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Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:1696-1702
Published online before print June 17, 2004, doi: 10.1161/01.ATV.0000135981.61827.9d
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:1696.)
© 2004 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Divergent Effects of the Catalytic and Bridging Functions of Hepatic Lipase on Atherosclerosis

Helén L. Dichek; Kun Qian; Nalini Agrawal

From the Department of Pediatrics, University of Washington, Seattle, Wash.

Correspondence to Helén L. Dichek, Department of Pediatrics, Box 356320, University of Washington, 1959 NE Pacific Street, Seattle WA 98195. E-mail hdichek{at}u.washington.edu


*    Abstract
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 *Abstract
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Objective— Increased expression of human hepatic lipase (HL) or a catalytically inactive (ci) HL clears plasma cholesterol in mice deficient in low-density lipoprotein receptors (LDLr) and murine HL. We hypothesized that increased expression of both HL and ciHL reduces atherosclerosis in these mice.

Methods and Results— Mice deficient in both LDLr and murine HL, alone or transgenically expressing similar levels of either human HL or ciHL, were fed a high-fat, cholesterol-enriched "Western" diet for 3 months to accelerate the development of atherosclerosis. Levels of plasma lipids, insulin, glucose, and liver enzymes were measured monthly, and aortic atherosclerosis was quantitated after 3 months. Plasma insulin, glucose, and liver enzyme levels did not differ significantly from controls. After 3 months, expression of HL reduced plasma cholesterol by 55% to 65% and reduced atherosclerosis by 40%. Surprisingly, expression of ciHL did not reduce plasma cholesterol or atherosclerosis.

Conclusions— High levels of HL, but not ciHL, delay the development of atherosclerosis in mice deficient in LDLr and mHL.

These studies demonstrate that high levels of catalytically active human hepatic lipase (HL) reduce atherosclerosis, whereas high levels of a catalytically inactive HL do not affect atherosclerosis in mice genetically deficient in low-density lipoprotein receptor and mouse HL.


Key Words: hepatic lipase • atherosclerosis • bridging function • fatty liver • mouse models


*    Introduction
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 *Introduction
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 down arrowDiscussion
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Human hepatic lipase (HL) plays a central role in lipid metabolism and atherosclerosis.1,2 HL is a secreted, multifunctional enzyme produced by the liver. In the liver, it binds to heparan sulfate proteoglycans (HSPG) on hepatocyte and endothelial cell surfaces and hydrolyzes triglycerides and phospholipids in lipoproteins yielding particles that are optimal for receptor-mediated uptake.2–5 HL functions as a bridge between lipoproteins and cell-surface HSPG, thereby facilitating receptor-mediated lipoprotein uptake by the low-density lipoprotein receptor (LDLr) and the LDLr-related protein (LRP).5–8 The bridging also facilitates selective cholesterol uptake by the scavenger receptor B1.9,10

The role of HL in atherosclerosis is controversial.11,12 Some studies support a pro-atherogenic role. For example, HL mediates production of small dense LDL (part of the atherogenic lipid profile). HL activity is elevated in males and postmenopausal women, both at increased risk for atherosclerosis.13–16 In rabbits transgenic for human apoB, HL expression results in atherogenic small dense LDL particles.17 In female apoE-deficient mice (an atherosclerosis model), atherosclerosis was reduced when the mouse (m) HL gene was deleted by gene targeting.18 Furthermore, adding mHL back to macrophages (using bone marrow transplantation) in the apoE-deficient and mHL-deficient mice actually increased atherosclerosis.19

Other studies support a protective role for HL. In humans, HL reduces atherogenic remnant lipoproteins and increases production of high-density lipoprotein (HDL)3 and pre-ß1 HDL, both avid acceptors of free cholesterol.20–24 In HL transgenic mice, aortic cholesterol was decreased.25 In mice overexpressing HL, cholesterol levels were significantly lower.5,26 Likewise, animal models overexpressing a catalytically inactive variant of HL (ciHL) (reflecting the bridging function of HL) also had lower cholesterol levels, suggesting that high levels of ciHL may be atheroprotective.10,27

We hypothesized that overexpression of HL (with catalytic and bridging functions) and ciHL (with the bridging function) will reduce atherosclerosis in both sexes. We transgenically overexpressed HL and ciHL in LDLr and mHL double-knockout mice and compared the effects of each transgene on lipid levels and atherosclerosis.


