RECENT STUDIES in this department

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1 Preventon of Coronary Atheroscleross by -Androgen Admnstraton n the Cholesterol-Fed Chck By JEREMAH STAMLER, M.D., RUTH PCK, M.D., AND LOUS X. KATZ, M.D. s are hghly effectve both prophylacteally and therapeutcally aganst cholesterol-nduced coronary atherogeness n cockerels. The relatve mmunty of premenopausal women to coronary scleross may be related to the presence of these ovaran hormones. Therefore, estrogens may be useful n the treatment of human coronary atheroscleross. Wth ths object n mnd, the elmnaton of the femnzng effects of estrogens would be hghly desrable. The present experments prove that concomtant exhbton of androgens and estrogens mantan masculne sex characterstcs wthout nterferng wth the benefcent effect of estrogens on the coronary arteres n cockerels. Downloaded from by on January, 9 RECENT STUDES n ths department demonstrate that estrogens are hghly.effectve both prophylacteally and therapeutcally aganst cholesterol-nduced coronary atherogeness n cockerels. " 3 * These fndngs ndcate that estrogens may be a key factor n the relatve mmunty of premenopausal women to clncal coronary scleross. 3 Further, they suggest that estrogens may be useful n the treatment of human coronary lesons. n any attempt to assess ths latter possblty, t would be hghly desrable to avod lkely femnzng effects of estrogens on male subjects. One possble way to attan ths objectve would be through combnaton of estrogen and androgen therapy. Wth ths n mnd, the present experment was prelmnarly undertaken, assessng the nfluence of ths combned hormonal regmen on the expermental lesons n cockerels. t attempted to supply adequate androgen for masculnzaton ether exogenously or endogenously, by admnstraton of testosterone or choronc gonadotrophnf respectvely. From the Cardovascular Department, Medcal Research nsttute, Mchael Reese Hosptal, Chcago,. Aded by Grants from the Natonal Heart nsttute (H-), the Chcago Heart Assocaton and the Mchael Reese Research Foundaton. Receved for publcaton Oct. 7,95. * n contrast, no effect on aorta atherogeness s observed. t Pregnant mare's serum. 94 Ths study also entaled an exploraton of the mechansm of estrogen nhbton of cholesterolnduced coronary atheroscleross. By analyzng the effects of gonadotrophn and androgen on estrogen-treated cholesterol-fed chcks, t permtted an evaluaton of the possblty that estrogen nhbton of coronary atherogeness s related to the concomtant estrogen-nduced suppresson of ptutary gonadotrophn-testcular androgen secreton. 4 Fnally, ths experment was desgned to determne the effects of exogenous testosterone and choronc gonadotrophn, per se, on cholesterol-nduced atherogeness, n the absence of concomtant estrogen exhbton. METHODS Two seres of experments were completed, utlzng a total of chcks, n accordance wth procedures establshed n ths department. 3 n both, one day old Hy-lne cockerels were obtaned from a commercal hatchery and reared n a battery brooder. Pror to ntaton of the expermental regmen, brds were mantaned on a commercal chck starter mash and tap water ad lb. The frst seres was dvded nto four expermental groups ( chcks per group) at the age of 9 weeks; the second, nto sx groups at 8 weeks of age (table ). The expermental det ngested by all groups was: n seres, mash supplemented wth per cent cholesterol plus 5 per cent cottonseed ol ( CO); n seres, mash supplemented wth per cent cholesterol plus 5 per cent cottonseed ol ( CO). The duraton of the experment was: n seres, fve weeks (age 9 to 4 weeks); n seres, seven weeks (age S to 5 weeks). Records of feed ntake and body weght were Crculaton Research. Volume, January 95S

