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Page 1: Estrogen and Raloxifene Activities on Amyloid-β-Induced Inflammatory Reaction

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Microvascular Research 61, 28–39 (2001)doi:10.1006/mvre.2000.2267, available online at http://www.idealibrary.com on

Estrogen and Raloxifene Activities on Amyloid-b-Induced Inflammatory Reaction

Tom Thomas,*,† Margie Bryant,* Linda Clark,* Amanda Garces,*nd Johannes Rhodin*

*Department of Anatomy, College of Medicine, University of South Florida, Tampa, Florida 33612; and†Woodlands Medical and Research Center, 3150 Tampa Road, Oldsmar, Florida 34677

Received July 11, 2000; published online November 14, 2000

The prevalence of Alzheimer’s disease (AD) in women isdouble that of men. Several studies indicate that use ofestrogen after menopause by women may reduce the riskof developing AD. The risk of estrogen-dependent tumorsassociated with estrogen replacement therapy hasprompted the use of alternatives, like the SERM ralox-ifene, which exert estrogen agonist effects on selectedtarget tissues. Whether SERMS provide cognitive andcardiovascular benefits comparable to those of estrogensis an active area of investigation in women’s health. Achronic inflammatory process is central to the pathologyof Alzheimer’s disease. Using an animal model we com-pared the anti-inflammatory activity of orally adminis-tered estrogens (2 mg/kg) and raloxifene (3 mg/kg) inovariectomized rats. Morphological analysis of Ab(1–40)-nduced inflammatory reaction featured adhesion andransmigration of leukocytes across the vessel wall, en-othelial disruption, and platelet activation. Estrogenhowed remarkable anti-inflammatory action, whereasaloxifene had no significant beneficial effect. Inhibitionf the inflammatory process may contribute to the re-orted efficacy of estrogen in the treatment ofD. © 2000 Academic Press

Key Words: menopause; estrogen; estrogen replace-

ment therapy; SERM; raloxifene; Alzheimer’s disease;cardiovascular disease; inflammation; amyloid-b; animalmodel; endothelium; platelet.

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INTRODUCTION

Menopause is a natural and physiological event af-fecting all women, with a mean age of onset of 51years. Due to the increase in longevity women arecurrently spending 30 or more years of their life spanbeyond menopause. About one third of the UnitedStates female population is currently postmenopausal.The classical features of menopausal symptoms areconsidered due to alterations in the synthesis andrelease of estrogen, progesterone, and androgens.Central nervous system manifestations of menopause,such as vasomotor symptoms, decreased sex drive,depression, and dementia, have been attributed to adeficiency of estrogen. Numerous observational and afew clinical trials have linked estrogen replacementtherapy (ERT)1 with the amelioration and possiblyprevention of vasomotor symptoms, osteoporosis and

1 Abbreviations used: Ab, amyloid-b protein; AD, Alzheimer’sdisease; ERT, estrogen replacement therapy; HDL, high-density li-poprotein; CEE, conjugated equine estrogens; HRT, hormone re-placement therapy; NFkB, nuclear factor k B; NO, nitric oxide;

SAIDs, nonsteroidal anti-inflammatory drugs; PARs, proteinase-ctivated receptors; SERMs, selective estrogen receptor modulators;NF-a, tumor necrosis factor-a; TRIP, Thomas–Rhodin inflamma-

tion profile.

All rights of reproduction in any form reserved.

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bone fractures, cardiovascular disease, stroke, Alzhei-mer’s disease, Parkinson’s disease, colon cancer, andretinal macular degeneration (Ernster and Cummings,1992; Halley et al., 1998; Sherwin, 1996; Strijks et al.,1999). Only 10–15% of women who might benefit fromhormone replacement therapy (HRT) receive it andmore than 60% of women placed on HRT discontinuethe treatment within 1 year. This is due to the fear ofthe increased risk of breast cancer and endometrialhyperplasia associated with long-term unopposedERT (Persson et al., 1996).

