mobilizing cholesterol for HDL formation by apoAI seen in several cultured cell types also occurs in vivo, providing increased cholesterol for HDL particle formation and decreasing the development of atherosclerosis. The decrease in formation of atherosclerotic lesions is likely attributable to the direct effect of enhanced cholesterol mobilization from tissues induced by tyrHDL and a secondary consequence of the increase in circulating HDL concentrations. Increased HDL may decrease atherosclerosis as a consequence of increased reverse cholesterol transport or any of the other potential protective actions of plasma HDL.25
The rise in circulating HDL-C levels in the treatment groups was attributed to increases in endogenous HDL-C induced by the injected tyrHDL or HDL rather than the injected HDL itself, because blood for HDL-C levels was drawn when residence time studies indicated more than 98% of injected HDL or tyrHDL was cleared from plasma. The increase in endogenous HDL-C in tyrHDL-treated mice and the less pronounced increase in control HDL-treated mice suggests tyrHDL may be mobilizing cholesterol through a pathway also used by HDL but in a more effective manner. The mechanism of enhanced cholesterol mobilization by tyrHDL is not yet known. Its action suggests involvement of the ABCA1 transporter, known to make more cholesterol available for removal by apoAI.26 We have found that the actions of tyrHDL are absent in cultured fibroblasts from patients with Tangier disease, suggesting at minimum that functional ABCA1 is required for the activity of tyrHDL. We are presently investigating the role of ABCA1 in the actions of tyrHDL.
We previously determined that the active components of tyrHDL are apoAI-AII heterodimers formed during tyrosyl radical oxidation of HDL and that isolated apoAI-AII heterodimers present on spherical reconstituted HDL particles were more potent on a microgram of protein per milliliter basis than intact tyrHDL at preventing the accumulation of LDL-derived cholesterol mass by cultured fibroblasts.20 The effects of tyrHDL seen in these studies are even more striking given that the apoAI-AII heterodimers in mouse tyrHDL represent only 14% of total HDL protein in these particles. Although we do not envision injecting patients with tyrHDL or attempting to induce tyrosyl radical oxidation of HDL in vivo, these results suggest that peptides based on the structure of tyrHDL apoAI-AII heterodimers, or small molecules mimicking the enhanced cholesterol mobilization of these heterodimers, might be highly effective at increasing HDL particle formation and preventing or decreasing atherosclerosis clinically.
The decline in endogenous HDL-C levels after the peak increases seen at 4 weeks in tyrHDL-treated and at 6 weeks in HDL-treated animals may be caused by changes in overall reverse cholesterol transport and HDL catabolism downstream of initial apolipoprotein-mediated lipid mobilization, as suggested in previous studies using ABCA1 transgenic mice.27 One example of this might be enhanced uptake of HDL-cholesterol via SR-BI in the liver. If the action of tyrHDL is ABCA1-dependent, our results suggest tyrHDL may enhance the expression or activity of hepatocyte ABCA1, known to be required to increase HDL levels in ABCA1-transgenic mice.6,28,29⇓⇓ Recent studies have provided additional evidence for a critical role of hepatocyte ABCA1 in the formation of HDL particles and determining circulating HDL levels.30,31⇓ The lack of a sustained increase in HDL levels in response to tyrHDL suggests that an ABCA1-dependent effect of tyrHDL in the liver may be transitory, as suggested by another recent report studying apoAI-dependent induction of ABCA1 activity of the liver.32 These findings and those of others using LXR agonists33,34⇓ suggest actual HDL-C levels may be an inappropriate marker to follow in response to agents that increase HDL particle formation and that, in addition to decreased atherosclerosis development, more definitive measures of enhanced reverse cholesterol transport are required.16 Proof of increased reverse cholesterol transport in response to increased HDL formation would require fecal sterol excretion studies such as those recently described using reconstituted HDL infusion in humans.35 Whether the decrease in atherosclerotic lesion development seen in response to tyrHDL requires a sustained increase in circulating HDL-C levels in response to tyrHDL is being examined in additional studies.
Anantharamaiah and colleagues36 have recently described the ability of daily intraperitoneal injections of a synthetic class A amphipathic peptide analog of apoAI for 16 weeks to decrease the development of atherosclerosis in C57Bl/6J mice fed an atherogenic diet. Shah and colleagues37,38⇓ have demonstrated the ability of reconstituted HDL containing apoAIMilano given in multiple or single injections intravenously to inhibit atherosclerotic lesion development and mobilize tissue cholesterol in apoE-deficient mice. To our knowledge, ours is the first report using the intraperitoneal injection route to study the effects of whole lipoproteins on endogenous lipid levels and atherosclerosis. Our results suggest effective absorption and striking effects on atherosclerosis development with even low levels of whole tyrHDL particles delivered intraperitoneally.
In summary, administration of tyrHDL induced significant but transient increases in endogenous HDL-C levels and decreased the development of atherosclerosis in a mouse model of human atherosclerosis. These results provide evidence that compounds increasing the availability of cellular cholesterol for HDL formation in vivo might be viable as therapies to treat and prevent atherosclerotic vascular disease.
This work was supported by grants from the Canadian Institutes of Health Research and Daiichi Pharmaceutical Inc. G.A.F. is a Scholar of the Alberta Heritage Foundation for Medical Research. The authors thank Dr Hugh Barrett for assistance with residence time determinations and Dr Renee LeBoeuf for helpful discussions.
- Received May 30, 2003.
- Accepted June 20, 2003.
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