35

Atherosclerosis, 91 (1991) 35-49

0 1991 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved 0021-9150/91/$03.50

ATHERO 04713

Doxazosin and cholestyramine similarly decrease fatty streak formation in the aortic arch of hyperlipidemic hamsters Mark C. Kowala ‘, John J. Nunnari 2, Stephen K. Durham 3 and Robert J. Nicolosi

’

I Department of Clinical Sciences, Unic’ersity of Lowell, Lowell, M4 01854 (U.S.A.), 2 Department of Pathology, Unicersity of Massachusetts Medical School, Worcester, MA 01655 (U.S.A.), and 3 Department of Interdisciplinan, Toxicology, University of Arkansas for Medical Sciences, Little Rock. AK 72205 (U.S.A.)

(Received 10 September, 1990) (Revised, received 5 December, 1990 and 19 June, 1991) (Accepted 31 July, 1991)

Summary

The effect of doxazosin, an alpha-l adrenergic inhibitor, on atherosclerosis was determined in hyperlipidemic hamsters. Control hamsters fed chow plus 0.05% cholesterol and 10% coconut oil were compared to chow baseline animals, and to those receiving either 10 mg/kg/day doxazosin, or 245 mg/kg/day cholestyramine in the atherogenic diet. During 8 weeks of treatment, plasma lipids, mean arterial pressure (MAP) and heart rate (HR) were measured, then the ascending aortic arch was examined en face. Numbers of subendothelial macrophage-foam cells/mm2 and their average size (pm21 were determined, and Oil red 0 staining (kg ORO/mm2) was quantitated to estimate lipid accumulation. Ultracentrifugation of control plasma demonstrated that low density lipoprotein (LDL) carried most of the cholesterol, and very low density lipoprotein (VLDL) was rich in triglycerides. Compared to controls, doxazosin and cholestyramine similarly decreased plasma total and LDL plus VLDL cholesterols, and total triglycerides on average by 46%, 61% and 45% respectively. High density lipoprotein cholesterol was unchanged. Doxazosin also reduced MAP by 18% without affecting HR. In all hamsters, foam cells and lipid accumulated in a lesion-prone area characterized by elevated endothelial cell density, and a thick intima of basement membrane-like material layered over “pads” of smooth muscle cells. Compared to controls, doxazosin and cholestyramine uniformly reduced the number of foam cells/mm2, foam cell size and OR0 staining on average by 66%, 29% and 56%, respectively. We conclude that doxazosin decreases plasma lipids and inhibits the development of the fatty streak to a similar level as cholestyramine treatment.

Key words: Alpha-1 adrenergic blockers; Doxazosin; Cholestyramine; sure; Macrophage; Foam cell; Oil red 0; Atherosclerosis

Correspondence to: Mark C. Kowala, Ph.D., Department of Pharmacology, Rm. F1.4801, Bristol Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543-4000. U.S.A.

Plasma cholesterol;

Blood pres-

36 Introduction The anti-hypertensive agent doxazosin inhibits alpha-l adrenergic receptors on arterial smooth muscle cells, decreases systemic vascular resistance [l], and reduces blood pressure in hypertensive patients [2-41. This treatment also increases the plasma high density lipoprotein (HDL)/total cholesterol ratio [5,6]. Despite the favorable impact of doxazosin on blood pressure and plasma cholesterol, its effect on atherosclerosis remains unknown. In early atherosclerosis, LDL and liposomes containing free cholesterol accumulate in the subendothelial space 17-91. LDL may undergo some modification [lo] or interact with subendothelial proteins [ 11,121. Increased numbers of monocytes are attracted to the artery wall by a variety of chemotactic factors [10,13,14], where they attach to the intact endothelial surface and migrate into the subendothelial space [15-181. The monocyte-derived macrophages phagocytose LDL particles, become loaded with cholesterol ester and transform into foam cells [lo]. Macrophage-foam cells accumulate in the intima to form fatty streaks [19,20] and they are also present in atherosclerotic plaques [21-231. The effect of doxazosin on the progression of the fatty streak was studied in the F,B hamster. Plasma lipids, blood pressure and foam cell accumulation were analyzed in chow baseline, cholesterol-fed controls and in doxazosin treated hamsters. The doxazosin group was also compared to hamsters treated with cholestyramine, to estimate the impact of lipid reduction alone on the fatty streak. The resin choIestyramine is not absorbed [24], it binds intestinal bile acids [25] and reduces plasma lipids [26,27] without reaching the circulation. This study extends the findings of our preliminary report [28], and suggests that doxazosin and cholestyramine similarly impede the progress of early atherosclerosis. Materials and methods Experimental animals