The undesirable side effects of and poor compli-ance with HRT have led to search for designer es-trogen-like molecules which retain the beneficialactions of estrogen, but are devoid of hormonaleffects on reproductive organs. Selective estrogenreceptor modulators (SERMS) are such a new classof compounds that interact with estrogen receptors,exerting estrogen agonist effects on some tissuesand antagonist effects on others (Mitlak and Cohen,1997). The first compound belonging to this class,tamoxifen citrate (Nolvadex), was originally devel-oped to treat breast cancer and currently is the mostwidely prescribed anti-neoplastic drug worldwide(Sato et al., 1996). The initial enthusiasm for SERMS

as considerably tempered when it was discoveredhat tamoxifen has proliferative or neoplastic effectsn the endometrium and increased the risk forhromboembolic events. The second generationERM raloxifene hydrochloride ( EVISTA) was alsoriginally developed to treat breast cancer and isurrently approved for the prevention of postmeno-ausal osteoporosis (Fuchs-Young et al., 1995).

Raloxifene has tissue specificity with estrogen an-tagonistic effects on breast and uterus, but estrogenagonistic effects on bone (Bryant et al., 1996). Ralox-ifene exerts a limited favorable effect on the plasmalipid profile, but unlike estrogen, it does not accordadditional cardioprotection by elevating HDL lev-els. A recent report (Clarkson et al., 1998) showedthat in postmenopausal monkeys, estrogen pro-duced a 70% reduction in coronary artery plaquesize, whereas raloxifene had no detectable effect.

Estrogen Inhibits Amyloid-b-Induced Inflammation

The cardiovascular benefits of raloxifene are cur-rently being explored. The undesirable side effectsof raloxifene include a higher incidence of hot

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flashes and an increased risk of venous thromboem-bolic events (Davies et al., 1999).

Alzheimer’s disease (AD) is the most prominentform of dementia and the rate of AD is currentlyapproaching epidemic proportions, affecting 50% ofindividuals beyond age 80. Women have a two- tothreefold higher age-specific prevalence of AD (Ri-pich et al., 1995). No identifiable cause has been

ttributed to the majority of cases of AD, and cur-ent therapies have met with only modest success. Aumber of reports have indicated that estrogen ther-py may retard the development and severity ofementia in postmenopausal women (Kawas et al.,997). At the same time, there are reports whichndicate that short-term estrogen therapy may notmprove symptoms of AD in women (Henderson etl., 2000). The association of several inflammatoryediators with lesions detected in the brains of

atients with neurodegenerative diseases, such aslzheimer’s disease, Parkinson’s disease, amyotro-hic lateral sclerosis, and multiple sclerosis, has led

o the suggestion that the neuronal damage ob-erved in these conditions may be the result of ahronic innate immune reaction in the brainMcGeer et al., 1996). Several factors, including thelzheimer peptide amyloid-b (Ab), can initiate the

classic complement cascade leading to the recruit-ment and activation of microglia which are of mono-cyte/macrophage lineage. Nonsteroidal anti-inflam-matory drugs (NSAIDS) have a protective effectagainst the development of AD (McGeer andMcGeer, 1995). But long-term use of NSAIDS isassociated with deleterious side effects. Using ananimal model (Rhodin et al., 1999), we recentlyshowed that conjugated estrogens have a protectiveeffect against Ab (1– 40)-induced inflammatory reaction(Thomas et al., 1999). There is no clear evidence ofenhancement of cognitive function by raloxifene(Nickelsen et al., 1999). Since an anti-inflammatoryaction would indicate a role in the prevention andtreatment of atherosclerosis and AD, we comparedthe in vivo anti-inflammatory action of conjugated

quine estrogens (Premarin) and raloxifene in ovari-ctomized female rats using morphological tech-iques.

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MATERIALS AND METHODS

Animals. The Institutional Animal Care and UseCommittee reviewed and approved the use of rats inthis research project according to the following proce-dures. Six-month-old female Sprague–Daley rats wereanesthetized intraperitoneally with 60 mg/kg of pen-tobarbital and ovariectomized by making a bilateral1.5-cm incision along the posterior abdominal wall.The ovarian fat pad which contains the ovarian arteryand vein and the oviduct was completely ligated andcut. The fat pad was removed along with the ovariesand the incision was closed using a wound applicator.The rats were kept warm without being given analge-sics and monitored every day for at least 3 weeksbefore the actual experiments. The degree of woundhealing and the rate of their recovering mobility wasassessed every day. Three weeks after the ovariec-tomy, the rats were divided into several experimentalgroups according to the section on Pharmacologicalagents below. Before abdominal surgery, the rats wereanesthetized intraperitoneally with 60 mg/kg pento-barbital. The anesthesia was monitored every 10 minby testing the animal’s corneal reflexes as well as theskin and muscular reflexes by pinching. A log of ob-servations was kept. The anesthesia was maintainedby giving additional small amounts of pentobarbital ifthe reflex monitoring so indicated. At the end of theexperiments, the rats were euthanized by an intraperi-toneal injection of 85 mg/kg pentobarbital.

