23

Effects of Estrogen and Progesterone onPlasma Lipoproteins and Experimental

Atherosclerosis in the Baboon (Papio sp.)Rampratap S. Kushwaha, Douglas S. Lewis, K. Dee Carey, and Henry C. McGill Jr.

We determined the effect of estrogen and progesterone on plasma cholesterol and lipoproteincholesterol concentrations and on arterial lesions in 24 ovariectomized and hysterectomizedbaboons fed a high-cholesterol/high-saturated-fat diet These baboons were divided into fourgroups: untreated control (C); estrogen, 100 /tg/kg/week injected l.m. (E); progesterone, 3mg/kg/day (P); and estrogen plus progesterone (E+P). The treatment regimen continued for 18months. Cholesterol levels in plasma and lipoproteins were measured before hormonetreatment and at 3, 10, and 18 months of treatment Postheparin plasma lipoprotein lipase(LPL) activity was also measured during the treatment After 18 months of hormone treatment,baboons were necropsied and arterial lesions were measured. Hormone treatment significantlyinfluenced plasma cholesterol (P>C>[E+P]>E) and very low density lipoprotein plus lowdensity lipoprotein (VLDL+LDL) cholesterol (P>C> [E+P] >E), with very little effect on highdensity lipoprotein (HDL) cholesterol concentration. The E+P group had a significantly higherHDL cholesterol concentration than did the P group. The (VLDL+LDL)/HDL cholesterolratios in the E and E+P groups were significantly lower than those in the P and C groups. LPLactivities were significantly lower in the E group compared with those in the E+P and P groups.Hormone treatment significantly influenced lesions in four (innominate, carotid, iliac, andabdominal aorta) of seven arteries. The P group had the most fatty streaks, and the E+P grouphad the least Multiple regression analysis suggested that hormone treatment influencedarterial lesions, both through effects on VLDL+LDL cholesterol and (VLDL+LDL)/HDLcholesterol ratio and through an effect independent of lipoproteins. {Arteriosclerosis andThrombosis 1991;11:23-31)

It is well known that premenopausal women haveless atherosclerosis and a lower incidence ofcoronary heart disease than do men and that the

incidence of coronary heart disease in women increasesafter natural or surgical menopause.1-2 Premenopausalwomen who have had bilateral ovariectomy demon-strate a higher incidence of myocardial infarction un-less estrogen therapy is begun immediately after theovariectomy.3'4 These associations have led to the con-cept that estrogen may protect against atherosclerosisand coronary heart disease. A number of studies inexperimental animals have shown a hypolipidemic ef-fect of estrogen.5-7 Our studies in cholesterol-fed rab-

From the Department of Physiology and Medicine, SouthwestFoundation for Biomedical Research, San Antonio, Tex.

Supported by grants HL-34982, HL-28972, and HL-41256 andcontract No. HV-53030 from the National Institutes of Health,Bethesda, Md.

Address for reprints: Rampratap S. Kushwaha, PhD, Depart-ment of Physiology and Medicine, Southwest Foundation forBiomedical Research, PO Box 28147, San Antonio, TX 78228-0147.

Received March 30, 1990; revision accepted June 22, 1990.

bits suggested that the hypolipidemic effect of estrogenwas mediated by a decrease in very low (VLDL) andintermediate (IDL) density lipoproteins.8 Estrogentreatment enhanced the uptake of cholesterol-richVLDL by isolated, perfused rabbit livers9 due to anincrease in the activity of the apolipoprotein (apo) B/Ereceptor.10-12 In human subjects with type III hyperli-poproteinemia, estrogen exerted a hypolipidemic effectand normalized lipoprotein levels by accelerating theremoval of cholesterol-rich VLDL.13 Estrogen treat-ment in premenopausal women increased apo A-I butdecreased hepatic lipase activity.14 Premenopausalwomen had lower levels of low density lipoproteins(LDL) and higher levels of high density lipoproteins(HDL) than did men at all ages beyond puberty.13

Thus, it appears that estrogen given alone produces anantiatherogenic lipoprotein profile. In contrast, oralcontraceptives containing a low dose of estrogen com-bined with a medium or high dose of progestins pro-duce the opposite effect,16-17 an observation suggestingthat progestin-containing agents produce an athero-genic lipoprotein pattern. The effects of estrogen and

by guest on May 6, 2018

http://atvb.ahajournals.org/D

ownloaded from

24 Arteriosclerosis and Thrombosis Vol 11, No 1, January/February 1991

TABLE 1. Compositions of Chow and High-Cholesterol/High-FatDiets

Nutrients

Carbohydrates (% cal)Protein (% cal)Fat (% cal)Energy (kcal/100 g diet)Cholesterol (mg/kcal)

Chow diet

622810

3290.03

HCHF diet*

402040

3771.7

HCHF, high-cholesterol/high-fat.*HCHF diet was prepared by mixing 81.4% (dry wt basis) of

Purina monkey meal 5-5045-6 (a special mix with no added fat,dehydrated alfalfa, sodium chloride, ascorbic acid, or retinylacetate) with lard (16.5%), sodium chloride (1.1%), retinyl acetate(0.005%), ascorbic acetate (0.2%), and cholesterol (0.74%).

progesterone on atherosclerosis may be mediated bytheir effects on lipoprotein levels and metabolism. Inaddition, estrogen and progesterone may directly affectthe arterial wall18-21 through estrogen and progester-one receptors.22-24 The present studies were conductedto determine if estrogen and progesterone at pharma-cological doses affect diet-induced atherosclerosis inbaboons and whether this effect is mediated by or isindependent of their effects on plasma lipoproteins.

MethodsAnimals and Diet

Twenty-four adult female baboons (Papio sp.),ranging from 4 to 9 years of age and with averageplasma and lipoprotein cholesterol responses to ahigh-cholesterol/high-saturated-fat (HCHF) diet (7weeks), were selected for these studies. Baboonsused for these studies had approximately twofoldincreases in plasma LDL levels and did not haveHDI^ and lipoprotein(a) (Lp[a]) in their plasma. Allthe animals were ovariectomized and hysterecto-mized after being maintained on the HCHF diet for8 weeks. After surgery, baboons were allowed torecover for 4 weeks after which they were started onhormone treatments. Baboons were maintained onthe HCHF diet during the recovery period and for theduration of hormone treatment. The composition ofthe diet was similar to that described earlier25 and isgiven in Table 1. The source of the saturated fat,which provided 40% of the total calories, was lard, andthe cholesterol content of the diet was 1.7 mg/kcal.