*    Methods
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 *Methods
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Genetically Modified Mice
All mice were backcrossed to at least 96% C57BL/6 background. LDLr-deficient (Ldlr–/–) mice transgenically expressing either human HL or human ciHL only in the liver were generated as described.27 These mice were bred with mice lacking mHL (hl–/–)28 (Jackson Laboratory) to yield Ldlr–/–hl–/–HL or Ldlr–/–hl–/–HLS145G mice (that were heterozygous for the HL or ciHL transgenes) and nontransgenic (control) Ldlr–/–hl–/– mice. All atherosclerosis studies compare end points in transgenic mice and nontransgenic littermates, which controlled for the presence of limited numbers (4%) of non-C57BL/6 genes.

Presence of the HL or ciHL transgenes and absence of the gene-targeted mouse LDLr and HL genes were determined by polymerase chain reaction analysis of tail DNA.10,29

Male and female mice were studied. All were homozygous for the wild-type mouse apoB gene. Mice were housed in a full-barrier facility with a 12-hour light-dark cycle. All studies were approved by the Institutional Animal Care and Use Committee of the University of Washington.

Expression of HL and ciHL
Plasma was obtained before and 10 minutes after tail-vein injection of heparin (150 U/kg body weight) and frozen at –80°C until assay for lipase activity (with glycerol[1-14C-trioleate]-labeled emulsion30) and Western blot.5 Analysis of HL immunostaining was with densitometric scanning.10

Lipoprotein Analysis
After a 4-hour fast, plasma from 3 to 5 mice was pooled, and 3 to 7 separate pools were analyzed for lipid levels for each genotype. Lipoproteins were fractionated by fast protein liquid chromatography (FPLC).5 Cholesterol and triglyceride levels in plasma and FPLC fractions were measured with standard enzymatic assays (cholesterol: Abbott Spectrum; triglycerides: GPO-PAP kit, Boehringer-Mannheim).

Apolipoprotein Analysis
Contiguous FPLC fractions were pooled, concentrated using Centricon-30 concentrators (Millipore), and stored at –80°C. For Western analysis, proteins were separated on either 4% SDS-PAGE (for apoB)31 or precast 12% Tris-glycine SDS-PAGE (BioRad) (for apoE and apoA1) as described.10 Western blots were visualized with horseradish peroxidase-goat antirabbit antibody and an enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech).

HDL Analysis
Plasma lipoproteins were separated by agarose gel electrophoresis (Titan gels; Helena Laboratories, Beaumont, Tex). One gel was stained with Fat Red 7B for lipoproteins; the other was analyzed by Western blot for apoA1.

Atherosclerosis Study
Mice were fed a chow diet until they were 10 to 16 weeks old. To accelerate development of atherosclerosis, 11 to 22 mice of each genotype were fed a Western diet [21% (w/w) fat, 0.15% (w/w) cholesterol; Harlan-Teklad] for 12 weeks. Mice were weighed, and fasting plasma and lipoprotein lipid levels were determined at baseline and at 4-week intervals. At 12 weeks, mice were deeply anesthetized with ketamine (225 mg/kg) and xylazine (23 mg/kg). Analysis of atherosclerosis was performed as described.32 Stained aortas were submerged in phosphate-buffered saline and photographed using a Nikon Coolpix 995 (Nikon) digital camera and stereomicroscope. Images were analyzed for Sudan IV staining with Adobe Photoshop (Adobe).

Biochemical Analyses
Levels of serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvate transaminase, and glucose were determined (Phoenix Central Laboratory) on fasted pooled plasma (3 to 5 mice per genotype). Plasma levels of glucose, insulin, and free fatty acids (FFA) were used as indicators of insulin resistance.33 Plasma insulin levels were measured in triplicate with a rat insulin enzyme-linked immunosorbent assay kit (Crystal Chem) that detects mouse insulin. FFA levels were measured in duplicate (3 to 7 mice per genotype) with the NEFA C kit (WAKO Chemicals).

