Effect of apolipoprotein E and A-IV phenotypes on the low density lipoprotein response to HMG CoA reductase inhibitor therapy

ATHEROSCLEROSIS Atherosclerosis 113 (1995) 157-166

Effect of apolipoprotein E and A-IV phenotypes on the low density lipoprotein response to HMG CoA reductase inhibitor therapy Jose M. Ordovas*a, Jose Lopez-Mirandaa, Francisco Perez-Jimenez”, Carmen Rodrigueza, Jong-Soon Parkb, Thomas Colec, Ernst J. Schaefer” ‘Lipid

Metabolism

Laboratory.

USDA

Human

Nutrition Research Center Boston, MA, USA bBristol-Myers Squibb, Princeton, c Washington University School of Medicine,

on Aging at TuJts University, NJ, USA St. Louis, MO,

711, Washington

St.,

USA

Received 8 August 1994; revision received 8 September 1994; accepted 12 September 1994

Our purpose was to assess the effect of apolipoprotein (apo) E and apo A-IV isoformvariation on low densitylipoprotein (LDL) cholesterolloweringresponseto the HMG CoA reductaseinhibitor, pravastatin.Plasmasamples were obtainedfrom participants(apoE, n = 97; apoA-IV, n = 144) in the PLAC-I (PravastatinLimitation of Atherosclerosis in Coronary Arteries Study-l). The mean LDL cholesterolreduction in thesesubjectswho were randomizedto pravastatin40 mg/day was28%. Subjectswith the APOE* allele(n = 12) had significantly (P = 0.04) greaterreductions at 36%than subjectshomozygousfor the APOE* allele(n = 66, 27%)or thosewith the APOE* allele(n = 19, 26%).No significanteffect of apo A-IV phenotypeon LDL cholesterolloweringin response to pravastatinwasnoted. A meta-analysis utilizing publisheddata from 4 previouslypublishedstudiesaswell asour own datawith a total sample sizeof 625subjectswascarriedout. This analysisindicatesthat the presenceof the APOE* allelewasassociated with a significantlygreater(P c 0.05) LDL-cholesterolloweringresponseat 37%than thosesubjectshomozygousfor the APOE* alleleat 35%,while thosewith theAPOE* allelehad a significantlylower response (P < 0.05),at 33%.These data are consistentwith the conceptthat apo E phenotypemodulatesthe LDL cholesterolloweringresponse observed with the useof HMG CoA reductaseinhibitors.

Keywords:Apolipopoprotein A-IV; Apolipoprotein E; Geneticpolymorphism;Low densitylipoproteins;Drug therapy; HMO CoA reductaseinhibitors; Pravastatin

1. Introduction There is an increased awareness in Western societies of the need for action to lower plasma * Corresponding author. Tel.: 617 556 3102; Fax: 617 556

3103.

total cholesterol and low density lipoprotein (LDL) cholesterol levels in order to decrease the high prevalence of coronary heart disease (CHD) in the population. To achieve this purpose, the current U.S. recommendations for the general population are to limit dietary fat to ~30% of calories, saturated fat to < lO%, and cholesterol to

0021-9150/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 002 l-9 150(94)05439-P