2 J. STAMLER, R. PCK AND L. N. KATZ 95 Downloaded from by on January, 9 mantaned throughout. The hormonal regmens are summarzed n table. Seral comb ndexes were obtaned to estmate over-all androgenc actvty (exogenous and/or endogenous). 4 n both seres, chcks were bled serally from a wng ven or by cardac puncture. ndvdual samples of heparnzed plasma were analyzed for total cholesterol and lpd phosphorus by the methods of Schoenhemer and Sperry 5 and Man and Peters respectvely. Samples of sera from ndvdual brds were also analyzed ultracentrfugally to determne concentratons of varous classes of lpoprotens. 7 * At the tme of sacrfce, complete autopses were performed. Aortas and hearts were evaluated for atherogencss by establshed gross and mcroscopc methods. " 3 Seres * * TABLE. Hormonal Regmens Estradol Benzoate mg. /brd/ dayt Testosterone Proponate mg./brd/ dayt Pregnant Mare's Serumt l.u./ chck/day -4-4 * Seres : cockerels age 9 to 4 weeks, per group, fed CO det. Seres : cockerels age 8 to 5 weeks, per group, fed CO det, f Parenterally, n ol. Parenterally, n salne. RESULTS All three groups of cockerels recevng estrogen were remarkably free of coronary atheroscleross (groups, 4 and, seres and, table ).f Ths estradol nhbton of cholesterol-nduced coronary atherogeness perssted when ether testosterone or choronc gonado- * We are ndebted to Drs. Lena Lews and rvne H. Page of the Cleveland Clnc Research Foundaton, where the ultracentrfuge studes were accomplshed. Ther fndngs wll be reported n detal n a separate communcaton. f n accord wth prevous observatons, " 3 none of the estrogen-treated groups exhbted any degree of nhbton of aorta atherogeness. n the four trophn was concomtantly admnstered (groups 4 and ). n seres, the dosage of testosterone was adequate to mantan masculnzaton of secondary sex characterstcs (combs and wattles) throughout the experment (seres, group 4, table ). Nevertheless, estrogen ant-atherogeness contnued to preval. The two usual effects of estradolfemnzaton of combs and nhbton of coronary atherogenessbecame dssocated, wth neutralzaton of the former and persstence of the latter. The estrogen-nhbton of coronary atheroscleross cannot be attrbuted to dfferences n TABLE.Mcroscopc Coronary Atherogeness* Control Androgen Androgen and Gonadotrophn Gonadotrophn and % of Brds wth Coronary Lesons % of Coronary Arteres Exhbtng LesonsAll Brds 57 * s -4 are combned data of Seres and. s 5- are from Seres only (see text). cholesterol ntake, snce all groups were smlar wth respect to feed ntake and rate of weght groups of Seres, the thoracc aortas were graded.,.8,.9,.7 respectvely for gross cholesterolnduced atheroscleross; n the sx groups of seres, they were graded.3,.3,.4,.3,.3 and.4 respectvely. Despte a greater percentage of cholesterol n the seres det ( per cent vs. per cent), coronary atherogeness was sgnfcantly less n the non-estrogeh treated groups of seres, as compared wth ther counterparts n seres. Ths fndng s n accord wth prevous observatons of an agecondtoned relatve mmunty of to week old cockerels to cholesterol-nduced atherogeness. 8 t Ths was the only estrogen-treated group n whch masculnzaton was successfully mantaned. Both the smaller doses of testosterone used n seres (group 4, table 3) and the level of gonadotrophn admnstered n seres (group, table 3) dd not acheve ths objectve. Further, n both seres, nether testosterone nor gonadotrophn reversed thefemnzng effect of estrogen on the cloaca. n all estrogen-treated cockerels (groups, 4, and ) the testes were small (table 3).