Tissue Triglycerides
Liver tissue from 3 chow-fed or 5 Western diet-fed mice of each genotype was dissolved in KOH, and liberated glycerol (to estimate triglyceride) was measured in quadruplicate with the Triglyceride GPO-PAP kit.34,35 Protein concentrations were measured in triplicate with the Lowry method (Sigma Diagnostics).

Liver Morphology
To determine whether hepatic HL or ciHL overexpression results in increased lipid uptake, livers were flash-frozen in liquid nitrogen (LN2), embedded in OCT compound, and 10-µ sections were cut on a cryostat (Leica). Tissue sections were stained with Oil Red O (ORO) to visualize neutral lipids.36 Nuclei were counterstained with hematoxylin. To distinguish lipid staining of macrophages (Kupffer cells) and hepatocytes, contiguous sections were stained with ORO or the macrophage-specific antibody MOMA-2 (Biosource International). Sections were photographed using a Nikon Coolpix camera and an inverted microscope (Leica DMIL, Leica Camera AG).

Statistical Analyses
Results are mean±SD, except in the FPLC tracings, which are mean±SE. Body weight, liver triglycerides, and levels of serum lipids, lipoproteins, FFAs, and insulin were analyzed by unpaired t tests. Because glucose levels were measured on 2 pooled samples, the individual values of each pool are shown in the Table . Apolipoprotein distributions were determined on pooled samples, and means of 2 pools are presented. All possible pair-wise comparisons were not made. Atherosclerosis was analyzed using Mann-Whitney rank sum test.


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  		Physical and Biochemical Characteristics of Ldlr–/–hl–/– Mice Alone or Expressing HL or ciHL


*    Results
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 *Results
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Hepatic Lipase Expression
Levels of immunoreactive human HL in Ldlr–/–hl–/–HL and Ldlr–/–hl–/–HLS145G mice were similar by Western blots of postheparin plasma (Figure I and other supplementary data, available online at http://atvb.ahajournals.org) and densitometric scanning analysis (1.04±0.23 AU, Ldlr–/–hl–/–HL versus 1.13±0.22 AU, Ldlr–/–hl–/–HLS145G, P=0.59). HL protein was absent from postheparin plasma of Ldlr–/–hl–/– mice. HL activity was undetectable in all preheparin plasmas (not shown). After heparin treatment, plasma HL activities equaled background in Ldlr–/–hl–/– and Ldlr–/–hl–/–HLS145G mice and were 20-fold higher than human postheparin plasma in Ldlr–/–hl–/–HL mice (Table).

Body Weight
All mice gained similar amounts of weight (70% to 90%) on the Western diet (Table). Males were 10% to 20% heavier than females.

Plasma Lipids
In chow-fed mice, HL expression reduced plasma cholesterol by 55% to 65%, and ciHL expression reduced cholesterol by 12% to 20% (Table I, available online at http://atvb.ahajournals.org). HL expression markedly reduced triglyceride levels (by {approx}65%), whereas ciHL expression had no significant effect.

In all mice on Western diet, plasma cholesterol increased 5- to 8-fold compared with mice on chow diet. After 12 weeks, HL expression reduced cholesterol by {approx}60% (Table IV, available online at http://atvb.ahajournals.org). ciHL expression initially reduced cholesterol by 25% to 30% [females: from 1305±113 to 873±78 mg/dL, P<0.002; males: 1493±199 to 1116±178 mg/dL, P<0.05 at 4 weeks and 1498±223 to 1098±82 mg/dL, P=0.02 at 8 weeks in females only] (Tables II and III, available online at http://atvb.ahajournals.org). The apparent cholesterol-lowering effect by ciHL was not sustained beyond 8 weeks. Thus, plasma cholesterol levels were not significantly different in ciHL-expressing mice and nontransgenic Ldlr–/–hl–/– mice at the end of the study.

The Western diet increased plasma triglycerides in all mice (up to 3-fold in females and 2- to 5-fold in males) (Tables II through IV). HL expression reduced triglycerides by 70% to 85% in both sexes. As expected, ciHL expression did not affect plasma triglyceride levels.

Plasma Lipoproteins
Plasma lipoproteins were fractionated by FPLC. In chow-fed control Ldlr–/–hl–/– mice, the FPLC profiles showed minimal very-low-density lipoprotein (VLDL) and broad intermediate-density lipoprotein (IDL)/LDL and HDL peaks (Figure 1). HL expression virtually eliminated the VLDL peak and markedly reduced the IDL/LDL and HDL peaks. ciHL expression reduced only the IDL/LDL peak (Figure 1, Table I). Lipoprotein triglyceride content was reduced by HL but not by ciHL (Table I).