Sixty-four male F,B hybrid Golden Syrian hamsters were used in this study (8 weeks old, averaging 90 g body weight, Biobreeders Inc.,

Fitchburg, MA). They were maintained according to the recommendations in the “Guide for the Care and Use of Laboratory Animals” prepared by the ILAR, NRC (DHEW Publications No. NIH 85-23, 1985). Animals were housed individually in hanging cages and were subjected to a 12-h day/night light cycle. Experimental design

First we determined the effect of a mild atherogenic diet on hamster plasma lipids. A group of 9 animals were fed chow containing 0.05% cholesterol and 10% coconut oil (w/w), and after 3 weeks, cholesterols and triglycerides in the VLDL, LDL and HDL fractions were assayed. In the atherosclerosis lesion study, four sets of hamsters were run together (n = 10 per set). The first group were fed chow (Purina no. 5001, St. Louis, MO) to provide baseline values for plasma lipids and for the accumulation of aortic lipids and foam cells. The second group were control hamsters fed the mild atherogenic diet consisting of chow supplemented with 0.05% cholesterol and 10% coconut oil w/w. Group three received the atherogenic diet plus 0.5% cholestyramine (Bristol Laboratories, Evansville IN), and the fourth group had 0.02% doxazosin (Pfizer Inc., Groton, CT) in the atherogenic diet. The average doses of doxazosin and cholestyramine per hamster were 10 and 245 mg/kg/day, respectively. All animals were treated for 8 weeks, and at 3, 5 and 7 weeks they were fasted for 20 h, then blood was collected via the retro-orbital sinus to measure plasma lipids. The three measurements of plasma total cholesterol, LDL plus VLDL cholesterols, HDL cholesterol and total triglycerides were averaged for each animal. Analysis of plasma lipids

The first group of 9 hamsters fed the mild atherogenic diet had their plasma total cholesterols and triglycerides measured with commercial enzyme kits on a Beckman System 700 automated analyzer. HDL cholesterol was assayed after lipoproteins containing apo B were precipitated from the plasma by the phosphotungstate method [29]. LDL plus VLDL cholesterol was

37 determined by subtracting the HDL value from the total. In addition the plasma lipoproteins of these 9 hamsters were separated by density gradient ultracentrifugation (UC) [30], in order to (a) determine the amount of cholesterol and triglycerides in the LDL, VLDL and HDL fractions, and (b) to verify HDL cholesterol measurements obtained by phosphotungstate precipitation. The density gradient consisted of potassium bromide (KEIr; 0.77 g), 2.0 ml of Sudan black (0.1 ml/100 ml ethylene glycol) and plasma (2.0 ml) mixed in a cellulose nitrate tube. Two salt solutions of densities 1.225 g/ml and 1.100 g/ml containing KBr and NaCl were overlaid. Distilled water (4 ml> was the final layer, and the samples were ultracentrifuged for 22 h at 40000 rpm in an SW 41 rotor. The labeled lipoprotein bands were aspirated and the lipids in each fraction were measured with the commercial enzyme kits. The four groups of hamsters in the lesion study had their plasma total, LDL plus VLDL and HDL cholesterols, and total triglycerides determined using the enzyme kits and the phosphotungstate precipitation method. Measurement of mean arterial pressure