Experimental Design

The experiment was conducted in two blocks.Baboons (n = 12) in each block were divided ran-domly into four treatment gTOups: untreated con-trol (C), estrogen (E), progesterone (P), and estro-gen plus progesterone (E+P). Each treatmentgroup in a block consisted of three baboons, for atotal of six baboons in each treatment group. Theuntreated control group received placebo (a fig barorally and cottonseed oil, the estrogen vehicle,injected i.m.). The E group was treated with £-es-tradiol 17-cypionate (The Upjohn Co., Kalamazoo,Mich.) in cottonseed oil, which was injected i.m.,

and was fed a fig bar daily. The P group receivedprogesterone (Sigma Chemical Co., St. Louis, Mo.)orally in a fig bar and was injected with the estradiolvehicle weekly. The E+P group received a combi-nation of /3-estradiol 17-cypionate and progester-one at the same doses that were given to theseparate E and P groups. In the first block, estrogenwas administered at a dosage of 100 /ig/kg the firstweek and was increased by 50 ^tg/kg/week until thebaboons were stabilized at a dose of 200 fig/kg/week. The progesterone in the first block wasstarted at 4 mg/kg/day and was raised to 6 mg/kg/day over a period of 6 weeks. After 7 months, thebaboons in the first block were left untreated for 4.5months to reduce the inflammation of their sex skin.Treatments were then resumed at reduced levels:estrogen at 100 jig/kg/week and progesterone at 3mg/kg/day. Baboons were treated for 3 weeks andthen taken off treatment for 1 week as describedpreviously.26 This cycle of treatment allowed the sexskin of the baboons to become less inflamed duringthe off-treatment period. The changes in hormonedoses of block 1 baboons were made to determinethe optimal doses of estrogen and progesterone.The hormone treatment was given to these baboonsfor a total of 18 months. The final doses of estrogenand progesterone in the first block and the sametreatment schedule were used for baboons in thesecond block. The second block of baboons was alsotreated with hormones for 18 months.

Lipid and apoprotein levels in plasma and lipopro-teins were measured periodically.27 Estrogen andprogesterone levels were also measured by radioim-munoassay (Radioassay Systems Labs, Inc., Carson,Calif.). As reported earlier,26 baboons in the E andE+P groups had similar levels of estradiol in theplasma, which were 10-fold higher than in the Cgroup (7.6±1.5 pg/ml in C versus 73.2±9.9 pg/ml inthe E group). Similarly, progesterone-treated ba-boons had a twofold increase (16.8±2.5 ng/dl in theC group versus 36.4±1.3 ng/dl in the P group) inplasma progesterone levels.26 After 18 months ofhormone treatment, baboons were euthanized andnecropsied, and arterial lesions were measured.

The protocol for this experiment was approved bythe Animal Research Committee of the SouthwestFoundation for Biomedical Research. The SouthwestFoundation is accredited by the American Associationfor Accreditation of Laboratory Animal Care and isregistered with the US Department of Agriculture.

Separation of Plasma Lipoproteins

Analysis of lipoproteins was performed at 3, 10,and 18 months after beginning the hormone treat-ment. Blood (10 ml) was collected in tubes contain-ing EDTA (1 mg/ml), and plasma was obtained bylow-speed centrifugation at 6°C. The plasma wastreated with sodium azide (2 mg/dl), chloramphen-icol (0.5 mg/dl), gentamycin sulfate (1 mg/dl), andphenylmethylsulfonyl fluoride (0.05 mmol/dl). Theplasma (2 ml), adjusted to a density of 1.30 g/ml,

by guest on May 6, 2018

http://atvb.ahajournals.org/D

ownloaded from

Kushwaha et al Sex Steroid Hormones and Atherosclerosis 25

was layered with solutions of different densities,from 1.21 to 1.006 g/ml, in SW41 Ti rotor tubes asdescribed earlier.28 The plasma was then ultracen-trifuged in an SW41 Ti rotor with a Beckmanultracentrifuge Model L8-70 or L8-70M (fromBeckman Instruments Inc., Palo Alto, Calif.) at39,000 rpm (175,000g) for 24 hours at 6°C. Afterultracentrifugation, the tube contents were frac-tionated by puncturing the tube at the bottom asdescribed previously.28 On the basis of density,lipoprotein fractions were pooled to representVLDL+IDL (d<1.019 g/ml), LDL (d=1.019-1.050g/ml), and HDL (d=1.050-1.21 g/ml).

Cholesterol Quantification in Plasmaand Lipoproteins

Cholesterol in plasma and lipoproteins was mea-sured by enzymatic method assay kits (Sigma).

Collection of Postheparinized Plasma and Assay ofLipoprotein Lipase Activity

Fasted baboons were injected with heparin derivedfrom porcine intestine (Elkins-Sinn, Inc., CherryHill, N.J.), 50 units/kg body wt, into the femoral vein.At 10 minutes after heparin injection, a 5.0-ml bloodsample was taken, placed on ice, and centrifuged torecover the plasma.

Postheparin plasma lipoprotein lipase activity wasassayed by a glycerol-based triglyceride substrate asdescribed by Nilsson-Ehle and Schotz.29 Activity wasdetermined by incubating the enzyme preparationwith 1.0 M and 0.15 M sodium chloride with 0.1 mldiluted substrate (containing 0.008 ml heat-inacti-vated baboon serum, 0.017 ml of the gh/cerol tri[9,10(n)3H]oleate stock (Amersham, ArlingtonHeights, 111.), and 0.075 ml 0.2 M Tris with 3% bovineserum albumin [Miles, Scientific, Naperville, 111.], pH8.0). After 30 minutes at 37°C, released fatty acidswere extracted and measured as described by Nils-son-Ehle and Schotz.29 Enzyme activity was ex-pressed as micromoles of free fatty acid released perhour per milliliter of plasma. Under the assay condi-tions, virtually all (>95%) of the lipase activity wasinhibited with 1.0 M sodium chloride.

