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There is substantial evidence that peptides derived from proteolyt-ic processing of the ß-amyloid precursor protein, including the amy-loid-ß peptide (Aß), are involved in the pathogenesis of Alzheimer’sdementia1. However, the mechanisms by which these peptides exerttheir pathogenic effects remain undefined2. Generation of reactiveoxygen species by Aß has been proposed as a potential link betweenAß and neurotoxicity3–6. Studies in isolated blood vessels and inendothelial cell cultures have suggested that peptides derived fromAPP processing produce deleterious vascular effects that may con-tribute to neurodegeneration in Alzheimer’s dementia7–10. Additionof micromolar quantities of synthetic Aß peptide to rings of rat aortacauses loss of vasodilation in response to acetylcholine and increasedcontractility in response to vasoconstrictors, effects caused by severedamage to the endothelial lining of the vascular rings7. These toxiceffects of Aß are prevented by addition of the superoxide-scavengingenzyme SOD. However, it is not known whether nanomolar eleva-tions in Aß comparable to those occurring in Alzheimer’s demen-tia result in superoxide-dependent alterations of cerebrovascularregulation in vivo. Therefore, we used transgenic mice overexpress-ing APP to explore whether elevation of the levels of APP and Aßalter cerebrovascular reactivity and, if so, whether the effect is medi-ated by reactive oxygen species.

RESULTSCrossing mice from a transgenic line Tg1130H expressing highcopy numbers of the human mutant APP11 with an outbred stockoverexpressing human SOD1 (ref. 12) provided littermates express-ing APP alone (APP+/SOD1–), SOD1 alone (APP–/SOD1+) or bothtransgenes (APP+/SOD1+), along with transgene-negative con-trols (APP–/SOD1–). For cerebrovascular studies, cerebral blood

flow (CBF) was continuously monitored in the exposed parietalcortex by a laser-Doppler probe. Resting CBF did not differ amongthe groups of mice studied (p > 0.05; data not shown). Topicalneocortical application of acetylcholine, a neurotransmitter thatincreases CBF by activating endothelial muscarinic receptors andreleasing endothelial nitric oxide13–15, increased CBF inAPP–/SOD1– mice (Fig. 1a). This increase was substantially atten-uated in littermates expressing APP but not SOD1 (p < 0.001).The reduction in acetylcholine-induced vasodilation was relatedto reactive oxygen species, because the altered response was notfound in mice overexpressing both APP and SOD1. Similarly, theincrease in CBF produced by bradykinin, a vasodilator that actsthrough specific endothelial receptors and cyclooxygenase prod-ucts15–17, or by the calcium ionophore A23187, a compound thatproduces endothelium-dependent vasodilation through receptor-independent mechanisms14,18, was also attenuated in APP+/SOD1–

mice (Fig. 1b and c). Again, this reduction in vasodilation was notfound in APP+/SOD1+ mice. In contrast to endothelium-depen-dent cerebrovascular responses, increases in CBF produced byvasodilators that do not act through the endothelium, such as thenitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) orhypercapnia19, were not reduced in APP+/SOD1– mice (Fig. 2).These endothelial-cell-independent responses did not differ sig-nificantly among APP+/SOD1+, APP–/SOD1+ and APP–/SOD1–

mice. Thus, APP overexpression influences only vasodilatorresponses mediated through the endothelium.

We then investigated the cerebrovascular effect of the throm-boxane A2 analogue U46619, a potent vasoconstrictor that actsdirectly on vascular smooth muscles20,21. The decrease in CBF elicit-ed by U46619 was exacerbated in mice overexpressing APP, but not

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SOD1 rescues cerebral endothelialdysfunction in mice overexpressingamyloid precursor protein

Costantino Iadecola1, Fangyi Zhang1, Kiyoshi Niwa1, Chris Eckman2, Sherry K. Turner3,Elizabeth Fischer4, Steven Younkin2, David R. Borchelt5, Karen K. Hsiao1 and George A. Carlson3

1 Department of Neurology, University of Minnesota, 420 Delaware Street S.E., Minneapolis, Minnesota 55455, USA2 Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, Florida 32224, USA3 McLaughlin Research Institute, 1520 23rd Street South, Great Falls, Montana 59405, USA4 Rocky Mountain Laboratory, National Institute of Allergy and Infectious Diseases, 903 South 4th Street, Hamilton, Montana 59840, USA5 Division of Neuropathology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, Maryland 21205, USA

Correspondence should be addressed to C.I. (iadec001@tc.umn.edu)

Peptides derived from proteolytic processing of the ß-amyloid precursor protein (APP), includingthe amyloid-ß peptide, are important for the pathogenesis of Alzheimer’s dementia. We found thattransgenic mice overexpressing APP have a profound and selective impairment in endothelium-dependent regulation of the neocortical microcirculation. Such endothelial dysfunction was notfound in transgenic mice expressing both APP and superoxide dismutase-1 (SOD1) or in APPtransgenics in which SOD was topically applied to the cerebral cortex. These cerebrovascular effectsof peptides derived from APP processing may contribute to the alterations in cerebral blood flowand to neuronal dysfunction in Alzheimer’s dementia.