Pharmacological agents. (1) Freshly preparedmyloid-b (1–40), 100 ng/gram body wt, was diluted

with 0.25 ml isotonic saline and infused for 2 min viaan aortic cannula, the tip of which was positioned nearthe exits of the mesenteric arteries, thus quickly reach-ing the mesenteric microvascular bed. This served todemonstrate the vascular effects of amyloid-b in

variectomized rats. (2) Premarin, 2 mg/kg/day, wasiven orally for 2 weeks, followed by an aortic infu-ion of saline, serving as a control experiment. (3)remarin, 2 mg/kg/day, was given orally for 2 weeks,

ollowed by an aortic infusion of amyloid-b, prepared

s in (1) above, serving to discover if Premarin had areventive action against the vascular effects of amy-

oid-b. (4) Premarin, 2 mg/kg/day, was given orally

for 2 weeks, followed by a 1-week washout of Prema-rin. Subsequently, the rats were given an aortic infu-sion of amyloid-b, prepared as in (1) above. Thisserved to investigate whether an assumed protectiveaction of Premarin was lost. (5) Raloxifene, 3 mg/kg/day, was given orally for 2 weeks, followed by anaortic infusion of saline, serving as a control experi-ment. (6) Raloxifene, 3 mg/kg/day, was given orallyfor 2 weeks, followed by an aortic infusion of amy-loid-ß, prepared as in (1) above. This served to dis-cover if raloxifene had an assumed preventive actionagainst the vascular effects of amyloid-b. (7) Ralox-ifene, 3 mg/kg/day, was given orally for 2 weeks,followed by a 1-week washout of raloxifene. Subse-quently, the rats were given an aortic infusion of amy-loid-b, prepared as in (1) above. This served to inves-tigate whether an assumed protective action ofraloxifene was lost.

Intravital and electron microscopy. In the anes-thetized ovariectomized rats, a cannula was intro-duced into the aorta via the femoral artery and its tippositioned near the exits of the mesenteric arteries. Amidline incision of the abdominal wall was then madeand part of the small intestine together with its mes-entery was pulled out and draped over a lucite ped-estal in a plastic cradle and mounted on the stage of alight microscope equipped with long distance work-ing objectives (5X, 25X, 40X, and 100X oil immersion).The mesenteric surface was kept moist by dripping awarm saline solution at intervals. When using the100X objective, immersion oil was applied to the mes-enteric surface. The mesentery is transparent, makingit possible to observe blood flow and interaction be-tween the formed elements of the blood and the vas-cular wall. The mesenteric field was video recordedfrom the very start of the experiments, continued for60–90 min with an Optronics camera (DEI-750C), andstored on a JCV BR-8000 S-VHS cassette recorder.Selected recordings were stored via a motion videodigitizing manipulation and the playback capturingcard Truevision Targa 2000 DTX and an IBM PCIcomputer and analyzed on a Sony SSM-2010 flatscreen monitor for live display at 750 lines resolution.

Thomas et al.

The observations of the vascular reactions were mon-itored continuously, starting before the administrationof the pharmacological agents and carried out to the

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end of the glutaraldehyde fixation. Subsequently, thevideo recordings were reviewed several times, andmeasurements and accounting of vascular events tab-ulated and evaluated.

At the end of the experiments, the mesentery wassuperfused for 15 min with glutaraldehyde and foran additional 15 min with osmic acid. Subsequently,the observed field was dehydrated, cut out, andembedded in Epoxy resin for transmission electronmicroscopy. Previously recorded microvascular ar-eas were retrieved in the embedded specimen, sec-tioned parallel to the long axes of the blood vessels,and analyzed in a Philips CM 10 transmission elec-tron microscope at magnifications ranging from6003 to 15,0003.