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  		Figure 1. FPLC profiles of pooled plasma cholesterol from fasted mice. Profiles of female (A) and male (B) mice on a chow or high-fat Western diet are shown. Pooled plasma (100 µL) from 3 to 5 mice was fractionated by Superose 6 chromatography, and fractions were assayed for cholesterol. Lipoprotein distributions are indicated by horizontal bars. Tracings represent the mean±SE of 3 to 7 FPLCs of each genotype. For Ldlr–/– hl–/–HLS145G, *values are at least P<0.05 compared with Ldlr–/–hl–/– mice. All fractions in Ldlr–/–hl–/–HL mice were significantly reduced, except for the HDL fractions in male mice fed a Western diet.

Analyses of plasma FPLC profiles indicated substantial changes in lipoprotein metabolism associated with the Western diet (Tables I through IV). Ldl–/–hl–/– mice showed a sharp peak in the VLDL and a broad peak in the IDL/LDL (Figure 1). HL expression markedly reduced cholesterol in all lipoproteins. Although ciHL expression significantly reduced some of the fractions in the LDL range, total LDL was not significantly reduced (Table IV). Also, ciHL expression did not affect VLDL or HDL. HL expression significantly reduced lipoprotein triglyceride in both sexes, whereas ciHL expression did not reduce triglycerides.

Apolipoprotein Distributions
In chow-fed Ldlr–/–hl–/– mice, apoB48 and apoB100 were distributed in VLDL, IDL/LDL, and large HDL (Figure 2). HL expression markedly reduced apoB48 and apoB100 in the VLDL, whereas ciHL expression had no effect. ApoE was associated with the IDL/LDL and HDL, regardless of genotype. ApoA1 was associated with the larger HDL in Ldlr–/–hl–/– mice (in the absence of HL hydrolysis) and with the smaller HDL in HL-expressing mice, reflecting the effect of HL-mediated hydrolysis to reduce HDL size. In ciHL-expressing mice (in the absence of HL hydrolysis), apoA1 was associated with the larger HDL, thus confirming the need for HL-mediated hydrolysis to drive the apoA1 distribution toward the smaller HDL.



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  		Figure 2. Apolipoprotein profiles of pooled plasma from female mice fed a chow diet. For each genotype, plasma from 3 to 5 mice was pooled, and 2 pools from each genotype were fractionated by FPLC. Contiguous fractions were pooled, concentrated, and analyzed by Western blotting. Apoprotein distributions were assessed by densitometric scanning and expressed as percent optical density units in each fraction divided by the sum of optical density units in all fractions. Horizontal bars indicate lipoprotein elution positions. The number of individual plasmas used in the plasma pools is indicated in the top right corner of each panel.

The apolipoprotein patterns in Western diet-fed mice paralleled those of mice on a chow diet (not shown), except that apoA1 was associated with large and small HDL.

HDL Analysis
HL expression substantially reduced neutral lipid staining and apoA1 staining of HDL. ciHL expression slightly reduced neutral lipid staining but had no effect on apoA1 staining of HDL (Figure II, available online at http://atvb.ahajournals.org).

Biochemical Studies
Because increased plasma FFA levels (from HL-mediated lipoprotein hydrolysis) promote insulin resistance, a condition associated with increased atherosclerosis risk, plasma insulin, glucose, and FFA levels were determined (as indicators of insulin resistance). All insulin levels increased on the Western diet. Although female levels remained within normal, male levels doubled (Table). Interestingly, all mice were mildly hyperglycemic at the beginning and remained so at 12 weeks. Unexpectedly, in HL-expressing male mice (on Western diet), FFA levels were significantly decreased, and insulin values increased, possibly reflecting suppression of lipolysis by insulin. In all mice, liver enzymes increased to the upper normal limit on the Western diet (not shown).