After 8 weeks animals from the lesion study were weighed. Non-fasted chow, control and doxazosin treated hamsters were anesthetized with methoxyfluorane (Pittman-Moore Inc., Washington Crossing, NJ) and then each animal had a catheter (PE-10, Clay Adams, Parsippany, NJ) inserted into the right carotid artery. MAP and HR were continuously monitored on a polygraph (Grass Instruments, Quincy, MA). The nose cone containing anesthetic was removed and the hamster gradually regained consciousness; when the animal first twitched following a gentle pinching of the foot, MAP and HR were recorded at this semiconscious state. The hamster was reanesthetized and this procedure was repeated twice more to give an average MAP and HR for a particular animal. Only one cholestyramine treated hamster had its blood pressure monitored. A separate group of four hamsters fed the atherogenic diet were catheterized as above for three days, in order to validate the temporary

blood pressure measurements of semiconscious animals from the lesion study. The carotid catheter was externalized from the back of the neck and attached to a tether and swivel apparatus (Alice King Chatham Medical Arts, Los Angeles, CA). The catheter was kept patent by continuous infusion of heparinized saline (10 U/ml) at a rate of 0.0038 ml/min. MAP and HR were monitored in the morning immediately after surgery and at 2-h intervals for another 8 h. This protocol was repeated on the second and third day and the measurements of the four animals in either an active or resting state were averaged. Quantitating fatty streaks

After the temporary blood pressure measurements, the hamsters of the lesion experiment had their aortas fixed by perfusing 10% formaldehyde (pH 7) into the left ventricle at a pressure of 120 mm Hg for 30 min. The right atrium was punctured for an outflow. The segment of ascending aortic arch that was dissected free started at a point 1 mm above the aortic valves, and extended 3-4 mm to the top of the arch near the branch of the right common carotid. The tissue was immersed in the same fixative for several days and then thoroughly cleaned of adventitial fat. The luminal surface was stained with hematoxylin and Oil red 0 (ORO; Sigma o-0625, St. Louis, MO) as described previously [17], and then the aortic arch was cut open, mounted on a glass slide under a coverslip and examined en face by light microscopy. Mononuclear cells attached to the endothelial surface and macrophage-foam cells on the surface and in the subendothelial space were counted at 400 X magnification, and divided by the area of the specimen to give adherent mononuclear cells and intimal foam cells per mm2 of aorta. The average size of the foam cell was determined by tracing the shape of loo-140 cells per specimen at 1000 X magnification with computer assisted image analysis (Sigma Scan, Corte Madera, CA). The total area of foam cells was divided by the number of cells measured to give average foam cell size in pm2. The quantity of aortic neutral lipid was determined indirectly by measuring the amount of OR0 staining using a modification of the method

38

of Nunnari et al. [31]. An area of the arch with the most intimal foam cells and extracellular lipid was cut out with a gel punch. OR0 was extracted with chloroform/methanol (2: 11, and the concentration of dye was determined from its absorbance value at 516 nm which was applied to a standard curve. The concentration of OR0 was divided by the area of the aorta, and the average background staining of the media was subtracted to give intimal OR0 pg/mm2. A segment of aorta adjacent to the extracted sample was embedded in glycol methacrylate (JB4 Polysciences, Warrington, PA), sectioned at 1.5 pm, stained with toluidine blue and examined by light microscopy. Additional control, doxazosin, cholestyramine and baseline hamsters were fixed by perfusion with 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Three baseline arches were stained by silver nitrate to outline endothelial cells [17], while others were routinely stained for photography en face. Rings of the aortic arch were post-fixed in 1.3% osmium tetroxide, dehydrated in graded alcohols and then embedded in Epon 812 (Fullam, Lantham, NY). Cross-sections, 1 Km thick, were stained with toluidine blue and photographed. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined with a Zeiss EM-1OC transmission electron microscope. A one way analysis of variance followed by a Student-Newman-Keuls multiple range test was used to compare plasma lipid, blood pressure, heart rate and fatty streak parameters of all four lesion groups simultaneously. Data were transformed where necessary to gain homogeneity between the groups.