Thomas–Rhodin inflammation profile (TRIP). Theinflammatory cascade involves the directed migrationof leukocytes into perturbed areas by a precisely reg-ulated communication between various cells, orches-trated by a number of molecular signals. To accuratelygauge the degree of inflammatory reaction, it is nec-essary to simultaneously monitor the activites of sev-eral components in the cascade.

We have developed a rating scale (TRIP) to quan-tify the myriad of cellular responses involved in theinflammatory process. This scale allows comparisonof the inflammatory response under various exper-imental and treatment conditions. Signs recordedintravitally include (1) the number of leukocytesmarginating and transmigrating during a 60-minobservation time in 1000-mm segments of arteriolesand venules. (2) The degree of platelet marginationin arterioles and venules. (3) The number of trappederythrocytes in arterioles and venules. (4) Mast cellactivation and degranulation or inhibition thereof.(5) Macrophage activation/inhibition, judged by de-gree of movements. (6)Mast cell/macrophage inter-action. This scale has not been used before. It is anattempt to quantify the approximate degree of reac-tion according to the following scale: No reaction, 0;mild reaction, 1; moderate reaction, 2; severe reac-tion, 3.

The exact number of marginating leukocytes in ar-

Estrogen Inhibits Amyloid-b-Induced Inflammation

terioles was based on an actual count, and its statisti-cal significance was calculated (Table 1). The numberof transmigrating leukocytes in arterioles was based

on estimation: mild, 2–4; moderate, 10–12; severe,15–20. The number of marginating leukocytes invenules was based on estimation: mild, 100; moderate,300; severe, 400. Other parameters were estimated:mild, 1; moderate, 2; severe, 3. Values for individualanimals were tabulated and the mean value foreach group was compared for treatment differences(Table 2).

RESULTS

Intravital Observations

Ab alone. Infusion of freshly prepared Ab(1–40) (thepredominant form of Ab in the circulation and cere-brovascular deposits) produced a rapid increase in theblood level of Ab at 30 min from a basal value of 1.6ng/ml. The majority (99%) of the administered Ab

was cleared from the circulation within 1 h (Sutton etal., 1999). Serum TNF-a levels were also significantlyelevated at 1 and 2 h post Ab infusion (Sutton et al.,999). Ovariectomized female rats (n 5 4) were in-used with 100 ng Ab/g body weight for 2 min. An

average segment of 1000 mm of arterioles was re-corded for at least 60 min. There were 10.5 6 1.7leukocytes that marginated and adhered to the endo-thelium, and about one third transmigrated and be-came lodged in the arteriolar wall (Figs. 1A–1C). Atcertain areas, often near the marginated leukocytes,platelets also marginated (Figs. 3A and 3B). Some

TABLE 1

Number of Leukocytes Adhering to the Endothelium during a60-Min Observation Time in a 1000-mm Segment of Arterioles

TreatmentNumber of WBCs stuck

[mean 6 SEM (n)]

Ab alone 10.5 6 1.7 (4)Premarin 1 Ab 3.6 6 0.8* (5)Raloxifene 1 Ab 7.3 6 1.9 (4)

Note. Statistical analysis was performed by t test: *P # 0.01Premarin vs Ab alone. Raloxifene vs Ab alone: no significant dif-erence.

tumbled over the endothelial surface, others becameattached, and some reached the subendothelial space.The degree of reaction was determined as severe. An

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inflamas perarin vs

equally long segment of postcapillary venules andmuscular venules was observed simultaneously. Ap-proximately 400 leukocytes marginated momentarilyand of these, about 70% transmigrated and becamelodged in the venular wall (Figs. 2A and 2B). Therewas considerable degranulation of mast cells (Fig. 3C),macrophage activity, and also mast cell/macrophageinteraction. The degree of reaction was determined assevere.