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s 300 mg/day (NCEP Step 1). In subjects who are still hypercholesterolemic on this diet, further restriction of saturated fat to <7% of calories and cholesterol to <200 mg/day (NCEP Step 2) is recommended prior to use of drug therapy [ 11. Candidates for drug therapy after diet treatment are those with LDL cholesterol values at or above 190 mg/dl, 160 mg/dl in the presence of two or more CHD risk factors, or 130 mg/dl in the presence of CHD [ 11. Pravastatin is an effective hypocholesterolemic agent that belongs to the new class of cholesterollowering agents known as HMG CoA reductase inhibitors, which inhibit HMG CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. This inhibition causes a decrease in intracellular cholesterol levels, resulting in upregulation of LDL receptors, enhanced LDL catabolism, and decreased plasma LDL cholesterol levels. These agents may also decrease very low density lipoprotein (VLDL) and LDL cholesterol by decreasing production (2,3]. The response to therapy with hypolipidemic agents shows considerable individual variation. These differences may be due to the interaction of environmental and genetic factors that affect drug bioavailability, receptor function or ligand structure. Two apolipoprotein gene loci, APOB [4] and APOE (4-101, have been studied in this regard, and based on previous studies, genetic variability at the APOE gene locus may predict some of the individual variability in response to hypolipidemic drugs. Other common apolipoprotein polymorphisms, such as the Glu to His substitution of amino acid 360 on apolipoprotein A-IV [l 11, have not been previously explored in this regard. Genetic variation at the APOE locus has been shown to be associated with alterations in lipoprotein metabolism [ 12,131. Apo E in serum is associated with chylomicrons, VLDL, and high density lipoproteins (HDL), and serves as a ligand for the LDL receptor and the LDL receptor-related protein (LRP) 114,151. When apo E deficiency is present, there is marked accumulation of cholesterol-enriched lipoproteins of density < 1.006 g/ml containing apo B-48 and apo A-IV, as well as apo B-100, and a markedly delayed clearance of apo B48 and apo B-100 within this lipoprotein class [ 161.

These data support the concept that apo E is important for the clearance of these lipoproteins [ 161. Genetic variation at the APOE locus results from three common alleles in the population, APOE*2, APGE*3 and APOE*4, with frequencies in Caucasian populations of approximately 0.08, 0.77, and 0.15, respectively [12]. These alleles code for the three major isoforms, apo E*2, apo E*3 and apo E*4, resulting in six different phenotypes (3/3, 3/4, 312, 414, 2l4, 212). Population studies have shown that plasma cholesterol, LDL cholesterol and apo B are highest in subjects carrying the apo E*4 isoform, intermediate in those with the apo E*3 isoform and lowest in those with the apo E*2 isoform [17-221. Genetic polymorphism for apo A-IV has been detected in humans and in other mammalian species [23-251. The effect of apo A-IV genetic variation on plasma lipid levels has been studied in several populations [26-301. In general, the APOA4*2 allele has been associated in Caucasians with higher levels of HDL cholesterol and/or lower triglyceride levels [30-321, although these findings are not unanimous [26,33-361. The precise function of apo A-IV is still unknown, its intestinal origin and the experimental evidence from a rare genetic mutation suggests a role in dietary fat absorption and chylomicron synthesis [37]. In vitro studies have shown that the activation of lipoprotein lipase by apo C-II is mediated by apo A-IV [38], and that apo A-IV serves as an activator of 1ecithin:cholesterol acyltransferase (LCAT) [39, 401. Apo A-IV-containing lipoproteins promote cholesterol efflux from cultured libroblasts [41] and adipose cells in vitro [42], and there is evidence showing that apo A-IV may be one of the ligands for the putative HDL receptor [42,43]. Therefore, the data suggest that apo A-IV plays an important role in fat absorption and reverse cholesterol transport. Apo A-IV polymorphisms have been documented to affect the response of LDL cholesterol to dietary intervention [44,45], but nothing has been reported about their association with drug responsiveness. In the present study, we have examined the impact of these polymorphisms on plasma lipid and lipoprotein response to treatment with pravastatin in subjects with moderate hypercholesterolemia,

J. M. Ordovas

et al. /Atherosclerosis

not consistent with the diagnosis of heterozygous or homozygous familial hypercholesterolemia. 2. MetWs

113 (1995)

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159

the third examination cycle of the Framingham Offspring Study 1471. Apo A-IV phenotype in the normal population was derived from 153 subjects who participated in dietary intervention studies as previously described [45].