Necropsy Procedure

After 18 months on hormone treatment, the ba-boons were immobilized with ketamine hydrochlo-ride (10 mg/kg), anesthetized with pentothal, andexsanguinated. The aorta and the coronary, iliac-femoral, brachial, and carotid arteries were openedlongitudinally, fixed with the adventitia adherent tochip board in 10% buffered formalin, stained withSudan IV, and packaged in plastic bags.30

Grading of Atherosclerosis

Fatty streaks were defined as Sudan IV-stainedintimal areas that were elevated slightly or not at allabove the surrounding intimal surface. Fibrousplaques were defined as firm, distinctly elevatedareas, regardless of whether their intimal area was

stained for lipid. These definitions are similar tothose used for grading human lesions.30 One of thecoinvestigators (H.C.M.) estimated the percentage ofintimal surface involved with each type of lesion ineach of the following arterial segments: the aorticarch from the aortic valve to the ligamentum arterio-sus; the thoracic aorta from the ligamentum arterio-sus to the origin of the celiac artery; the abdominalaorta from the origin of the celiac artery to the iliacbifurcation; the entire innominate artery; the left andright carotid arteries from their origins to and includ-ing their trifurcations; the left and right brachialarteries from their origins to the branchings at theelbows; the left and right iliac-femoral arteries fromtheir origins to the branchings of the femoral arteriesat the knee; and the right, circumflex, and leftanterior descending coronary arteries. Values forpaired arteries were averaged for statistical analysis.

These evaluations were made independently foreach segment and in random order without knowl-edge of the treatment group from which the speci-men was derived. Independent regrading of all 24specimens by the same grader indicated an accept-able intraobserver variability. The intraclass correla-tion coefficients between the two grades were 0.839for the aortic arch, 0.866 for the innominate artery,0.918 for the brachial arteries, 0.948 for the carotidarteries, 0.860 for the iliac-femoral arteries, 0.899 forthe thoracic aorta, and 0.876 for the abdominal aorta.

All the lesions encountered were simple fattystreaks as delineated by Sudan IV staining, except forone small atypical plaque in the abdominal aorta ofone animal that was not considered a result of theexperimental treatment. The coronary arteries con-tained no lesions, and, therefore, no analyses arepresented for them.

Statistical Analyses

The main effects of hormone treatment on choles-terol concentrations in plasma, VLDL+LDL, andHDL, (VLDL+LDL)/HDL cholesterol ratio, andpostheparin lipolytic activity were analyzed sepa-rately by analysis of variance (ANOVA).31 ANOVAwith repeated measures, with pretreatment and 3months of treatment as the trial factors, was used todetermine the effect of initial hormone treatment onplasma and lipoprotein cholesterol. Multiple com-parisons of group means from equal sample sizeswere done with Duncan's multiple-range test,32 andmeans from unequal sample sizes were comparedwith Tukey's honestly significant difference test.33

Serum lipid concentrations and lipoprotein lipaseactivity were log transformed before analysis to bet-ter meet the distributional assumption that variancesare normally distributed among the treatment groupsfor the ANOVA.32 The assumption of normal distri-bution of variance among groups appeared not to beviolated after examination of residuals. All resultsfrom transformed data are presented as the meanplus and minus 95% confidence intervals.

by guest on May 6, 2018

http://atvb.ahajournals.org/D

ownloaded from

26 Arteriosclerosis and Thrombosis Vol 11, No 1, January/February 1991

TABLE 2. Mean Plasma and Lipoprotein Cholesterol Concentrations by Diet

Diet

Chow

HCHF

Significance level

n

24

24

Totalcholesterol

(mg/dl)

103

(97-110)

179

(167-191)

0.0009

VLDL+LDLcholesterol

(mg/dl)

36

(31-41)

67

(59-77)

0.0009

HDLcholesterol

(mg/dl)

65

(59-71)

108

(97-119)

0.0009

(VLDL+LDL)/HDLcholesterol

(ratio)

0.55

(0.45-0.67)

0.62

(0.52-0.76)

0.098

Values are expressed as mean, with 95% confidence intervals in parentheses. There were no significant differencesbetween treatment groups. See text for explanation of abbreviations.

The percentages of intimal surface area involvedwith fatty streaks were also analyzed by ANOVA. Anexamination of residuals of the variance of lesiondata indicated that variances appeared to be nor-mally distributed among all treatment groups, withthe exception of the abdominal aorta. The percent-ages of intimal surface area involved with fattystreaks in the abdominal aorta were transformedusing square roots to better meet the normal distri-bution assumptions for the ANOVA. Associations oflesions with steroid hormone treatment, VLDL+LDL cholesterol, and (VLDL+LDL)/HDL choles-terol ratio as predictor variables were analyzed bymultiple linear regression. We have reported effectsat p<0.1 to achieve a better balance between Type Iand Type II statistical errors.

ResultsEffect of Challenge Diet on Plasma and LipoproteinCholesterol Concentrations BeforeHormone Treatment

Plasma and lipoprotein cholesterol values for ba-boons on the chow and challenge diets before treat-ment are given in Table 2. Plasma cholesterol con-centration was increased by 75% after feeding theHCHF diet. There were similar increases in LDL andHDL cholesterol. The (VLDL+LDL)/HDL choles-terol ratio was not influenced by the challenge diet.There were no significant differences among thetreatment groups in plasma and lipoprotein choles-terol values before hormone treatment.

Effect of Initial Hormone Treatment on Plasma andLipoprotein Cholesterol Concentrations

The effects of 3 months of each hormone treat-ment on plasma and lipoprotein cholesterol concen-trations are described in Table 3. There was asignificant treatment by time (pretreatment and at 3months' treatment) interaction effect on plasma cho-lesterol, VLDL+LDL cholesterol, and HDL choles-terol concentrations and on the (VLDL+LDL)/HDLcholesterol ratio. Total plasma cholesterol decreasedsignificantly in baboons receiving estrogen and estro-gen plus progesterone therapy, whereas total plasmacholesterol did not change in baboons receivingprogesterone and placebo control. LDL cholesteroldecreased in both the E (not significant) and E+P(significant) groups but increased in C (significant)and P (not significant) groups. HDL cholesteroldeclined significantly in the E, E+P, and P groups.(VLDL+LDL)/HDL cholesterol ratios significantlyincreased in the C and P groups but not in the E andE+P groups.