TABLE

Results

The four groups of hamsters in the lesion experiment gained weight equally during the study as shown by the 8 week measurements; (mean f SD) baseline 105 f. 16 g, control 109 & 15 g, cholestyramine 107 k 8 g and doxazosin 103 + 7 g* The first group of 9 hamsters fed the atherogenie diet had a moderate level of hypercholesterolemia (287 + 48 mg/dl; Table 1) and hypertriglyceridemia (141 & 67 mg/dl). Density gradient ultracentrifugation demonstrated that 53% of the plasma cholesterol was carried in LDL, while HDL contained approximately 32% of this lipid and the remaining 15% was present in the VLDL fraction. HDL cholesterols determined by the density gradient method were similar to values obtained by phosphotungstate precipitation (Table 1). Ultracentrifugation also indicated that VLDL carried 75% of the plasma triglycerides. In the lesion study, plasma total, LDL plus VLDL and HDL cholesterols and total trigylcerides of baseline hamsters were predictably low, whereas controls had far greater lipid levels in each fraction (Table 2). Compared to the control group, cholestyramine treatment resulted in a decrease of total and LDL plus VLDL cholesterol of 43% and 56%, respectively, while the level of HDL cholesterol remained unchanged. Plasma triglycerides decreased by 42% (Table 2). Similarly, doxazosin produced a decline in plasma total and LDL plus VLDL cholesterols, and total triglycerides of 48%, 65% and 47%, respectively, whereas HDL cholesterol was normal. Cholestyramine and doxazosin lipid levels were significantly greater than those of the baseline frac-

1

CHOLESTEROL LEVELS (MEAN f SD) IN PLASMA LIPOPROTEIN PLUS 0.05% CHOLESTEROL AND 10% COCONUT OIL

Fractions separated by apo B precipitation Fractions separated by ultracentrifugation

FRACTIONS

FROM

F,B HAMSTERS

FED CHOW

Total

LDL

VLDL

LDL + VLDL

HDL

hug/d0

(mg/dl)

(mg/dl)

(mg/dl)

(mg/dl)

197+47

90+

189 + 45

88+10

_

287 f 48 277 f 59

147f41

42+21

8

39 TABLE

2

PLASMA LIPID LESION STUDY

VALUES

(MEANkSDl

Chow Baseline Control 0.5% Cholestyramine 0.02% Doxazosin ” P < 0.05 compared h P < 0.05 compared ’ P < 0.05 compared

TABLE

OF

BASELINE,

CONTROL

AND

EXPERIMENTAL

HAMSTERS

Total cholesterol

LDL + VLDL cholesterol

HDL cholesterol

Total triglycerides

(mg/dl)

(mg/dll

(mg/dl)

(mg/dl)

116+ 14 a 322 k 69 183rt31 h 168+39’

58 f 9 h,c 234 + 57 103+32’ 82+31’

58+ 7a 881t26 so+ 8 86+12

87& 22” 216+ 102 l26+ 26 h 114* 12h

to the control, cholestyramine to the control group. to the cholestyramine group.

and doxazosin

OF

THE

groups.

3

MEAN ARTERIAL HAMSTERS

MAP (mm Hg) HR tbeats/min.)

PRESSURE

AND

HEART

RATE

(MEAN*

SD)

OF

CONTROL

AND

DOXAZOSIN

Chronic measurements conscious unrestrained

in hamsters

Temporary measurements in semiconscious hamsters

Active Cl

Resting

Controls

Doxazosin

l42+ 11 402 f 40

121+11 396 & 39

142+11 363 i 24

117* 10 h 349 ri_30

” Active means eating, drinking, grooming h P < 0.05 compared to the semiconscious

TREATED

and climbing. control group.

tions, except for the comparison between baseline and doxazosin LDL plus VLDL cholesterol (Table 2). Chronic monitoring in conscious hamsters indicated that the MAP ranged between 121 and