2.1. Subjects and study design

Apolipoprotein E isoforms were determined in 97 randomly selected subjects (87 males and 10 females, mean age 56.8 years), participants in the pravastatin arm of the PLAC I study [46]. Apolipoprotein A-IV isoforms were determined in 144 subjects participating in this study. PLAC I was a randomized, double-blind, placebo-controlled, multicenter trial. Subjects undergoing baseline coronary angiography were screened for inclusion. Men and postmenopausal or surgically sterile women <75 years old were eligible for enrollment into the study if they met the following criteria: recent coronary angiography after either myocardial infarction or at the time of percutaneous transluminal coronary angiography (PTCA), provided that the angiography did not reveal only normal coronary arteries; or undergoing diagnostic coronary angiography for chronic or unstable angina that revealed at least one angiographically documented stenosis >50% in a major coronary artery; serum LDL cholesterol > 130 and < 190 mg/dl and triglycerides < 350 mg/dl. Subjects with any of the following conditions were excluded: uncontrolled hypertension, endocrine disease, type III hyperlipoproteinemia, congestive heart failure, debilitating noncardiac chronic disease, significant renal or hepatic disease, chronic pancreatitis, dysproteinemia, porphyria, lupus erythematosus, poorly controlled or insulin-dependent diabetes mellitus, likelihood of coronary artery bypass graft surgery or PTCA to the qualifying coronary artery within 6 months, history of cerebrovascular disease, significant gastrointestinal disease, excessive ethanol consumption, hypersensitivity to HMG CoA reductase inhibitors, treatment with corticosteroids, estrogens, androgens, fish oil, barbiturates, antiacids or other lipid altering drugs. Following dietary assessment and counseling, subjects who qualified were randomized to either pravastatin 40 mg qd or to placebo. APOE allelic frequency in the normal population was assessed in 2457 subjects participating in

2.2. Laboratory

methods

Blood samples were collected in 0.1% EDTA. Plasma was isolated by centrifugation at 2500 rev./mm at 4°C for 20 min. Total cholesterol and triglycerides were determined at the Central Laboratory (Lipid Research Center, Washington University School of Medicine, St. Louis, MO) using enzymatic reagents [48]. Assays were standardized through participation in the Centers for Disease Control-National Heart, Lung, and Blood Institute Standardization program. ApoA-IV and apo E phenotyping were carried out by isolectric focusing of whole plasma followed by immunoblotting as previously described using specific anti-human apo A-IV and apo E antisera [31]. 2.3. Statistical analyses

The SAS statistical program (SAS Institute, Cary, NC) was used to perform statistical analyses. One-way analysis of covariance (ANCOVA) was utilized to analyze the effect of apo E and apo A-IV isoforms on LDL cholesterol lowering. ANCOVA was also used to adjust for baseline lipid values since degree of lipid-lowering effect is highly correlated with baseline lipid level. Z-score analysis of pooled data from previously published studies as well as our own data [4,6-81 was carried out as previously described [49]. 3. Results 3.1. APOE and APOA4 allele distribution

The relative frequencies of the APGE*2, E*3 and E*4 alleles were 0.070, 0.793 and 0.137, respectively. No significant differences were observed between the expected frequencies derived from the Framingham Offspring Study control population [47] (E*2, 0.08; E*3, 0.78; E*4, 0.14) and observed values (x2 = 0.865, P = 0.649). The relative frequencies of the APOA4*1 and APOA4*2 alleles were 0.946 and 0.054, respective-

160

J.M. Ordovas et al. /Atherosclerosis 113 (1995) 157-146

ly. The frequency of the APOA4*2 allele was lower than expected [45] (0.19; x2 = 3.744); however this difference was not statistically significant (P = 0.053). 3.2. Effect of apo E and apo A-Wpolymorphisms on baseline lipid concentrations and response to pravastatin