Premarin 1 Ab. Ovariectomized female rats (n 5

5) received a daily oral dose of 2 mg Premarin/kgbody weight for 2 weeks and then were infused with100 ng Ab/g body weight for 2 min. During anobservation time of 60 min, 3.6 6 0.8 leukocytesmarginated and adhered in a 1000-mm segment of

rterioles (Figs. 1D and 1E). Platelets did not mar-inate. An average of 100 leukocytes marginated in

1000-mm segment of postcapillary venules and

TABLE 2

TRIP Inflammation Profile

Note. Comparison of components of the4, 5, and 4 animals. Statistical analysis wPremarin vs Ab alone. **P # 0.001 Prem

muscular venules. About 20% of these leukocytesbecame lodged in the wall (Fig. 2C). There was littlemast cell degranulation (Fig. 3D) and no macro-

phage activity. The overall degree of reaction wasdetermined as mild.

Premarin washout 1 Ab. When Premarin treat-ment was discontinued for 1 week, the animalsshowed significant reaction to Ab.

Raloxifene 1 Ab. Ovariectomized female rats(n 5 4) received a daily dose of 3 mg raloxifene/kg

ody weight for 2 weeks and were then infused with00 ng Ab/g body weight for 2 minutes. During an

observation time of 60 min, 7.3 6 1.9 leukocytes mar-ginated and adhered in a 1000-mm segment of arte-rioles (Figs. 1F and 1G). Only a few platelets margin-ated. The degree of reaction in arterioles wasdetermined as mild to moderate. An average of 400leukocytes marginated in segments of postcapillaryvenules and muscular venules. Of these, 70–80% be-came lodged in the venular wall (Fig. 2D). This reac-tion was determined as severe. Pronounced degranu-

matory cascade. Values represent mean offormed by one-way ANOVA: *P # 0.05Ab alone and raloxifene.

Thomas et al.

lation of mast cells and macrophage activity werenoted. The overall reaction in venules was determinedas severe.

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FIG. 1. Arterioles. (A) Ab alone. In these closely apposed mesenteric microvessels, a leukocyte (1) is adhering to the arteriolar endothelium2), whereas the leukocyte (3) in the postcapillary venule is marginated and rolling slowly. The direction of blood flow is indicated by arrows.ideo recording. Original magnification, 15003. (B) Ab alone. The monocyte (1) is firmly attached to the endothelial (2) cell surface of this

precapillary arteriole. It is drop-shaped, caused by he force of the blood flow. Arrows indicate flow direction. Erythrocytes (3) are also seen.Electron micrograph. Original magnification, 80003. (C) Ab alone. The cytoplasm of this arteriolar endothelial cell displays vacuoles (1) of

arious sizes, a typical early sign of cytoplasmic damage. The smooth muscle cell (2) is not damaged, and there is still no plasma leakageccumulating between the endothelial cell and the smooth muscle cell. Electron micrograph. Original magnification, 11,5003. (D) Premarin 1

b. It is apparent that Premarin completely blocks the inflammatory reaction in this arteriole. This is a completely normal arteriolar wall. Thereare no monocytes adhering to the endothelial surface (1), and platelets (2) do not marginate. There is no plasma leakage accumulation betweenthe endothelial cell and the smooth muscle cells (3). Electron micrograph. Original magnification, 66003. (E) Premarin 1 Ab. The cytoplasmof the arteriolar endothelial cell (1) is not damaged. Organelles seen are microtubules (2), one small microvesicular body (3), and micropino-cytotic vesicles (4). The cytoplasm of the smooth muscle cell (5) is completely normal. Electron micrograph. Original magnification, 18,0003.(F) Raloxifene 1 Ab. On the average, four to five leukocytes would become attached to the endothelial surface in a 1000-mm stretch of arterioles.

33Estrogen Inhibits Amyloid-b-Induced Inflammation

his is a marginated monocyte (1), adhering to the endothelial cell (2). A platelet (3) is also marginated. Infrequently, but not seen here, smallmounts of plasma was observed accumulating between the endothelial cell and the smooth muscle cells (4). Electron micrograph. Originalagnification, 60003. (G) Raloxifene 1 Ab. The cytoplasm of the arteriolar endothelial cell (1) shows a limited degree of vacuolization (2), often

eading to cytoplasmic disruptions and shedding of cell fragments (3). Electron micrograph. Original magnification, 16,0003.