142 mm Hg, depending on the level of physical activity, while HR was relatively stable (Table 3). In the lesion experiment, temporary MAP for semiconscious controls averaged 142 mm Hg, whereas the blood pressure of the doxazosin

.. Fig. 1. Chow baseline hamster. Ascending aortic arch stained with silver viewed en face. Blood flow is from left to right. (A) Along the inner curvature, there are many small polygonal endothelial cells having the appearance of cobblestones ( x 290). (B) The outer curvature of the same specimen. where elongated endothelial cells are less dense and are aligned in the direction of blood flow. Medial smooth muscle cells are lightly outlined ( x 290).

41 TABLE

4

FATTY STREAK TAL HAMSTERS

Chow Baseline Control 0.5% Cholestyramine 0.02% Doxazosin ’ ” c d

P < 0.05 compared P < 0.05 compared n=6. n=o.

PARAMETERS (MEAN + SD) IN THE AORTIC OF THE LESION STUDY

ARCH

Adherent mononuclear cells/mm*

lntimal foam cells/mm2

3irl 3+2 3+2 3+1

5+ 7” 91+45 34klO h 29+21 b

to the control, cholestyramine to the control group.

OF BASELINE,

Foam cell size (pm*)

CONTROL

AND

EXPERIMEN-

Intimal Oil red 0 (pg/mm’l

and doxazosin

group was 18% lower. Heart rate for both groups were similar (Table 3). Although the MAP for baseline hamsters averaged 134 k 8 mm Hg and was similar to the controls, baseline HR was significantly less at 326 k 13 beats/minute (mean _t SD; P < 0.0.9. The MAP and HR of one cholestyramine hamster was close to the averages of the baseline group. In baseline hamsters, silver stained en face specimens revealed that the inner curvature of the aortic arch was comprised of many small, polygonal endothelial cells in a well defined area. The outer curvature of the arch had large, elongated cells aligned in the direction of the blood flow (Fig. 1A and B). Hematoxylin and OR0 staining indicated that endothelial mitotic figures and mononuclear cells attached to the luminal surface were rare in all areas. There were few small subendothelial foam cells and extracellular

69* 7” 149i_ 18 114+ 19 h 98k21 b

0.001 0.055 0.022 0.027

+ + * +

0.008 3.r 0.028 0.013 h 0.009 b,d

groups.

lipid droplets along the inner curvature of the arch. Control en face specimens demonstrated that the number of mononuclear cells attached to the endothelial surface was low, however all animals had many plump foam cells and extracellular lipid droplets located along the the inner curvature beneath the area of the polygonal endotheha1 cells (Fig. 2A and B). Therefore this region was regarded as lesion-prone. Quantitative analysis showed that the atherogenic diet dramatically increased the number of intimal foam cells/mm*, foam cell size and intimal OR0 staining compared to chow alone (Table 4). Endothelial lipid droplets and mitotic endothelial cells were scarce in the lesion-prone region. In cholestyramine treated hamsters the number of adherent mononuclear cells was similar to controls, whereas intimal foam cells/mm’ de-

4-

Fig. 2. Control hamster fed 0.05% cholesterol and 10% coconut oil. En face specimen of the aortic arch stained with Oil red 0. Blood flow is left to right. (A) At low power many lipid inclusions are visible along the inner curvature t x25). (B) Higher magnification details numerous intimal macrophage-foam cells engorged with lipid. Arrowheads indicate some of the individual foam cells (X 885). Fig. 3. Lesion-prone

area of the aortic arch from a hamster treated with cholestyramine. (A) Far less intimal Fig. 2A t x 25). (B) Foam cells are fewer and smaller than those of controls (X 885).

Fig. 4. Aortic arch stained with Oil red 0 from a hamster intimal lipid compared to Fig. 2A (X 25). (B) The number

lipid is present

than in

treated with doxazosin. (A) Low magnification illustrates the decrease in and size of the intimal foam cells are less than those of controls ( x 885).