Both men and women showed a similar magnitude of response to pravastatin treatment in each apo E subgroup and they were analyzed together in terms of responsiveness. There were significant differences in baseline LDL cholesterol concentrations between subjects carrying the APOE* allele (E2/2 and E2/3 phenotypes) (154 mg/dl) and those carrying the APOE* allele (E3/4 and E4/4 phenotypes) (171 mg/dl, P = 0.01) or those homozygous for the APOE* allele (166 mg/dl, P = 0.03). Total cholesterol showed a similar trend, although the differences were not statistically significant. No baseline differences were found among apo E phenotype subgroups in HDL cholesterol and triglyceride concentrations (Table 1). Table 1 shows that subjects carrying the APOE* allele were more responsive in terms of cholesterol reduction following treatment with pravastatin (-25%) than APOE*3/APOE*3 subjects (-18%, P = 0.03) and APOE* subjects (-18%, P = 0.06). A similar effect was also observed for LDL cholesterol; APOE* subjects showed a 36% decrease from baseline compared with 27% decrease for APOE*3/APOE*3 (P = 0.04) and 26% decrease for APOE* subjects (P = 0.04). No statistically significant difference was found between APOE* and APOE* subjects. No differences were observed between any pair of APOE group comparisons with regard to HDL cholesterol and triglyceride alterations in response to pravastatin. ApoA-IV polymorphism was not associated with different lipid or lipoprotein levels or response to pravastatin in this population (Table 2). We carried out a meta-analysis by pooling the data from this and previous studies examining the effect of apo E phenotypes on the response to HMG CoA reductase inhibitor therapy [4,6-81, including a total of 625 subjects (183, APOE*4; 384, APOE*3/APOE*3 and 58 APOE*2). Fig. 1

shows percent LDL cholesterol reduction in all studies combined by APOE allele. There is a gradual increase in response from APOE* (including apo E3/4 and apo E4/4 phenotypes) to APOE* (apo E3/3) and APOE* (apo E2/3 and apo E2/2). Fig. 2 shows the Z-score analysis of the percent difference between the APOE* and APOE* subgroups and the APOE*3/APOE*3 subgroup. APOE* carriers were significantly more responsive than APOE*3/APOE*3 subjects (P < 0.05), whereas the presence of the APOE* allele was associated with a significantly lesser response (P c 0.05). 4. Discussion In population studies, a significant difference in plasma total cholesterol and LDL cholesterol concentrations has been observed among the different apo E phenotype groups. The APOE* allele has been associated with the highest concentrations, while the APOE* allele has been associated with the lowest levels of these parameters. Several studies have addressed the effect of genetic variation at the APOE locus on the response to changes in dietary fat and cholesterol [SO-551. In some of these studies, the APOE* allele has been associated with increased response of plasma total cholesterol and LDL cholesterol to diet therapy [55,56], but not in others [57,58]. The effect of apo E phenotype on the variability of plasma lipid response to drug therapy has also been examined for probucol [9,10] and for other HMG CoA reductase inhibitors [4,6-81. The interaction reported between APOE genetic variation and different types of hypocholesterolemit therapy may be explained in part using the following model derived from metabolic studies [12,13]: VLDL and chylomicrons from subjects carrying the apo E*4 isoform are apo E-enriched, resulting in enhanced binding to the hepatic B/E receptor and possibly the chylomicron remnant receptor, with enhanced uptake of lipoprotein remnants, resulting in an increased intracellular pool of cholesterol. Down-regulation of the enzyme HMG CoA reductase and of the hepatic LDL receptor and elevation of serum LDL cholesterol levels occurs as a consequence of these ef-

cholesterol

HDL

E*4 E*3 E*2 E*4 E*3 E*2 E*4 E*3 E’2 E*4 E*3 E*2

Allele

concentrations

I9 66 I2 I9 66 I2 I9 66 12 19 66 12

n

according

I93 192 171 127 122 I04 40.5 45.7 39.2 164 157 177

24 23 22 19** 16 20*** 7.5 8.5 8.4 70 75 58

238 235 223 171 I66 154 39.4 41.4 38.2 170 174 190

ct f * f f f * f f f f A

Post

zt 25 * 26 f 18 f 26 zt 23 f I8 ZIZ 6.2 f 10.8 zt 7.6 f 125 f 81 * 88

E phenotypes

Pre

to the apolipoprotein

-18.2 - 18.4 -25.0 -25.6 -27.4 -35.7 2.88 9.84 2.02 -I 1.9 -12.2 -9.2

(9%

A4*llA4+1 A4*2

I

I

I

according

125 I7

I26 18 125 I7 I25 17

n

to apolipoprotein

All data are presented as mean f SD. aC1, confidence intervals; UL, upper limit; LL, lower limit. bA4*2 includes APDA4*2/APOA4*1 and APOA4*2/APDA4*2 *Difference between pre- and postreatment periods statistically