Effect of Long-term Hormone ReplacementTherapy on Plasma and LipoproteinCholesterol Concentrations

The effects of 18 months of hormone replacementtherapy on mean plasma and lipoprotein cholesterolconcentrations are reported in Table 4. Plasma cho-lesterol concentrations in the E group were signifi-cantly lower than in the P group. The E group had

TABLE 3. Mean Plasma Cholesterol and Lipoprotein Cholesterol Levels After Initial Sex Hormone Replacement Therapy

Treatment

Control

Estrogen

Estrogen+

progesterone

Progesterone

n

6

6

6

6

Totalcholesterol (mg/dl)

Pretreatment

160

(143-180)

187

(152-231)

188

(166-211)

181

(155-211)

Posttreatment

166

(137-203)

139*

(108-178)

152*

(132-176)

184

(158-213)

VLDL+LDLcholesterol (mg/dl)

Pretreatment

59

(34-100)

68

(55-85)

70

(56-87)

73

(56-96)

Posttreatment

78*

(60-101)

59

(48-71)

51*

(39-67)

87

(57-133)

HDLcholesterol (mg/dl)

Pretreatment

93

(72-121)

117

(88-187)

101

(85-120)

105

(82-135)

Posttreatment

86

(68-109)

79*

(58-109)

87*

(74-102)

89*

(68-116)

LDL/HDLcholesterol (ratio)

Pretreatment

0.63

(0.3-1.33)

0.58

(0.42-0.81)

0.60

(0.46-0.77)

0.69

(0.45-1.06)

Posttreatment

0.93*

(0.67-1.24)

0.74

(0.61-0.90)

034

(0.4-0.73)

0.98*

(030-1.93)

*p<0.05 between pretreatment and posttreatment (3 months) values. 95% confidence intervals in parentheses. See text for explanationof abbreviations.

by guest on May 6, 2018

http://atvb.ahajournals.org/D

ownloaded from

Kushwaha et al Sex Steroid Hormones and Atherosclerosis 27

TABLE 4. Effect of Long-term Hormone Therapy on Plasma Cholesterol and Lipoprotein Cholesterol Concentrations

Treatment

Control

Estrogen

Estrogen +progesterone

Progesterone

n

6

6

6

6

Plasmacholesterol

(mg/dl)

161(133-195)

144*(129-160)

159(143-176)

171(160-184)

VLDL+LDLcholesterol

(mg/dl)

77(58-101)

55t(47-66)

60(46-77)

88§(68-114)

HDLcholesterol

(mg/dl)

80(62-104)

87(76-99)

98(88-108)

76 |(59-98)

(VLDL+LDL)/HDLcholesterol

(ratio)

0.95(034-1.40)

0.63*(0.52-0.77)

0.61(0.47-0.81)

1.16*(0.71-1.89)

95% confidence intervals in parentheses. See text for explanation of abbreviations.•Significantly different (p<0.05) from progesterone group.tSignificantty different (p<0.05) from control group.^Significantly different (p<0.05) from estrogen and estrogen+progesterone groups.§Significantly different (p<0.05) from estrogen and estrogen+progesterone groups.|Significantly different (p<0.06) from estrogen+progesterone group.

significantly lower VLDL+LDL cholesterol than didthe P and C groups. The E+P group also hadsignificantly lower VLDL+LDL cholesterol than theP group. The E+P group had a higher HDL choles-terol concentration than the P group. The(VLDL+LDL)/HDL cholesterol ratio was also influ-enced by the hormone treatment. The E+P grouphad a significantly lower (VLDL+LDL)/ HDL cho-lesterol ratio than did the P and C groups. The Egroup also had a (VLDL+LDL)/HDL cholesterolratio significantly different from P and C groups.

The changes in VLDL+LDL cholesterol and the(VLDL+LDL)/HDL cholesterol ratio during the ex-periment are summarized in Figures 1 and 2, respec-tively. During the treatment period (3-21 months),VLDL+LDL cholesterol and (VLDL+LDL)/HDL

S 12 19TIME (MONTHS)

F I G U R E 1. Line plot showing mean values forVLDL+LDL-C (mg/dl) as a function of time (months) inovariectomized and hysterectomized baboons in control (o),estrogen (•), estrogen plus progesterone (A), and progesterone(A) groups. Each group had six baboons. Mean valuesdenoted by arrows A and B are for the chow and high-cholesterol/high-fat diet, respectively, before ovariectomy andhysterectomy. Baboons were ovariectomized and hysterecto-mized and treated with hormones after the lipoprotein mea-surement denoted by arrow B. All baboons were maintainedon the high-cholesterol/high-fat diet throughout the hormonetreatment period. VLDL+LDL-C, very low plus low densitylipoprotein cholesterol

cholesterol ratio were analyzed for treatment effectwith time as a trial factor by repeated-measuresANOVA. There was no significant time-by-treatmentinteraction on VLDL+LDL cholesterol concentra-tions. In contrast to VLDL+LDL cholesterol, therewas a significant time-by-treatment interaction on(VLDL+LDL)/HDL cholesterol ratios. The P and Cgroups increased their (VLDL+LDL)/HDL choles-terol ratios during hormone treatment, but the E andE+P groups did not.

Effect of Hormone Replacement Therapy onPostheparin Plasma Lipoprotein Lipase Activity

The effect of hormone replacement therapy onpostheparin plasma lipoprotein lipase activity in dif-ferent treatment groups is reported in Table 5.Baboons treated with estrogen had significantly lowerplasma lipoprotein lipase activity than that in theE+P and P groups. Hepatic triglyceride lipase activ-ity measured at pH 8.8 and in the presence 1.0 M

0.000

F I G U R E 2. Line plot showing mean values for(VLDL +LDL)/HDL-C ratio in ovariectomized and hysterec-tomized baboons in control (o), estrogen (•), estrogen plusprogesterone (A), and progesterone (A) groups. Each grouphad six baboons. Details of the hormone treatments aredescribed in Figure 1. (VLDL+LDL)/HDL-C, ratio of verylow plus low density lipoprotein to high density lipoproteincholesterol

by guest on May 6, 2018

http://atvb.ahajournals.org/D

ownloaded from

28 Arteriosclerosis and Thrombosis Vol 11, No 1, January/February 1991

TABLE 5. Effect of Steroid Treatment on Postbeparinlzed PlasmaLJpoproteln LJpase Activity

LPL activityTreatment n (/imol FFA/hr/ml plasma)

ControlEstrogenEstrogen+progesteroneProgesterone

6665

21.114.630.9*31.3*

(14.6-30.5)(11.6-18.5)(19.5-49.1)(23.7-40.7)

LPL, lipoprotein lipase; FFA, free fatty acids.95% confidence intervals in parentheses.'Significantly different from estrogen-treated group atp<0.010.

sodium chloride was very low and did not differbetween treatment groups (data not presented).