Fig. 5. Baseline hamster. Toluidine blue stained cross section through the inner curvature of the aortic arch. The wide intima consists of extracellular hyaline material overlying a “cushion” of smooth muscle cells, which are surrounded by a thin sheath of elastin (arrowheads) (X 420).

Fig. 6. Control hamster. A cross-section through the lesion prone area stained with toluidine blue. Two foam cells are located in the subintima (arrowheads). The thick extracellular matrix and smooth muscle cell “pads” appear similar to the baseline hamster in Fig. 5 (X 420).

Fig. 7. Qoxazosin treated hamster. A cross-section demonstrates that the intima of the lesion-prone area resembles the control in Fig. 6 (X 420).

creased by 63%, their average size was 23% smaller and intimal of OR0 staining fell by 60% (Fig. 3A and B; Table 4). Compared to controls, hamsters in the doxasozin group had low numbers of adherent mononuclear cells, 68% fewer foam cells/mm2, a 34% decrease in the size of these cells and a 51% reduction of OR0 staining (Fig. 4A and B; Table 4). In both drug treated groups, intimal foam cells localized predominantly along the inner curvature of the aortic arch. When cholestyramine and doxazosin treated hamsters were compared to the baseline animals, the chow-fed group had on average 84% fewer

foam cells/mm 2, the cells were 35% smaller and OR0 staining was 96% less (Table 4). Semi-thin sections of baseline arches demonstrated that the region with high endothelial cell density had a thickened subendothelial space, containing hyaline material layered over smooth muscle cell “cushions”. These cells were surrounded by a thin sheath of elastin (Fig. 5). By electron microscopy the endothelial lining was undulating with a villous surface and the subjacent extracellular matrix was composed of dense granular material (Fig. 8). In other regions of the arch, the endothelium rested on the internal elastic lamella. In the lesion-prone area of control arches, a few macrophage-foam cells were imbedded within the hyaline matrix. Eight weeks of hyperlipidemia did not alter the thickness of the matrix nor change the morphological appearance of the smooth muscle cell “pads” (Fig. 6). By electron microscopy, scattered intimal foam cells bulged into the endothelium resulting in an attenuated lining. The subendothelial space contained clumps of elastin adjacent to the smooth muscle cells, reticular basement membrane-like material and many clear droplets with small myelin figures inside (Fig. 9). Two months of treatment with either cholestyramine or doxazosin did not markedly affect the architecture of the lesion-prone intima, except for reducing the number of foam cells; cross sections from both treated groups appeared similar to those of controls. In the doxazosin group there were a few foam cells, and the intimal smooth muscle cell masses were located beneath a thick layer of hyaline (Fig. 7). Electron microscopy demonstrated that the extracellular matrix consisted of multiple strands of reticular material, elastin and small droplets containing myelin figures (Fig. 10). Discussion The hamster model

The F,B hamster has several useful features such as a sensitivity to dietary cholesterol and saturated fat, that leads to a moderate level of hyperlipidemia. Most of the plasma cholesterol and triglycerides were carried by the LDL and

.h

Fig. 8. A transmission electron micrograph (TEM) of the lesion-prone intima from a chow-fed hamster. The undulating endothelium (E) has a villous surface. The subintima contains a thick layer of dense granular material, one mononuclear cell CM) and a single smooth muscle cell “pad” (S) surrounded by elastin ( x 6900).

VLDL respectively, which is similar to the lipoprotein profile of humans. HDL cholesterols determined after precipitating apolipoprotein B were close to the ultracentrifugation values. Thus rapid analysis of lipoprotein subfractions from whole plasma was possible. The increased level of LDL cholesterol in response to the atherogenic diet was probably a result of reduced hepatic LDL receptor activity [32]. Other advantages of the hamster are the frequency and the size of the atherosclerotic lesions. Fatty streaks consistently develop along the inner curvature of the aortic arch, hence the term lesion-prone area. During severe hypercholesterolemia fibro-fatty plaques ultimately evolve in the same region ([33], M.C. Kowala, unpublished observations). Since the site of lesion development is predictable, it allows meaningful studies on atherosclerosis prevention or regression. In addition, the small size of both types of lesions are suitable for morphometric analyses.