cholesterol

HDL

Triglycerides

cholesterol

LDL

A4* l/A4* A4*2 A4+ l/A4* A4*2 A4* l/A4* A4*2

concentrations

Plasma cholesterol

Table 2 Lipid and lipoprotein

f f f f + *

----

subjects. significant,

-..-

--

25 29 23 24 9.7 Il.6 84 66

---

-

.

(95%

=

0.773

0.115 0.969

0.091

0.953 0.033 0.612 0.043

E*3

CI,

_

_-_^

treatment

0.827 0.773 -

0.877 0.115 -

0.040 0.043 -

0.062 0.033 -

E*2

(40 mg qd)

P-value

(40 mg qd)

c

0.117

0.264 0.494 0.249

_.___

A4* 1/A4* 1

Pairwise

treatment

0.249 0.117

0.494

0.264

A4*2*

different

b

from

^

E3;

apo E2/3 and apo E2/2 subjects.

P-value

of pravastatin

P = 0.033; ***Significantly

(-20.9k17.5)’ (-26.0/-17.4)* (-30.2/-35.9)* (-35.9/-24. I)* (3.7/9.0)* (3.6/18.8)* (-15.9/-4.6)* (-34.7/-8.1)*

-19.20 -21.9 -28. I -30.2 6.30 12.3 -10.4 -22.5

f f f f * f f f

E2,

of pravastatin

from

189 182 121 I16 43.9 44.7 151 127

I2 month

different

% Change LL/UL)”

and after

0.969 0.827

0.091 0.877

0.612 0.040

0.953 0.062

E*4

Pairwise

I2 months

apo E3/3 and apo E*2 includes

Post

before

P < 0.05.

23 26 I8 I8 8.7 10.2

phenotypes

163 zt 66 I63 zt 80

233 231 I66 I65 41.2 40.0

Pre

A-IV

and after

CI, LUUL)”

(-22.7/-13.5)* (-20.8/-l 5.9)* (-30.2/-l9.5)* (-31.6/-l9.1)* (-30.6/-24. I)’ (-42.3/-28.3); (-3.8/10.0) (6.0/13.9)* (-6.2/l 1.0) (-25.8/4.6) (-19.9k3.8)’ (-26.9/12.8)

% Change

in males and females before

All data are presented as mean * SD. E*4 includes apo E3/4 and apo E4/4 subjects, E*3 includes %, confidence intervals; UL, upper limit; LL, lower limit. *Difference between pre- and postreatment periods statistically significant, P < 0.05; **Significantly P=O.Oll.

Triglycerides

cholesterol

LDL

Plasma cholesterol

Table 1 Lipid and lipoprotein

7.

162

J.M. Ordovas et al. /Atherosclerosis 113 (1995) 157-166 E”3

It.2

n=384

n-58

i

Fig. 1. Percent changes in plasma LDL cholesterol levels in subjects on HMG CoA reductase inhibitor therapy according to APOE allele (males and females, data from 5 studies).

fects. Conversely, apo E*2 has been associated with delayed clearance of remnants resulting in accumulation of remnants in the serum and a decreased conversion of VLDL to LDL. Subjects with the apo E2/2 phenotype are more likely to have dysbetalipoproteinemia (591. The resulting decrease in remnant uptake by subjects carrying

the apo E*2 isoform results in a lowering of the hepatic free cholesterol content and upregulation of hepatic HMG CoA reductase and the LDL receptor activity. These changes lead to an increase in the removal of circulating LDL. In addition to effects on lipoprotein clearance, subjects with different apo E phenotypes have been

0.6 I

1 TOTAL NUMBER

APOE*

AWE.2

-0.6 --

-0.0 -

J

Fig. 2. Standardized difference between percent LDL cholesterol reduction in APOE*U+ (APOE*4/APOE*4 and APOE*3/APOE*4) and APOE*2/+ (APOE*Z/APOE*2 and APOE*2/APOE*3) subgroups and the corresponding value of the APOE*3/APOE*3 subgroup (data from five studies).