Effect of Hormone Replacement Therapy onArterial Lesions

The percent of intimal surface area involved withlesions in major arteries is reported in Table 6.Hormone replacement therapy significantly influ-enced the extent of these lesions in the innominate,carotid, and iliac arteries and the abdominal aorta.The effects of hormone treatment on fatty streaks inthe brachial artery (/j=0.094) and aortic arch(/>=0.134) approached statistical significance. Thehighest percentage of surface involved by lesions wasobserved in baboons treated with progesteronealone. In contrast, the least extensive fatty streaksoccurred in baboons treated with a combination ofestrogen and progesterone. In the innominate artery,the surface area involved with fatty streaks wassignificantly more in the P group than in the othertreatment groups. The extent of lesions in the carotidartery in the E and E+P groups was significantly lessthan that in the C or P group. In the iliac artery, theextent of lesions was significantly higher in the Pgroup compared with the C and E+P groups. Ba-boons treated with estrogen and progesterone hadsignificantly less atherosclerosis in the abdominalaorta than did the P group. There were no measur-able lesions in the coronary arteries of baboons inany treatment group.

Regression Analysis of LesionsPlasma VLDL+LDL cholesterol was positively cor-

related with lesions in the innominate (/-=0.679,p=0.mi), carotid (r=0559, p=0.006), iliac (r=0.554,P=0.005), and brachial (r=0.506,/>=0.014) arteries andin the abdominal aorta (r=0.658, p=0.001). Similarly,the plasma (VLDL+LDL)/HDL cholesterol ratio waspositively associated with lesions in the innominate(r=0.691,p=0.009), carotid (r=0.465, p=0.025), iliac(r=0.583, />=0.003), and brachial (r=0398, p=0.06)arteries and in the abdominal aorta (r=0.702,/?=0.0009). To determine whether the observed effectsof hormone replacement therapy on lesions in theinnominate, brachial, carotid, and iliac arteries and inthe abdominal aorta (Table 6) resulted from the ob-served treatment effects on VLDL+LDL cholesteroland the (VLDL+LDL)/HDL cholesterol ratio, weanalyzed the extent of lesions by multiple linear regres-sion analysis. The (VLDL+LDL)/HDL cholesterolratio, when added with hormone treatment, signifi-cantly increased the prediction of lesions for the ab-dominal aorta (p<0.05) and for the innominate(p<0.025) and iliac (/J<0.025) arteries but not for thecarotid and brachial arteries. Hormone treatment,when added with (VLDL+LDL)/HDL cholesterol ra-tio, significantly increased the prediction of lesions inthe carotid (p<0.05), brachial (p<0.1), innominate(p<0.05), and iliac (p<0.1) arteries and the abdominalaorta (p<0.l). When adjusted for hormone therapy,the (VLDL+LDL)/HDL cholesterol ratio still wasassociated with lesions in the innominate (partial cor-relation coefficient=0.538, p=0.008) and iliac (partialcorrelation coefficient=0.524, /?=0.009) arteries andabdominal aorta (partial correlation coefficient=0.478,p=0.018) but not in the carotid (partial correlationcoefficient=0.185) and brachial (partial correlation co-efficient=0.199) arteries. Similar results were obtainedwith VLDL+LDL cholesterol as the predictor in placeof the (VLDL+LDL)/HDL cholesterol ratio.

DiscussionSummary of Results

The present study demonstrates that hormonetreatment in pharmacological doses influences the

TABLE 6. Mean Arterial Lesions After 18 Months of Hormone Treatment

Treatment

ControlEstrogenEstrogen+

progesteroneProgesteroneEffect of treatment

n

66

65

Aortic arch

4.7±1.210.2±4.5

2.7±1.211.6±3_5p=0.134

Innominate

9.0±2.24.3 + 1.3

3.3 ±1.318.0±3.7'p-0.001

Percent of surface area with

Arteries

Brachial

8.8±3.46.5±1.6

4.3±2.018.8±7Jp=0.094

Carotid

4.4±1.51.9±0.7

1.7±0.79.0±2.8tp=0.013

fatty streaks

Iliac

1.9±0.93.0±0.7

1.3±0.65.6±1.5tp=0.037

Thoracic

25.2±5.127.2±10.0

21.8±5.736.7±13.8p=0.705

Aorta

Abdominal

13.8±2.911.2±1.8

6.5±1.426.3±8J§p=0.035

Values are mean±SEM.'Significantly different from control, estrogen, and estrogen+progesterone groups atp<0.05.tSignificantly different from estrogen and estrogen+progesterone groups atp<0.05.^Significantly different from control and estrogen+progesterone group atp<0.05.{Significantly different from estrogen and estrogen+progesterone groups atp<0.05.

by guest on May 6, 2018

http://atvb.ahajournals.org/D

ownloaded from

Kushwaha et al Sex Steroid Hormones and Atherosclerosis 29

development of arterial lesions in ovariectomizedand hysterectomized baboons. The least extensivelesions were observed in the group treated with acombination of estrogen and progesterone. Proges-terone treatment alone, on the other hand, resultedin more extensive arterial lesions. During hormonetreatment, progesterone-treated baboons also hadgreater plasma cholesterol (P>C>[E + P]>E),VLDL+LDL cholesterol (P>C>[E+P]>E), and(VLDL + L D L ) / H D L c h o l e s t e r o l r a t i o(P>C>E>[E+P]), but lower HDL cholesterol(P<C<E<[E + P]) than the other treatmentgroups. Regression analysis suggested that hor-mone therapy influenced arterial lesions partly byaffecting VLDL+LDL cholesterol and the(VLDL+LDL)/HDL cholesterol ratio, but hor-mone therapy also affected lesions independently ofplasma lipoprotein levels.