Plasma lipid reductions and mechanisms

In hamsters, cholestyramine treatment decreases LDL cholesterol without affecting the HDL fraction which agrees with studies in humans [26,27] and other animals [34-361. Cholestyramine reduces LDL cholesterol by sequestering bile acids in the intestine, which in turn increases fecal sterol excretion [25] and thus depletes the entero-hepatic pool of circulating cholesterol. The liver compensates by upregulating LDL receptor activity and increases the fractional catabolic rate of plasma LDL cholesterol [34-371. The mechanism behind the decrease of VLDL triglycerides remains unclear, one possibility is that elevated hepatic B/E receptor activity could enhance the catabolism of VLDL particles as we have previously suggested [381. Doxazosin in hamsters produces a lipid response similar to cholestyramine by significantly decreasing LDL cholesterol and VLDL triglycerides, without affecting HDL cholesterol. This

Fig. 9. TEM from a control hamster. The subendothelial foam cell (F) bulges towards the lumen resulting an attenuated endothelial lining. The intimal “cushion” of two smooth muscle cells is surrounded by islets of elastin. There is reticular basement membrane-like material adjacent to the endothelial cell (E), and clear droplets containing myelin figures (arrowheads) scattered in the subendothelial space ( X 11450).

effect differs slightly from that of humans where LDL cholesterol declines as HDL cholesterol becomes elevated [5,6]. Chow-fed hamsters treated with doxazosin showed a decrease in cholesterol in all lipoprotein fractions [391, whereas rats receiving a cholesterol-free diet 1401 and hypercholesterolemic monkeys [41] responded in a simi-

lar manner to doxazosin as the hamsters. Although serum levels of doxazosin were not assayed in this study, equivalent doses of this drug in hamsters (10 mg/kg/day) and in rats (16 mg/kg/day), produced average amounts of 10 ng/ml (A. Swindel, unpublished observations) and 69 ng/ml [42] respectively. This is within the

Fig. 10. TEM of the lesion-prone reticular basement membrane,

region from a doxazosin-treated hamster. The subendothelium droplets of lipid with myelin figures and a prominent “cushion”

range of lo-75 ng/ml found in the serum of treated hypertensive patients [43]. There are several possible mechanisms behind the lipid lowering effect of doxazosin. Alpha-1 adrenergic inhibitors of the quinolazine series appear to inhibit cholesterol absorption through the gastrointestinal system [44,45]. Another mechanism is a diminished synthesis of cholesterol, since doxazosin treatment inhibits this pathway in cultured Hep G2 cells and in hamsters [46,47]. In response to doxazosin, the liver may compensate by secondarily upregulating the LDL receptor and increasing its catabolism of circulating LDL cholesterol. Supporting evidence comes from studies where doxazosin treated Hep G2 cells have elevated LDL receptor activity [46]. Whether upregulation of the LDL receptor by doxazosin is a primary effect, or secondary to a

contains a foam cell (F), strands of of smooth muscle cells ( X 6900).

decrease in cholesterol synthesis or absorption remains to be determined. Enhanced lipoprotein lipase activity [48] or decreased VLDL synthesis may account for the reduction of VLDL triglycerides. Arterial blood pressure

The MAP and HR of semiconscious hamsters were similar to those of conscious active animals, which validated the measurements made by the temporary indwelling catheters. Both methods indicated that blood pressure in the hamster was higher than those of rats [49]. Alpha-l adrenergic inhibition significantly decreased MAP in nonfasted hamsters. This result contrasts a preliminary study, which failed to show a response in fasted animals [28] probably as a result of low levels of doxazosin in the circulation. Similar

46 pressure reductions have been reported in treated humans [3,4] and other animals [41,42,50]. The decrease in blood pressure was probably a result of doxazosin interacting with smooth muscle cell alpha-l adrenergic receptors, which in turn inhibited norepinephrine induced vasoconstriction of small arteries [l]. The lesion-prone area