3 9 CORONARY ATHEROSCLEROSS PREVENTON Downloaded from by on January, 9 gan. Nether s the mmunty to coronary atherogeness n the estrogen-treated groups attrbutable to dfferences n degree of hypercholesterolema, snce smlar levels of plasma total Seres TABLE 3.Comb ndexes and Tesls Weghts. Control.. Control. 5. Gonadotrophn. Gonadotrophn plus Weeks of Expermental Regmen TABLE 4.Mean Plasma Lpds for Entre Experment, Seres 7/f. Control. 5. Gonadotrophn. Gonadotrophn plus Total Cholesterol mg.% * Lpd Phosphorus mg.% * Tests Weght grans C/P Rato * Range of values. t Mean value for entre experment,.e., mean of ndvdual bleedngs of brds at, 3 and 7 weeks on expermental regmen. cholesterol prevaled throughout the experment n all groups of a gven seres (table 4). n accord wth prevous fndngs, estrogen admnstraton effected a marked elevaton of plasma phospholpd levels, wth consequent depresson of the plasma total cholesterol/ lpd phosphorus (C/P) ratos toward normal despte the concomtant hypercholesterolema (table 4). Ths estrogen nfluence on hyperphospholpema tended to persst n the groups gven testosterone or gonadotrophn together wth estradol (groups 4 and, table 4). However, n the androgen-estrogen treated chcks of seres (group 4), plasma phospholpd levels tended to fall (wth resultant rses n C/P ratos) durng the latter weeks of the experment, when testosterone dosage attaned the level of. to 3. mg/brd/day. Thus, the mean plasma lpd phosphorus levels of the group 4 cockerels were 57.7 and 5.3 at the thrd and seventh expermental weeks respectvely, and the C/P ratos were 7 and 7 respectvely. Only ths group exhbted ths apparent testosterone-nduced reversal of estrogen hyperphospholpema. Further work s needed to verfy ths phenomenon and assess ts sgnfcance, partcularly n relaton to the problem of the role of C/P ratos n coronary atherogeness. Exhbton of testosterone or gonadotrophn alone, wthout estrogen, had no sgnfcant effect on plasma lpd levels or atherogeness (coronary or aorta) (groups 3 and 5, tables and 4). n both seres, exogenous testosterone exhbton apparently effected an over-all hyperandrogensm, judgng from the data on comb ndces (group 3, table 3). These hgh comb ndces n the testosterone-treated cockerels were assocated wth large testes n seres and small testes n seres (table 3). t s not clear from the data avalable whether ths dvergency n results s related to chck age, testosterone dosage and/or other factors. Choronc gonadotrophn alone produced antcpated effects, that s, ncreases n both comb ndces and testes weghts (group 5, table 3). As already ndcated, the hormone-nduced hyperandrogensm n groups 3 and 5 (prmarly exogenous n the former, endogenous n the latter) was wthout effect on plasma lpd patterns or atherogeness. DSCUSSON AND CONCLUSONS Prevously ' 3 t was shown that estrogen s effectve both prophylactcally and therapeutcally aganst cholesterol-nduced coronary atherogeness n cockerels. The fndngs of the present experment are addtonal confrmaton

4 J. STAMLER, R. PCK AND L. N. KATZ 97 Downloaded from by on January, 9 of the estrogen prophylactc effect. Further, they demonstrate that coronary lesons are also nhbted by combned admnstraton of estrogen and androgen, a regmen that avods the usual femnzng effects of estrogen on secondary sex characterstcs. Studes are now n order to assess the possble utlty of both regmensestrogen alone, and estrogen plus androgenfor the prophylaxs and/or therapy of coronary atheroscleross n man. Such an nvestgaton s now beng pursued. Prevously we presented the tentatve conclusonbased on the demonstraton of estrogen nhbton of expermental coronary atherosclerossthat estrogen secreton may be a key factor responsble for the relatve mmunty of premenopausal women to coronary atheroscleross. " 3 Ths concept poses the queston: s the relatve susceptblty of the human male to coronary atheroscleross due to absence (relatve) of estrogen or to presence of androgen, or to both (that s, a low rato estrogen/androgen)? The fndngs of the present study, together wth other observatons on castrated cockerels, 9 may afford a partal answer to ths problem. They demonstrate that n the absence (relatve) of estrogen, cholesterol-nduced coronary atherogeness proceeds n cockerels, whether they are hyperandrogenc,* euandrogencf or hypoandrogenc.j