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in APP+/SOD1+ mice (Fig. 1d). SOD1 overexpression alone did notalter responses to vasodilators and vasoconstrictors (Figs. 1 and 2).These results indicate that APP overexpression leads to attenuationof endothelium-dependent cerebrovascular vasodilation, and toenhancement of vasoconstriction by mechanisms involving reactiveoxygen species.

Expression of SOD1 transgenes also provides protection againstAPP-induced premature death22. Twenty-five of thirty-oneAPP+/SOD1– mice died within 150 days of birth, whereas 19APP+/SOD1+ mice survived for at least 1 year, and 1 mouse died at167 days. This difference in survival is statistically significant(p < 0.001; Fisher’s exact test).

To examine the possibility that the nor-mal response to endothelium-dependentvasodilators in APP+/SOD1+ mice resultedfrom reduced Aß levels, we measuredhuman Aß peptides 1–40 and 1–42 in thebrains of APP+/SOD1– and APP+/SOD1+

mice11,23. In APP+/SOD1+ mice, levels ofboth forms of Aß were slightly, but signifi-cantly, lower than those of APP+/SOD1–

mice (Fig. 3). To determine whether the lackof endothelial dysfunction in APP+/SOD1+

mice could be attributed to this reductionin Aß, we studied a line of wild-type humanAPP transgenic mice (Tg6209) that express-es less transgene-encoded protein thanTg1130H (ref. 11). Despite their lower Aßlevels (Fig. 3), Tg6209 mice showed alter-ations in endothelium-dependent cere-brovascular responses indistinguishablefrom those of Tg1130H APP+/SOD1– mice(percent CBF increases: acetylcholine, APP–

27 ± 1, APP+ 8 ± 1; bradykinin, APP–

44 ± 4, APP+ 18 ± 2; A23187, APP– 49 ± 2,APP+ 14 ± 5; n = 5–6 per group). Thus, theprotection exerted by SOD1 overexpressionin APP mice cannot be attributed to SOD1-related reduction in Aß concentration.

Synthetic Aß has been reported to pro-duce endothelial damage in isolated arter-ies7–9. There is no evidence forcerebrovascular abnormalities in Tg1130H

mice at the light-microscope level, with the only consistent pathol-ogy being hypertrophic corticolimbic astrocytic gliosis11. We there-fore used electron microscopy to determine whether endogenousAß produced cerebral endothelial damage in vivo. The morpholo-gy of endothelial cells was not grossly altered in APP+/SOD1– mice(Fig. 4). Therefore, in contrast to previous investigations in whichhigh concentrations of synthetic Aß were used in isolated vessels7,we find that the disturbance in endothelium-dependent vasodila-tion in the transgenic mice results from perturbation of endothelialcell function and not from endothelial cell destruction.

The observation that endothelium-dependent cerebrovascularresponses are intact in transgenic mice expressing both APP and

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Fig. 1. Effects of endothelium-dependent vasodilators and vasocontrictors on cerebral blood flowin nontransgenic mice (APP–/SOD1–) and in littermates overexpressing APP (APP+/SOD1–), SOD1(APP–/SOD1+) or both APP and SOD1 (APP+/SOD1+). (a) Acetylcholine (10 µM). (b) Bradykinin(50 µM). (c) A23187 (3 µM). (d) The vasoconstrictor U46619 (1 µM). Data were collected from5–8 mice per group. *p < 0.001–0.003, analysis of variance and Tukey’s test.

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Fig. 2. Effect of endothelium-independent vasodilators S-nitroso-N-acetylpenicillamine (SNAP) or hypercapnia (pCO2 = 50–60 mmHg) in nontrans-genic mice and in littermates overexpressing APP, SOD1 or both APP and SOD1.

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SOD1 implicates reactive oxygen species in the mechanism of theendothelial dysfunction. To study the possible source of superoxide,we tested whether the cerebrovascular dysfunction could be reversedby local treatment of the cerebral cortex with SOD, an enzyme thatdoes not easily cross cell membranes24. In APP+ mice (Tg6209),SOD superfusion completely reversed the attenuation of the CBFincrease evoked by acetylcholine without affecting the response tohypercapnia (Fig. 5). In APP– littermates, SOD superfusion did notinfluence vasodilator responses to acetylcholine or hypercapnia(Fig. 5). SOD superfusion did not affect resting CBF in either groupof mice (p > 0.05; data not shown).