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Raloxifene washout 1 Ab. In this group of ani-mals the raloxifene was discontinued for 1 week. The

FIG. 2. Venules. (A) Ab alone. In this postcapillary venule, thereining, making the wall of the venule look abnormally thick. Some lo the venular wall (which is not in focus). This is a typical inflammf blood flow. Video recording. Original magnification, 25003. (B)here is one circulating monocyte (2). Another monocyte (3) is adheubendothelial space and another (5) is in the process of reaching thow. Electron micrograph. Original magnification, 26003. (C) Premostcapillary venule. There are no marginating leukocytes at the sueached the subendothelial space, and one (3) reached the paravenituation corresponds to the modest reaction recorded in controlaloxifene 1 Ab. It is apparent that raloxifene does not entirely bloore severe than that in C, and several granulocytes (1) have tran

ecoming marginated. No leukocytes have reached the paravenular0003.

margination of leukocytes and platelets in arterioleswas mild to moderate, whereas the leukocyte margin-ation and transmigration in venules were determined

as moderate to severe. The mast cell degranulationand macrophage activity were mild to moderate.

ronounced transmigration of leukocytes (1) across the endothelialtes (2) were observed to roll slowly, and still others (3) were stuckreaction after amyloid-b administration. Arrow indicates directionne. Detail of the wall of a venule. Among the free erythrocytes (1)the endothelial surface. Several granulocytes (4) are lodged in the

venular connective tissue space. Arrow indicates direction of bloodAb. Premarin blocks completely the inflammatory reaction in thisf the endothelial cell (1). A small number of granulocytes (2) have

onnective tissue space with its bundles of collagen fibers (4). Thisiments. Electron micrograph. Original magnification, 70003. (D)inflammatory reaction in this postcapillary venule. The reaction isted, and one monocyte (2) and several associated platelets (3) arective tissue space (4). Electron micrograph. Original magnification,

Thomas et al.

is a peukocyatory

Ab aloring toe paraarin 1

rface oular cexper

Controls. One group of ovariectomized rats (n 5

) received a daily dose of 2 mg Premarin/kg bodyeight for 2 weeks. A second group of rats (n 5 4)

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der, prry casc

received a daily dose of 3 mg raloxifene/kg bodyweight for 2 weeks. Subsequently, the rats in bothgroups received an infusion of 0.25 ml of saline for 2

FIG. 3. (A) Ab alone. In this arteriole, there is a slight thickening ofplatelets (2), some adhering momentarily, others being stuck forchanging situation. Arrow indicates direction of blood flow. Video rmicrograph of the identical area recorded in A (rectangle). One pprobably were tumbling at the moment of fixation. The endothelial c(4) is not; cytoplasmic process of fibroblast (5). Electron micrograactivated, degranulated mast cell. The central nucleus (1) is surrounOther granules (3) rest in loosely fitted pockets of the cytoplasm. T(4). Electron micrograph. Original magnification, 25003. (D) Premardistinct granules (2) closely packed, and a well demarcated cell borto an arteriolar wall (4). Obviously, Premarin inhibits the inflammatoOriginal magnification, 90003.

Estrogen Inhibits Amyloid-b-Induced Inflammation

min. There was no reaction recorded in relation toarterioles, venules, mast cells, and macrophages in therats of either group in the absence of Ab.

Electron Microscopic Observations

Ab alone. The cytological changes that appeared

scular wall (1) and a distinct margination and adhesion of activatedl minutes. The video recording clearly demonstrated the rapidlyng. Original magnification, 20003. (B) Ab alone. This is an electron(1) is attached to the endothelium (2), whereas the other two (3)sm is slightly damaged and vesiculated, but the smooth muscle celliginal magnification, 11,5003. (C) Ab alone. This shows a highlya large number of granules, most of which are widely scattered (2).border shows an intricate network of microvilli and cell processesb. This shows a nonactivated mast cell with its central nucleus (1),

ovided with short microvilli (3). The mast cell is in close proximityade brought on by amyloid-b administration. Electron micrograph.

the vaseveraecordilateletytopla

ph. Orded byhe cellin 1 A

in the walls of arterioles after an infusion of Ab invariectomized female rats were similar to thoseeported earlier in male rats (Thomas et al., 1999).