Effect of Estrogen on Lipoprotein Levelsand Composition

The effect of estrogen treatment in this study wasmainly on VLDL + LDL cholesterol and(VLDL+LDL)/HDL cholesterol ratio. Estrogentreatment only slightly increased HDL cholesterolconcentrations. The minimal effect of estrogen treat-ment on HDL cholesterol concentration may be dueto the naturally occurring high concentration of HDLin the baboon. These results are consistent with thoseobserved in humans and experimental animals. Wal-lentin and Varenhorst34 reported that patients withprostatic carcinoma, who were treated daily withethinyl estradiol orally and with polyestradiol phos-phate i.m. once a month for 8 weeks, had lowerplasma cholesterol concentrations. The HDL choles-terol increased by 53%, and the LDL cholesteroldecreased by 25%. Krauss et al35 found a significantlyhigher level of HDL (predominately HDL2) in post-menopausal estrogen users compared with nonusers.Cauley et al36 also reported that menopausal womenusing estrogens had significantly higher total HDLand HDL2 cholesterol concentrations than did con-trols, without any difference in HDL3. Schaefer etal14 reported that estrogen-treated premenopausalwomen had increased plasma HDL2 cholesterol lev-els with no change in LDL levels. We found thatHDL increased while LDL decreased during estro-gen treatment of a normolipidemic postmenopausalwoman.37

A number of studies conducted in several animalspecies have shown a hypolipidemic effect of estro-gen. Moskowitz et al38 reported lower serum cho-lesterol levels and less-severe coronary atheroscle-rosis in estrogen-treated rats fed an atherogenicdiet. Similarly, Prichard et al5 have shown a lipid-lowering and antiatherogenic effect of estrogen incholesterol-fed, male, White Carneau pigeons.Chao et al6 and Davis and Roheim7 have reportedthat administration of large amounts of ethinylestradiol to male rats produced a profound hypo-lipidemia that involved all major lipoprotein

classes. Our studies in rabbits showed that thehypolipidemic effects of estrogen were mediated bya decrease in VLDL and IDL alone.8

Effect of Progesterone on Lipoprotein Levelsand Composition

Progestins with strong androgenic or antiestrogeniceffects have been shown to decrease HDL2 selectivelyin premenopausal women, and this decrease wasassociated with increased hepatic lipase activity.39'40

Plasma HDL2 concentration was inversely correlatedwith postheparin plasma hepatic lipase activity inpostmenopausal women.41 This observation suggestedthat the effects of progesterone may be mediated byhepatic lipase, as suggested for anabolic steroids.42 Inwomen43-43 and cynomolgus monkeys,46-48 oral con-traceptives containing mainly progestins increasedplasma triglycerides and decreased plasma HDL, es-pecially HDL2. In cynomolgus monkeys treated withcontraceptives containing both estrogen and proges-terone, the HDLa, subtraction was decreased, with anincrease in H D L ^ (as determined by gradient gels).

The main purpose of these studies was to inves-tigate the effects of estrogen and progesterone,alone and in combination, on lipoprotein levels andcomposition and on arterial lesions in baboons. Ourstudies suggest that progesterone, when given aloneto ovariectomized and hysterectomized baboons,increases VLDL+LDL cholesterol and the(VLDL+LDL)/HDL cholesterol ratio. In addition,most of the HDL in progesterone-treated baboonswas in HDL3 range.26 The combined treatment withprogesterone plus estrogen produced changes op-posite to those observed in baboons treated withprogesterone alone. Estrogen treatment decreasedplasma postheparin lipoprotein lipase activity, butwhen estrogen was combined with progesterone,lipoprotein lipase activity was increased as it waswith progesterone only. Thus, progesterone alsocounterbalanced the effect of estrogen in reducinglipoprotein lipase activity. These results suggestedthat estrogen and progesterone modulated lipopro-tein levels and composition independently of eachother in baboons. In some cases, estrogen andprogesterone counterbalanced each other's effectson lipoprotein levels and metabolism, for example,apo B production49 and apo A-I catabolism.26 Es-trogens and estrogen plus progesterone produced alipoprotein pattern that was antiatherogenic, whileprogesterone alone resulted in a lipoprotein patternthat was atherogenic.

Effect of Estrogen and Progesterone onAtherosclerotic Lesions

Although studies in experimental animals haveshown an antiatherogenic effect of exogenous estro-gens,5-8-18-21 there are no reports on the effects ofprogesterone on atherosclerosis. Our study demon-strates that progesterone therapy increased athero-sclerosis compared with controls, estrogen treatment,and estrogen plus progesterone treatment. Our ex-

by guest on May 6, 2018

http://atvb.ahajournals.org/D

ownloaded from

30 Arteriosclerosis and Thrombosis Vol 11, No 1, January/February 1991

periment provides some evidence that the observedeffects of hormone therapy on experimental athero-sclerosis may be independent of lipoproteins. In thebrachial and carotid arteries, hormone treatmentpredicted lesions independently of eitherVLDL+LDL cholesterol or the (VLDL+LDL)/HDL cholesterol ratio. In the abdominal aorta andinnominate artery, the association of (VLDL+LDL)/HDL cholesterol ratio and VLDL+LDL cho-lesterol with lesions was reduced after adjusting forhormone therapy. However, because of the smallnumbers of baboons in each group, the relativecontributions of each hormone therapy and lipopro-teins to atherosclerosis could not be determined. Thepresence of an independent effect of hormone ther-apy on atherosclerosis as suggested in our study isconsistent with similar studies in other animal mod-els. Estrogen treatment retarded arterial lesion de-velopment in rabbits without an effect on plasma andlipoprotein cholesterol concentrations.518"21 Like-wise, intravaginal ring treatment of female cynomol-gus monkeys was associated with plaques that werelarger than those in monkeys treated with oral con-traceptives (containing both estrogen and progester-one) despite a similar reduction in plasma HDLcholesterol in both groups.49 Thus, it appears thatestrogens have an antiatherogenic effect that is inde-pendent of reductions in plasma and lipoproteincholesterol concentrations. This antiatherogenic ef-fect of estrogens may involve a direct effect on thearterial wall, as suggested by some investigators.18"21

The primary effect of estrogen deficiency and estro-gen replacement on coronary atherosclerosis in othermodels has been described.3745-47 In our studies,lesion development was not noticed in coronaryarteries in any treatment group. The beneficial effectof estrogen-replacement therapy was detected mainlyon the innominate, brachial, iliac, and carotid arter-ies and the abdominal aorta.

Although progesterone produces an atherogeniclipoprotein profile, it is not known whether proges-terone influences atherosclerosis independently oflipoprotein levels. In our studies, progesteronetreatment resulted in extensive arterial lesions.However, baboons given progesterone in combina-tion with estrogen had the least fatty streak involve-ment. This result suggests that estrogen reverses theatherogenic effect of progesterone. It is also un-likely that increased lipoprotein lipase due to pro-gesterone treatment plays a role in the lipoproteinchanges and atherosclerosis observed in the proges-terone treated baboons. Lipoprotein lipase waselevated to the same degree in baboons treated withprogesterone and with estrogen plus progesterone,but the estrogen plus progesterone-treated grouphad much more extensive lesions than did theprogesterone-treated group.