DISCUSSIONWe have demonstrated that APP overexpression in living mice leadsto a profound and selective alteration in endothelial vascular regu-lation. The cerebral vascular reactivity to acetylcholine, bradykininand A23187 are all reduced in APP mice, suggesting that APP over-expression leads to a global impairment in endothelium-dependentresponses rather than to an alteration of a specific endothelial recep-tor or vasodilator mechanism. The impairment in endothelium-dependent vasodilation would be expected to render cerebral bloodvessels more susceptible to vasoconstriction. Consistent with thishypothesis, the response to the vasoconstrictor U46619 is enhancedin mice overexpressing APP.

The fragment of APP that is involved in the mechanisms ofthis endothelial dysfunction remains to be identified. The obser-vation that the abnormality in endothelium-dependent vasodi-lation is present both in Tg1130H and Tg6209 mice, despite atwofold difference in Aß(1–40) levels, suggests that this APP frag-ment may not mediate the endothelial dysfunction. However,Aß(1–42) is not substantially reduced in Tg6209, and this APPfragment could conceivably be involved. Transgenic mice withdeletions and mutations of candidate APP regions will have tobe studied to determine which APP domain is responsible forvascular dysfunction.

Although insufficient to account for SOD1-mediated protec-tion against vascular dysfunction and against premature death,the reduction in Aß peptide concentration by SOD1 expressionis noteworthy. It is not clear whether SOD1 alters the processing ofAPP so that less Aß is produced or whether Aß peptide is removedmore efficiently. Both the short (1–40) and more amyloidogeniclong (1–42) forms of Aß peptide are reduced by SOD1 overex-pression. The ratio of the two forms is unchanged, a result more

compatible with a reduction in normal processing than with alter-ations in peptide catabolism.

The finding that the endothelial dysfunction can be rescued byexogenous SOD or prevented by SOD1 overexpression suggests thatreactive oxygen species are involved in mediating the effect. Super-oxide anions could be produced either intracellularly, for example,by activation of the receptor for advanced glycation end products5, orextracellularly by the endothelial NADH-dependent oxidoreductase,which is membrane bound25. The finding that both SOD1, expressedintracellularly in endothelial cells of SOD1 transgenics26, and exoge-nous SOD, which does not cross cell membranes24, are protectivesuggests that superoxide is not generated within endothelial cells.This hypothesis is supported by two observations in APP transgen-ics. First, endothelial cells do not show histochemical markers orultrastructural evidence of free-radical-mediated damage (G. Perryand K.K. H., unpublished observations, ref. 3 and Fig. 4). Second,the endothelial dysfunction is not irreversible because it can be ‘res-cued’ by exogenous SOD. However, vascular production of reactiveoxygen species remains to be demonstrated in APP transgenics.

There is ample evidence that reactive oxygen species result incerebral endothelial dysfunction, and they are thought to be respon-sible for loss of endothelium-dependent vasodilation in atheroscle-rosis, diabetes and hypertension27–30. However, the mechanisms ofthis effect are not understood31. Considering that a commondenominator in the vasodilator effect of acetylcholine, bradykininand A23187 is an increase in endothelial calcium31, superoxide andother radicals could perturb endothelial calcium homeostasis andimpair production of endothelial vasoactive factors.

Overexpression of APP in the FVB/N strain of mice leads to pre-mature death11,22. The observation that SOD1 protects against thelethal effects of APP overexpression could provide a link betweenreactive oxygen species and early mortality22. Although SOD1expression prevents both cerebrovascular dysfunction and prematuredeath, the relationship, if any, between the two phenomena remainsto be determined. Although FVB/N mice could be more sensitiveto induction of superoxide-dependent cerebrovascular dysfunction

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Fig. 3. Brain Aß levels in Tg1130H-derived APP+/SOD1– orAPP+/SOD1+, and in transgene-positive FVB/N Tg6209 mice. *p < 0.01from Tg1130/SOD1–; #p < 0.001 from both Tg1130/SOD1– andTg1130/SOD1+ (analysis of variance and Tukey’s test).

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Fig. 4. Electron micrographs of thin sections from parietal cortex of mice.The morphology of endothelial cells (arrowheads) in transgene-negativecontrol mice (a) is comparable to that of APP–/SOD1+ (b), APP+/SOD1–

(c and d) or APP+/SOD1+ mice (e and f). Scale bar, 1.0 µm.

a c e

b d f

Tg1130/SOD1- (n = 6)Tg1130/SOD1+ (n = 9)

Tg6209/SOD1- (n = 8)

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than other mouse strains, it is also possible that the involvement ofreactive oxygen species is not related to their cerebrovascular effects.