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Several organelles, including mitochondria and en-doplasmic reticulum in the endothelial cells, weredamaged, and vacuoles of varied sizes appeared inthe cytoplasm, which also was rarefied and oftendisrupted (Fig. 1C). The smooth muscle cells werenot damaged. The walls of postcapillary venulesand muscular venules contained a varied number ofleukocytes, lodged in the subendothelial space (Fig.2B). Degranulated mast cells showed freely dis-persed granules and some still remaining within thecell (Fig. 3C).

Premarin 1 Ab and Premarin 1 saline (controls).hese two groups of ovariectomized female rats dis-layed perfectly normal cytological details in endothe-

ial as well as in smooth muscle cells. This mimickedhe results of similar experiments in male rats (Figs.D and 1E).Premarin washout 1 Ab. Changes occurred in

the walls of arterioles as well as venules ofovariectomized female rats which were very simi-lar to the damage and cytological alterationsthat appeared after infusion of Ab alone. Also,hese findings were similar to those observed in

ale rats.Raloxifene 1 Ab. Marginated and adhering leuko-

cytes in arterioles were identified as monocytes (Fig.2D). None was found to have transmigrated the en-dothelial layer. The endothelial cells of arterioles con-tained bloated mitochondria and a varied number ofsmall to medium size vacuoles (Fig. 1G). There wassome cytoplasmic disruption and fragmentation. Thesmooth muscle cells had a normal appearance. Therewas no plasma leakage in the subendothelial space.The walls of postcapillary venules and muscularvenules contained numerous leukocytes, some mar-ginated and adhering to the surface of the endothe-lium (Fig. 2D). However, the majority of leukocytes(monocytes and polymorphonuclear leukocytes) werelodged in the subendothelial space.

Raloxifene washout 1 Ab. The cytological alter-ations were similar to those in rats infused with Ab

alone.

Raloxifene 1 saline (controls). The endothelialand smooth muscle cells of arterioles and venulesdisplayed a completely normal cytology.

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TRIP

As summarized in Table 1, there was a significantdifference in the number of leukocytes adhering to theendothelium in a 1000-mm segment of arterioles inPremarin-treated animals vs animals treated with Ab

alone. In animals treated with raloxifene vs animalstreated with Ab alone, there was no significant differ-ence.

As summarized in Table 2, comparing several com-ponents of the inflammatory cascade, including leu-kocyte adhesion in venules, platelet margination, andmast cell degranulation, Premarin demonstrated anoverall anti-inflammatory activity superior to that ofraloxifene.

DISCUSSION

The inflammatory process is considered a major fac-tor contributing to neuronal damage in neurodegen-erative diseases, such as Alzheimer’s disease, Parkin-son’s disease, amyotrophic lateral sclerosis, andmultiple sclerosis (McGeer and McGeer, 1998; Tor-reilles et al., 1999). A chronic innate immune reaction

f brain in response to a variety of noxious stimuli cannitiate a self-sustaining autodestructive sequence in

hich the cell response injures bystander neurons androduces further lesions (McGeer et al., 1996). Inflam-atory activity in the blood vessel wall is currently

hought to play a prominent role in the endothelialysfunction that leads to atherosclerosis, coronary

hrombosis, and acute coronary events (Ross, 1999).here is significant overlap between the inflammationnd coagulation pathways; when the endothelium isisrupted during inflammation, release of procoagu-

ant factors would activate the coagulation process.here is considerable evidence to indicate that leuko-yte adhesion and infiltration following cerebral isch-mia and reperfusion can exacerbate neuronal damageGarcia et al., 1994). This would suggest that inhibitionf the inflammatory process would accord protection

Thomas et al.

gainst vascular and neurodegenerative diseases. Us-ng an animal model of vascular inflammation andovel morphologic techniques, we report the unique

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ability of conjugated equine estrogens to inhibit theinflammatory process, whereas the SERM raloxifenehad no significant protective effect (Figs. 1 and 2).