AcknowledgmentsWe thank Maggie Garcia and Michael Bernard for

providing technical assistance for these studies.

Manuscript preparation and editorial assistance wasprovided by Jo Fletcher.

References1. Oliver MF, Boyd GS: Effect of bilateral ovariectomy on

coronary-artery disease and serum-lipid levels. Lancet 1959;2:690-694

2. Kannel WB, Hjortland MC, McNamara PM, Gordon T:Menopause and risk of cardiovascular disease: The Framing-ham Study. Ann Intern Med 1976;85:447-452

3. Robinson RW, Higano N, Cohen WD: Increased incidence ofcoronary heart disease in women castrated prior to the meno-pause. Arch Intern Med 1959;104:908-913

4. Rosenberg L, Hennekens CH, Rosner B, Belanger C, Roth-man KJ, Speizer FE: Early menopause and the risk ofmyocardial infarction. Am J Obstet Gynecol 1981;139:47-51

5. Prichard RW, darkson TB, Lofland HB: Estrogen in pigeonatherosclerosis: Estradiol valerate effects at several dose levelson cholesterol-fed male White Cameau pigeons. Arch Pathol1966;82:15-27

6. Chao Y-S, Windier EE, Chen GC, Havel RJ: Hepatic catab-olism of rat and human lipoproteins in rats treated with17a-ethinyl estradiol. / Biol Chem 1979^54:11360-11366

7. Davis RA, Roheim PS: Pharmacologically induced hypolipi-demia: The ethinyl estradiol-treated rat. Atherosclerosis 1978;30:293-299

8. Kushwaha RS, Hazzard WR: Exogenous estrogens attenuatedietary hypercholesterolemia and atherosclerosis in the rabbit.Metabolism 1981;30:359-366

9. Floren C-H, Kushwaha RS, Hazzard WR, Albers JJ: Estro-gen-induced increase in uptake of cholesterol-rich very lowdensity lipoproteins in perfused rabbit liver. Metabolism 1981;30:367-375

10. Kovanen PT, Brown MS, Goldstein JL: Increased binding oflow density lipoprotein to liver membranes from rats treatedwith 17a-ethinyl estradiol. J Biol Chem 1979;254:11367-11373

11. Windier EET, Kovanen PT, Chao Y-S, Brown MS, Havel RJ,Goldstein JL: The estradiol-stimulated lipoprotein receptor ofrat liver: A binding site that mediates the uptake of ratlipoproteins containing apoproteins B and E. / Biol Chem1980;255:10464-10471

12. Iozzo RV, Kushwaha RS, Wight TN, Hazzard WR: Cellularand subcellular distribution of 125-I-labeled very low densitylipoproteins in the liver of normal and estrogen-treated rab-bits. Am J Pathol 1982;107:6-15

13. Kushwaha RS, Hazzard WR, Gagne C, Chait A, Albers JJ:Type III hyperlipoproteinemia: Paradoxical hypolipidemicresponse to estrogen. Ann Intern Med 1977;87:517-525

14. Schaefer EJ, Foster DM, Zech LA, Lindgren FT, Brewer HBJr, Levy RI: The effects of estrogen administration on plasmalipoprotein metabolism in premenopausal females. / Endo-crinol Metab 1983^7:262-267

15. Heiss G, Tamir I, Davis CE, Tyroler HA, Rifkind B, SchonfeldG, Jacobs D, Frantz ID: Lipoprotein-cholesterol distributionsin selected North American populations: The Lipid ResearchClinics Program Prevalence Study. Circulation 1980;61:302-315

16. Lipson A, Stoy DB, LaRosa JC, Muesing RA, Cleary PA,Miller VT, Gilbert PR, Stadel B: Progestins and oral contra-ceptive-induced lipoprotein changes: A prospective study.Contmception 1986;34:121-134

17. La Rosa JC: The varying effects of progestins on lipid levelsand cardiovascular disease. Am J Obstet Gynecol 1988;158:1621-1629

18. Hough JL, Zilversmit DB: Effect of 17-beta estradiol on aorticcholesterol content and metabolism in cholesterol-fed rabbits.Arteriosclerosis 1986;6:57-63

19. Souadjian JV, Kottke BA, Titus JL: Estrogen effect on spon-taneous artherosclerosis: Experimental studies in WhiteCarneau pigeons. Arch Pathol 1968;85:463-467

20. Hanash KA, Kottke BA, Greene LF, Titus JL: Effects ofconjugated estrogens on spontaneous atherosclerosis inpigeons. Arch Pathol 1972;93:184-189

by guest on May 6, 2018

http://atvb.ahajournals.org/D

ownloaded from

Kushwaha et al Sex Steroid Hormones and Atherosclerosis 31

21. Subbiah MTR: Effect of estrogens on the activities of cho-lesteryl ester synthetase and cholesteryl ester hydrolases inpigeon aorta. Steroids 1977^0:259-276

22. Stumpf WE, Sar M: Autoradiographic localization of estrogen,androgen, progestin, and glucocorticosteroid in "target tis-sues" and "nontarget tissues," in Pasqualini JR (ed): Receptorsand Mechanisms of Action of Steroid Hormones. Part I. NewYork, Marcel Dekker, Inc, 1976, pp 41-84

23. McGill HC Jr, Sheridan PJ: Nuclear uptake of sex steroidhormones in the cardiovascular system of the baboon. Ore Res1981;48:238-244

24. Horwitz KB, Horwitz LD: Canine vascular tissues are targetsfor androgens, estrogens, progestins, and glucocorticoids. /Clin Invest 1982;69:750-758

25. Williams MC, Kushwaha RS, McGill HC Jr: Quantitation ofbaboon lipoproteins by high-performance gel exclusion chro-matography. Lipids 1987;22:366-374

26. Kushwaha RS, Foster DM, Murthy VN, Carey KD, BernardMG: Metabolic regulation of apoproteins of high densitylipoproteins by estrogen and progesterone in the baboon(Papio sp.). Metabolism 1990;39:544-552

27. Laurell C-B: Quantitative estimation of proteins by electro-phoresis in agarose gel containing antibodies. Anal Biochem1966;15:45-52