J.M.

Ordovas

et al. /Atherosclerosis

reported in some studies to have variable efficiencies of intestinal cholesterol absorption [60]. In the present study, baseline levels of LDL cholesterol were the highest in apo E*4 carriers and lowest in apo E*2 subjects, but only the latter difference was statistically significantly different from apo E+3/E*3 subjects. No significant differences were reported in baseline LDL cholesterol for the different apo E subgroups in previous clinical studies using HMG CoA reductase inhibitors [4,6-81. This may be due to the fact that in previous studies most of the subjects were familial hypercholesterolemia (FH) heterozygotes; it is conceivable that defects at the LDL receptor locus may obscure the smaller LDL cholesterol raising effect associated in population studies with the apo E*4 isoform. Another explanation is that subjects participating in clinical studies were already on lipid lowering diets. Because the APOE* allele is associated with greater response to lipidlowering diets, it is possible that diet therapy eliminated most of the pre-existent difference that may have been present as a result of apo E genetic variation [55,56]. In the present study, subjects carrying the APOE* allele were more responsive to pravastatin in terms of LDL cholesterol lowering than subjects in other apo E subgroups. These results are consistent with the theoretical model previously discussed, indicating that subjects with the APOE* allele may have higher hepatic HMG CoA reductase activity which may be susceptible to greater inhibition by pravastatin. This effect will result in a greater decrease in cholesterol biosynthesis and greater upregulation of LDL receptor activity. According to the same model, the response to pravastatin could be impaired in subjects who carry the APOE* allele. The accelerated clearance of apo E*4-containing particles will result in hepatic cholesterol loading and suppression of HMG CoA reductase activity, which may result in a blunted response to pravastatin. We and others have previously noted that subjects with the APOE* allele are more responsive to diet therapy than those carrying the APOE* allele or those homoxygous for the APOE* allele [55,56,61]. Because the subjects studied here already had been placed on lipid lowering diets, it is

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possible that the drug effect may have been attenuated in APOE* subjects as a result of the diet effect. Only one of the previous studies shows a significant interaction between APOE genetic variation and LDL cholesterol response to HMG CoA reductase inhibitors [6], while a non-significant trend was noted in other studies [4,7,8]. The metaanalysis of the combined data shows a significant difference in response associated with the APOE locus, with subjects carrying the APOE* allele having a significantly lower response, and those carrying the APOE* allele having a significantly greater response to treatment. An interaction between APOA4 genetic variation and response to drug therapy has not been previously reported. In this study we have not detected any significant differences in LDL cholesterol lowering response to pravastatin associated with apo A-IV polymorphisms. This data suggests that the mechanisms associated with the previously shown hyporesponsiveness of the apo A4*2 isoform to diet therapy may be independent of those related to drug therapy [44,45]. In conclusion, the data presented indicate that apo E genetic variation has a significant impact on the lowering of LDL cholesterol in response to drug therapy using HMG CoA reductase inhibitors, with APOE* carriers being more responsive than APOE* carriers or APOE*3/ APOE* subjects. In addition, a meta analysis, based on all data available in the literature, indicates that this response follows a gradient with APOE* subjects being the least responsive, APOE*3/APOE*3 subjects having an intermediate response, and APOE* subjects being the most responsive with regard to LDL cholesterol lowering with HMG CoA reductase inhibitors. Acknowkdgements This work was supported by NIH grant HL39326 from the National Institutes of Health and contract 53-3KO6-5-10 from the USDA Department of Agriculture Research Service. Dr. Lopez-Miranda was supported by a fellowship from the Spanish Ministry of Health, Madrid, Spain.

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