Tissue Triglycerides
On chow diet, compared with control, ciHL expression was associated with a significant ({approx}20%, P<0.05) increase in liver triglyceride content (Table). There was a trend toward increased liver triglyceride content associated with HL expression. These results indicate that ciHL increases liver triglyceride accumulation. A Western diet increased liver fat content by {approx}10-fold in all genotypes, with the highest values associated with ciHL-expression.

Liver Morphology
To assess whether hepatic overexpression of HL or ciHL results in increased liver lipid uptake and content, 2 livers of each genotype were stained with ORO and counterstained with hematoxylin. On a chow diet, livers of HL-expressing mice displayed small (1 to 2 µ) ORO-staining lipid droplets that lined the cell periphery (Figure 3). Surprisingly, ciHL-expressing mouse livers had large (4 to 8 µm) ORO-staining lipid globules throughout the cytoplasm in addition to lipid droplets. Liver lipid staining in control (Ldlr–/–hl–/–) mice was slightly less than in HL-expressing mice. Frozen sections of livers of Ldlr+/+hl+/+ mice (fed chow diet) stained with ORO displayed less lipid staining than Ldlr–/–hl–/– mice (not shown). Twelve weeks on a Western diet resulted in large ORO-staining lipid globules (hepatic steatosis) in livers from all 3 genotypes (Figure 3). Macrophage staining was minimal and not apparently associated with lipid accumulation (not shown).



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  		Figure 3. Liver histology in male mice. Frozen sections from mice fed a chow diet (top) or a Western diet for 12 weeks (bottom) were stained with Oil Red O to visualize fat and hematoxylin to visualize nuclei. Size bars indicate 20 µm.

Atherosclerosis Study
To examine the effects of HL and ciHL expression on the development of atherosclerosis, we performed en face analysis of aortas stained with Sudan IV. In female mice, HL expression reduced atherosclerosis by 40% (from 6.2±0.7 [Ldlr–/–hl–/– mice], n=15, to 3.7±1.2 [Ldlr–/–hl–/–HL mice], n=13, P=0.006). Surprisingly, expression of ciHL failed to reduce atherosclerosis. Thus, the stained aortic surface area was 7.2±1.8 (n=16) in Ldlr–/–hl–/–HLS145G female mice (Figure 4).



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  		Figure 4. Atherosclerosis in mice after 12 weeks of a Western diet. A, En face mounted aortas were stained with Sudan IV to visualize lipid-laden atherosclerotic areas in both sexes. Note reduced staining in Ldlr–/–hl–/–HL mice. B, Ratio of areas stained by Sudan IV to total aortic area. Note that HL expression reduced atherosclerosis by 40% in both female (P=0.006) and male mice (P=0.001).

The effects in male mice were similar: HL expression reduced atherosclerosis by 40%. Specifically, the stained surface area decreased from 12.2±0.8 (Ldlr–/–hl–/– mice, n=20) to 7.5±1.3 (Ldlr–/–hl–/–HL mice, n=11, P=0.001). ciHL expression failed to reduce atherosclerosis as demonstrated by a stained surface area of 11.9±1.6 (n=22) in Ldlr–/–hl–/–HLS145G male mice (Figure 4).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This study demonstrates that overexpression of HL reduces atherosclerosis in a mouse model of LDLr and mHL deficiency. Surprisingly, similar levels of ciHL did not reduce atherosclerosis. The most likely explanation for the reduction in atherosclerosis is the considerable (60% to 70%) decrease in atherogenic apoB-containing lipoproteins associated with high levels of HL. Previous studies established a clear relationship between lower levels of these lipoproteins and reduced atherosclerosis.32

In our study, atherosclerosis was reduced despite a {approx}30% reduction in the atheroprotective HDL fraction. We speculate that the lower HDL levels resulted from hydrolytic processing that, in turn, generated cholesterol-poor HDL subspecies, including HDL3 and pre-ß HDL.22,23 These HDL subspecies efficiently absorb free cholesterol from the periphery, thereby removing 1 substrate for atherosclerosis formation.12 Consistent with this speculation, our results indicate that HDL in HL-expressing mice differ from HDL in ciHL-expressing and control mice. Specifically, HDL in HL-expressing mice were smaller and showed reduced lipid staining and apoA1 staining compared with HDL in ciHL-expressing and control mice.