Therefore the presence of estrogen, per se, wth a relatvely hgh rato of estrogen/androgen, appears to be the sne qua non for ant-atherogenc actvty. Pror to age 5, coronary atherogeness s more common n males than n females, apparently because men are lackng (relatvely) n estrogens and have a low estrogen/androgen rato and not prmarly because of ther relatvely hgh level of androgen secreton. Further expermental and clncal nvestgaton evaluatng ths concept s essental. An addtonal proposton s suggested by the fndngs of the present experments: Antatherogeness s present n all estrogen-treated groups, regardless of concomtant male hor- * Testosterone or gonadotrophn-treated ntact brds of the present experment (groups 3 and 5). t Untreated ntact brds of the present experment (group ). t Untreated castrate brds." mone admnstraton. Wth adequate doses of testosterone, t s possble to overcome the hypo-androgensm due to estrogen nhbton of anteror ptutary gonadotrophn-testcular androgen secreton. Nevertheless, coronary atherogeness contnues to be nhbted. Therefore, estrogen ant-atherogeness apparently s not a consequence of estrogen suppresson of anteror ptutary gonadotrophn-testcular androgen secreton. t s not a consequence of estrogennduced hypoandrogensm. The mechansm of estrogen ant-atherogeness remans to be elucdated. Fnally, one further tentatve concluson: Snce t s apparently possble wth androgen to effect a dssocaton of the femnzng and ant-atherogenc actons of estrogen, the mechansms of these two nfluencesas already ndcatedare apparently dfferent. Therefore, we may antcpate the eventual acquston of an estrogen-lke compound whch produces mnmal femnzaton and maxmal nhbton of coronary atherogeness. SUMMARY. The phenomenon of estrogen prophylactc nhbton of coronary atherogeness s further confrmed.. The combned admnstraton of estrogen and androgen, wth resultant preventon of estrogen femnzaton, s at least as effectve as estrogen alone n the prophylactc nhbton of cholesterol-nduced chck coronary atherogeness. 3. Nether androgen nor choronc gonadotrophn alone has a sgnfcant nfluence on cholesterol-nduced coronary or aorta atherogeness n ntact cockerels. ACKNOWLEDGMENTS We are greatly ndebted to Dr. Abbott Allen of the Scherng Corporaton, Bloomfeld, N. J., for generous supples of estradol, testosterone and choronc gonadotrophn (pregnant mare's serum); and to Dr. Kenneth G. Kohlstaedt of the Llly Laboratory for Clncal Research, ndanapols, nd., and to Dr. Kenneth W. Thompson of Organon, nc., Orange, N. J., for generous supples of testosterone and estradol respectvely. The successful accomplshment of ths study was made possble only by the competent work of the able group of techncans on the department's athero-

5 98 CORONARY ATHEROSCLEROSS PREVENTON scleross research team: Chrstne Bolene-Wllams, Marlyn Dudley, Dolores Fredman, Phlp Johnson, John Morrs and Ronald Wallace. REFERENCES PCK, R., STAMLER, J., RODBARD, S., AND KATZ, L. N.: The nhbton of coronary atheroscleross by estrogens n cholesterol-fed chcks. Crculaton : 7, 95.,,, AND : -nduced regresson of coronary atheroscleross n cholesterol-fed chcks. Crculaton. n press. 3 KATZ, L. N., AND STAMLER, J.: Expermental Atheroscleross. Sprngfeld,., C. C Thomas, n press. 4 PARKES, A. S., AND EMMENS, C. W.: Effect of androgens and estrogens. Vtamns and hormones : 3, 944. SCHOENHEMER, R., AND SPERHY, W. M.: A mcromethod for the determnaton of free and combned cholesterol. J. Bol. Chem. : 745, 934. MAN, E. B., AND PETERS, J. P.: Gravmetrc determnaton of serum cholesterol adapted to the man and Gldea fatty acd method, wth a note on the estmaton of lpd phosphorus. J. Bol. Chem. : 85, GOFMAN, J. W., JONES, H. B., LNDGREN, F. T., LYON, T. P., ELLOTT, H. A., AND STRSOWER, B.: Blood lpds and human atheroscleross. Crculaton :, 95. * RODBARD, S., KATZ, L. N., BOLENE, C, PCK, R., LOWENTHAL, M., AND GROS, G.: The age factor n hypercholesterema and atheromatoss n the chck. Crculaton 3: 87,95. 8b, PCK, R., BOLENE-WLLAMS, C, AND KATZ, L. N.: Age-condtoned spontaneous regresson of atheroscleross n the cholesterol-fed chck. Crculaton : 459, 95. 'PCK, R., RODBARD, S., STAMLER, J., AND KATZ, L. N.: nfluence of gonadectomy on cholesterolnduced aorta and coronary atherogeness n young chcks. Crculaton : 47, 95. Downloaded from by on January, 9