The role of platelets in thrombogenicity has beenwell characterized. Endothelial disruption by Ab cantrigger platelet adhesion and aggregation. Plateletsalso mediate a pathogenic role in inflammationthrough the production of chemotactic leukotrienes(Evangelista et al., 1999) and may further exacerbatevascular disruption by releasing Ab stored in plate-lets. Raloxifene was particularly ineffective in inhibit-ing Ab-induced platelet activation (Fig. 2D) and this

ay contribute to the increased incidence of thrombo-mbolytic events associated with raloxifene therapy.he amelioration of platelet activation by conjugatedquine estrogens (CEE) was an unexpected finding, asRT has been also associated with the risk of throm-oembolic events.Mast cells play an important role in acute inflam-ation by releasing stored and newly synthesized

nflammatory mediators on activation (Young, 1997).he proteases secreted from activated mast cells, par-

icularly tryptase, contribute to the inflammatory pro-ess by the activation of proteinase-activated receptorsPARs) and microvascular leakage (He et al., 1999).

CEE showed severe inhibition of mast cell activationand degranulation (Figs. 3C and 3D), whereas ralox-ifene exhibited only mild inhibition of mast cell acti-vation.

The lack of a protective effect by raloxifene in ourstudies is similar to the findings of Clarkson et al.(1998), in which CEE produced a significant reductionin coronary artery plaque size, whereas raloxifene hadno effect on coronary artery plaque size in postmeno-pausal monkeys. Another study in ovariectomizedrabbits has previously demonstrated that raloxifeneinhibited the aortic accumulation of cholesterol (Bjar-nason et al., 1997). Several previous studies haveshown a protective effect of estrogen in AD, eventhough short-term therapy may not be beneficial(Henderson et al., 2000). AD is a slow progressive

isorder and neuroprotective agents may not be ableo rescue the neurons once the cellular damage is

Estrogen Inhibits Amyloid-b-Induced Inflammation

xtensive. Therefore, early intervention with cytopro-ective agents like estrogen may be the appropriateourse of action.

The mechanism underlying the anti-inflammatoryction of CEE remains to be established. Oxidativetress induced by Ab (Thomas et al., 1997) or other

agents may activate the transcription factor NFkB (nu-clear factor k B), leading to expression of cellularadhesion molecules and proinflammatory cytokinesinvolved in the adhesion and transmigration of leuko-cytes (Kupatt et al., 1997; Sutton et al., 1999). Theactivation of NFkB is closely regulated by endotheli-um-derived nitric oxide (NO) (Lefer and Lefer, 1996).There is considerable evidence to support the conten-tion that estrogen stimulates NO production in theendothelium and enhances vasodilation and bloodflow in both the peripheral and cerebral vasculature(Dubal et al., 1998; Hashimoto et al., 1995; Kleimert etl., 1998; Karas et al., 1999). Thus it is reasonable topeculate that the anti-inflammatory activity of CEEbserved in our animal model could be partly medi-ted through enhanced NO-dependent mechanisms.strogens may also inhibit the inflammatory reactiony decreasing the production of proinflammatory cy-okines by repressing the induction of TNF-a (An etl., 1999; Sutton et al., 1999). Other genomic as well as

nongenomic membrane actions of estrogen cannot beruled out. CEE is composed of a number of constitu-ents with different modes of action which may actsynergistically to ameliorate the various molecularevents contributing to the pathology of vascular andneurodegenerative diseases. There is considerable ev-idence implicating inflammation in the etiology andpathophysiology of ischemic/thrombotic vasculardiseases. The anti-inflammatory actions of CEEmay contribute to the reported beneficial effectsof estrogens in the amelioration of cardiovascularand neurodegenerative diseases. To the spectrum ofactions mediated by CEE, we will have to add thecontribution of the anti-inflammatory property. Cur-rently available SERMS, such as raloxifene, may haveonly a limited range of targeted activity and may notprovide the broad range of benefits accorded by CEE.The long-term clinical benefits of selective estrogenreceptor modulators (SERMS) relative to estrogen

replacement therapy, especially with regard to car-diovascular disease and dementia, remain to beestablished.

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ACKNOWLEDGMENTS

This study was supported by a grant from Wyeth-Ayerst Phar-maceuticals. None of the authors own any stocks or have anyfinancial interest in any of the companies whose products are dis-cussed in this report. Dr. Thomas and Dr. Rhodin have receivedresearch grants and honoraria for presentations from Wyeth-AyerstPharmaceuticals.

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