Our observation that HL decreases mouse atherosclerosis differs with a previous report.18 In that study, mice with combined mHL and apoE deficiencies had less atherosclerosis than apoE-deficient mice with mHL, suggesting that mHL promotes the development of atherosclerosis. One explanation for the differences in atherosclerosis might be the {approx}20-fold difference in HL activities in these studies: increased HL activity reduces atherogenic lipoproteins, thereby reducing atherosclerosis development. Another explanation could be differences in the HL and mHL proteins. Thus, HL, located mainly at cell surfaces, binds HSPG strongly, whereas mHL mainly circulates in plasma and binds HSPG weakly.37 "Bound" HL may capture lipoproteins for hydrolytic processing and hold processed lipoproteins near receptors for more efficient uptake. Circulating mHL hydrolyzes lipoproteins but cannot assure proximity to receptors for removal. Yet another explanation for the differences in atherosclerosis may be differences in genetic backgrounds (apoE deficiency versus LDLr deficiency) in these studies.

Unexpectedly, high levels of ciHL did not reduce atherosclerosis. This was surprising because previous metabolic studies demonstrated that ciHL reduced atherogenic lipoproteins without significantly affecting HDL.10,27,38 Those findings predicted that ciHL would protect against atherosclerosis. However, those mice also expressed mHL. Endogenous mHL activity may suffice to modify lipoprotein particles and optimize their clearance through receptor-mediated8,10,39–41 and other pathways.42

Also, the previous metabolic studies of ciHL were mainly conducted in chow-fed mice that have markedly different lipoprotein lipid compositions compared with mice fed a Western diet. In fact, the Western diet-induced lipoprotein changes paralleled those reported in genetically intact C57BL/6 mice fed a semisynthetic atherogenic diet (similar to the Western diet).43 The diet-induced changes may result from increased lipoprotein production and decreased lipoprotein clearance by the liver.

The lack of an effect of ciHL on atherosclerosis may relate to its failure to sustain reduced levels of atherogenic lipoproteins. We reasoned that liver-specific transgene expression might increase liver lipid accumulation and impair hepatic lipid catabolism (hepatotoxicity). We evaluated for hepatotoxicity by examining biochemical parameters (glucose, insulin, and liver enzymes) and biochemical and morphological analyses of liver tissues (triglyceride content and ORO staining). We found that all mice had mildly elevated glucose levels (on both diets) that were genotype-independent. However, because previous studies reported abnormal glucose levels in C57BL/6 mice fed a high-fat diet,34 we attribute the increased glucose levels to the C57BL/6 genetic background rather than to an effect of liver-specific transgene expression.

Plasma insulin levels were normal in all mice on the chow diet and all female mice on the Western diet. Male mice on the Western diet had increased insulin levels, independent of genotype. Although males were slightly heavier than females (by 10% to 15%), it is not clear that this could explain their increased insulin levels. Liver enzymes, normal in all mice on a chow diet, increased to the upper normal limit during the study. These relative increases in liver enzymes likely reflect increased intrahepatic fat accumulation from the Western diet.

Another explanation for the lack of an effect of ciHL on atherosclerosis may relate to the quantity and quality of hepatic lipid accumulation. Thus, whereas the small lipid droplets in the livers of HL-expressing mice may reflect normal endogenous synthesis of triglycerides from FFAs derived from fully processed lipoproteins, we speculate that the larger lipid globules in the livers of ciHL-expressing mice reflect accumulation of incompletely processed triglyceride-rich remnant lipoproteins from ciHL-facilitated liver uptake.

Finally, we found that the effects of HL and ciHL on atherosclerosis are independent of gender: in mice of both sexes, atherosclerosis was reduced by HL expression but not by ciHL expression.

In summary, we demonstrate that high levels of HL expression can delay the development of atherosclerosis. However, ciHL expression failed to reduce atherosclerosis despite its initial promise as an efficient lipid-lowering agent. We discovered that liver ciHL expression promotes liver fat accumulation. The fat accumulation is enhanced by feeding a Western diet and confounds the beneficial cholesterol-lowering properties of ciHL by inducing hepatic steatosis.


*    Acknowledgments
 
This study was supported by National Institutes of Health Grant RO1HL-69775 (H.L.D.) and by the J. David Gladstone Institutes.

Received April 27, 2004; accepted June 4, 2004.


*    References
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 *References
 

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