In the hamster the region susceptible to early atherosclerosis occurs along the inner curvature of the aortic arch, and is characterized by small polygonal endothelial cells, and a thick intima consisting of basement membrane-like material layered over smooth muscle “pads”. During hyperlipidemia, droplets containing myelin figures are located within the intima, and these structures possibly represent aggregates of lipoproteins. Resistant areas of the arch have large elongated endothelial cells aligned in the direction of the blood flow, and the endothelium rests directly on the internal elastic lamella. Polygonal endothelial cells exist in regions of low or increasing shear stress [51], and low shear occurs along the inner curvature of arteries [52,53]. In low or increasing shear regions of rat aortas, mononuclear cells preferentially emigrate into the subendothelial space [51]. During hypercholesterolemia, ORO-positive lipid accumulates in the same area [54]. In rabbits, LDL is selectively trapped and metabolized in the aortic arch [55]. Thus the hamster lesion-prone area is possibly influenced by low shear, and is associated with a morphologically unique intima that favors lipid and leukocyte accumulation. It may in part explain the susceptibility of this region to the formation of fatty streaks. In our lesion experiment, all four groups had few mononuclear cells attached to the luminal surface, which suggest that in control hamsters most adherent cells probably emigrated into the intima. Prevention of the fatty streak

Cholestyramine and doxazosin treatment similarly lowered plasma LDL cholesterol and VLDL triglycerides and in turn reduced foam cell number, the size of these cells, and intimal OR0 staining approximately to the same degree. Therefore doxazosin and cholestyramine proba-

bly lowered plasma lipids and inhibited early atherogenesis. This result differs from a study in severe hypercholesterolemic rabbits (total cholesterol 1600-1800 mg/dl), where doxazosin diminished the area of the fatty streak and aortic cholesterol ester content without affecting plasma total cholesterol (A. Swindel, unpublished observations), thus suggesting an effect independent of plasma lipids. Doxazosin also reduced aortic collagen synthesis in spontaneously hypertensive rats, beyond its influence on blood pressure [42]. Whether doxazosin had a direct impact on atherosclerosis in the hamster study remains unclear. Treatment with either drug may have produced the maximum possible reductions in the three fatty streak parameters, therefore an additional effect of doxazosin would remain undetected. However there was a significant difference in the progression of fatty lesions between doxazosin treated hamsters and the baseline animals, indicating some potential for further decreases in the doxazosin group. The data suggest that if doxazosin had also directly inhibited lesions, the lipid reduction may have precluded any evidence to indicate this effect. Since elevated blood pressure is associated with accelerated atherosclerosis, it was anticipated that the decline of MAP with doxazosin would produce a further decrease in the fatty streak, when compared to the cholestyramine group. One explanation why this failed to occur is that mild hypotension may not protect the arterial intima of individuals that were originally normotensive. Another possibility is that the level of blood pressure may not entirely influence for example, the extent of mononuclear cell diapedesis. In normotensive rats fed chow, mononuclear cells accumulate in the aortic intima as a result of aging [56], and even in experimental models of hypertension, aggravated leukocyte emigration appears to be independent of blood pressure 1.571. To summerize, in hamsters with elevated LDL cholesterol and VLDL triglyceride, macrophagefoam cells rapidly accumulate along the inner curvature of the aortic arch. This lesion-prone region has a high density of polygonal endothelial cells, and a wide subendothelial space containing a thick matrix of basement membrane-like mate-

47 rial layered over “pads” of smooth muscle cells. Hyperlipidemic hamsters treated with either doxazosin or cholestyramine had parallel reductions of LDL cholesterol, VLDL triglyceride, intimal foam cell number, foam cell size and intimal lipid stained with ORO. We conclude that doxazosin inhibits early atherosclerosis to a similar degree as cholestyramine.

in atherogenesis.

12

I3 I4

Acknowledgement IS

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