28. McGill HC Jr, McMahan CA, Kushwaha RS, Mott GE, CareyKD: Dietary effects on serum lipoproteins of dyslipoproteine-mic baboons with high HDL!. Arteriosclerosis 1986;6:651-663

29. Nilsson-Ehle P, Schotz MC: A stable radioactive substrateemulsion for assay of lipoprotein lipase. / Lipid Res 1976;17:536-541

30. Guzman MA, McMahan CA, McGill HC Jr, Strong JP, Tajda C,Restrepo C, Eggen DA, Robertson WB, Solberg LA: Selectedmethodological aspects of the International Atherosclerosis Proj-ect. Lab Invest 1968;18:479-497

31. Draper NR, Smith H: Applied Regression Analysis. New York,John Wiley & Sons, Inc, 1981, p 709

32. Steel RDG, Torrie JH: Principles and Procedures in Statistics.New York, McGraw-Hill Book Co, 1960, p 481

33. Sokal RR, Rohlf RJ: Biometry. San Francisco, WH FreemanCo, 1969, pp 235-246

34. Wallentin L, Varenhorst E: Plasma lipoproteins during anti-androgen treatment by estrogens or orchidectomy in men withprostatic carcinoma. Horm Metab Res 1981;13:293-297

35. Krauss RM, Lindgren FT, Wingerd J, Bradley DD, Ramcha-ran S: Effects of estrogens and progestins on high densitylipoproteins. Lipids 1979;14:113-118

36. Cauley JA, LaPorte RE, KuUer LH, Bates M, Sandier RB:Menopausal estrogen use, high density lipoprotein cholesterolsubtractions and liver function. Atherosclerosis 1983;49:31-39

37. Hazzard WR, Haffner SM, Kushwaha RS, Applebaum-Bowden D, Foster DM: Preliminary report: Kinetic studies on

the modulation of high density lipoprotein, apolipoprotein,and subtraction metabolism by sex steroids in a postmeno-pausal woman. Metabolism 1984;33:779-784

38. Moskowitz MS, Moskowitz AA, Bradford WL Jr, Wissler RW:Changes in serum lipids and coronary arteries of the rat inresponse to estrogen. Arch Pathol 1956;61:245-263

39. Krauss RM: Effects of progestational agents on serum lipidsand lipoproteins. / Reprod Med 1982;27:503-510

40. Tikkanen MJ, Nikkila EA, Kuusi T, Sipinen S: Differenteffects of two progestins on plasma high density lipoprotein(HDL2) and postheparin plasma hepatic lipase activity. Ather-osclerosis 1981;4O:365-369

41. Kuusi T, Saarinen P, NikkilS EA: Evidence for the role ofhepatic endothelial lipase in the metabolism of plasma highdensity lipoprotein in man. Atherosclerosis 1980^6:589-593

42. Haffner SM, Kushwaha RS, Foster DM, Applebaum-BowdenD, Hazzard WR: Studies on the metabolic mechanisms ofreduced high density lipoproteins during anabolic steroidtherapy. Metabolism 1983;32:413-420

43. Bradley DD, Wingerd J, Petitti DB, Krauss RM, RamcharanS: Serum-high-density-lipoprotein cholesterol in women usingoral contraceptives, estrogens, and progestins. N Engt J Med1978;299:17-20

44. Wahl P, Walden C, Knopp R, Hoover J, Wallace R, Heiss G,Rifkind B: Effect of estrogen/progestin potency on lipid/lipoprotein cholesterol. N Engl J Med 1983;308:862-867

45. Krauss R: Contraceptive steroid effects on serum lipoproteinsand lipoprotein subclasses, in Gregoire AT, Blye RP (eds):Contraceptive Steroids: Pharmacology and Safety. New York,Plenum Publishing Corp, 1986, pp 321-338

46. Koritnik D, Clarkson T, Adams M: Cynomolgus macaques asmodels for evaluating effects of contraceptive steroids onplasma lipoproteins and coronary artery atherosclerosis, inGregoire AT, Blye RP (eds): Contraceptive Steroids: Pharma-cology and Safety. New York, Plenum Publishing Corp, 1986,pp 303-319

47. Adams MR, Rudel LL, Clarkson TB, Nelson CA, Thau RB,Moo-Young AJ: Influence of a levonorgestrel-containing con-traceptive vaginal ring on plasma lipids and lipoproteins incynomolgus monkeys. Contraception 1983;28:253-266

48. Adams MR, Clarkson TB, Koritnik DR, Nash HA: Contra-ceptive steroids and coronary artery atherosclerosis in cy-nomolgus macaques. Fertil Steril 1987;47:1010-1018

49. Kushwaha RS, Foster DM, Barrett PHR, Carey KD: Effect ofestrogen and progesterone on metabolism of apoprotein B inbaboons. Am J Physiol 1990;258:E172-E183

KEY WORDS • cholesterol • coronary artery disease • highdensity lipoproteins • lipoprotein lipase • lipoproteins •low density lipoproteins • baboon

by guest on May 6, 2018

http://atvb.ahajournals.org/D

ownloaded from

R S Kushwaha, D S Lewis, K D Carey and H C McGill, Jratherosclerosis in the baboon (Papio sp.).

Effects of estrogen and progesterone on plasma lipoproteins and experimental

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 1991 American Heart Association, Inc. All rights reserved.

Avenue, Dallas, TX 75231is published by the American Heart Association, 7272 GreenvilleArteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/01.ATV.11.1.231991;11:23-31Arterioscler Thromb Vasc Biol. 

http://atvb.ahajournals.org/content/11/1/23World Wide Web at:

The online version of this article, along with updated information and services, is located on the

  http://atvb.ahajournals.org//subscriptions/

at: is onlineArteriosclerosis, Thrombosis, and Vascular Biology Information about subscribing to Subscriptions:

  http://www.lww.com/reprints

Information about reprints can be found online at: Reprints: 

document.Permissions and Rights Question and AnswerFurther information about this process is available in theis being requested is located, click Request Permissions in the middle column of the Web page under Services.Clearance Center, not the Editorial Office. Once the online version of the published article for which permission

can be obtained via RightsLink, a service of the CopyrightArteriosclerosis, Thrombosis, and Vascular Biology Requests for permissions to reproduce figures, tables, or portions of articles originally published inPermissions:

by guest on May 6, 2018

http://atvb.ahajournals.org/D

ownloaded from