The findings also raise the possibility that cerebrovascularendothelial dysregulation may contribute to the neuronal dysfunc-tion observed in APP+ mice11. Vascular dysregulation may impairbrain function by altering the balance between flow-dependent sub-strate delivery and energy demands imposed by neural activity. Inaddition, the endothelial dysfunction may disrupt the blood-brainbarrier and impair the delivery to the brain of critical substrates. Insupport of this possibility, subtoxic concentrations of Aß reduce thetransfer of glucose across endothelial cells, an effect likely to be medi-ated by reactive oxygen species10. In addition, the vascular dysreg-ulation induced by APP overexpression makes the brain moresusceptible to the effects of cerebral ischemia32. Thus, cerebrovas-cular dysfunction resulting from APP overexpression renders thebrain more vulnerable to injury and could amplify the pathogenicprocess in Alzheimer’s dementia.

It is well established that Alzheimer’s dementia and aging areassociated with alterations in cerebral hemodynamics and cere-brovascular regulation33,34. Resting CBF is decreased and the vas-cular reactivity to selected vasodilator stimuli is reduced inAlzheimer’s dementia33,35,36. There is evidence that endothelium-dependent vascular relaxation is impaired in isolated cerebral ves-sels from elderly humans and in cerebral arterioles from agedrats37,38. Although the CBF alterations in Alzheimer’s dementia couldbe secondary to neuronal degeneration or to morphological alter-ations in cerebral blood vessels, these factors are less likely to beinvolved in the cerebrovascular changes occurring early in the diseasewhen pathological changes have not yet occurred39. Our results sug-gest that alteration of endothelial regulation of the cerebral circula-tion could be an important factor in the mechanisms of thecerebrovascular dysfunction in Alzheimer’s dementia and aging.Furthermore, our finding that exogenous SOD rescues the endothe-lial dysfunction in APP transgenics adds further support to the valid-ity of antioxidant treatment in Alzheimer’s dementia40.

METHODS

Transgenic mice. The APP and SOD1 transgenic mice used in these studieshave been described11,12. Tg1130H mice overexpress the human APP695isoform with the V717I familial Alzheimer’s dementia mutation, along withV721A and M722V changes. Tg6209 express wild-type human APP695.Both transgenes have a 3’-myc epitope tag. A hamster prion protein gene-derived cosmid vector41 was used to drive expression of APP in these trans-genic lines, both of which were produced on an inbred FVB/N background.

SOD1 mice (line Tg(SOD1)76) were constructed with a 12-kb genomic clonecontaining the entire human gene12. Offspring of Tg(APP) × Tg(SOD1)mice were typed for the presence of the transgenes as described22. All exper-iments were done on littermates that shared genetic background except forthe transgene(s) being overexpressed. This experimental design eliminatesthe possibility that the observed effects are a consequence of differences ingenetic background rather than expression of the transgene.

Because SOD1 activity is a reflection of copper concentrations42 andbecause APP is a copper-binding protein that reduces Cu2+ to Cu+ (ref.43), we examined the effects of expression of each protein on the other.To verify that APP overexpression did not influence SOD1 activity in dou-ble transgenics, we measured SOD1 activity in whole blood ofAPP–/SOD1+ and APP+/SOD1+ mice using the Bioxytech SOD1-525 spec-trophotometric assay44. In SOD1– mice (n = 6), SOD1 activity was 3.6 ±0.4 units, significantly (p < 0.05) less than the 9.2 ± 1.5 units inAPP–/SOD1+ mice (n = 5) or 10.3 ± 0.6 units in APP+/SOD1+ mice (n =5). APP–/SOD1+ and APP+/SOD1+ were not significantly different fromone another (p > 0.05, t-test). Therefore, APP overexpression did notinterfere with the elevation in SOD1 activity in SOD1+ mice. Similarly,western blots did not show any obvious change in APP expression as aconsequence of SOD1 overexpression.

Determination of cerebral blood flow. Techniques used for studying thecerebral circulation in mice were similar to those described32. Mice wereanesthetized with halothane (maintenance 1%), intubated and artifi-cially ventilated with an oxygen-nitrogen mixture. End-tidal CO2, mon-itored by a CO2 analyzer (Capstar-100, CWI, Ardmore, Pennsylvania),was maintained at 2.6–2.7% (pCO2 = 33–35 mmHg)32. Arterial pO2was 161 ± 8, and the pH 7.41 ± 0.03. The parietal cortex was exposed,and the site was superfused with Ringer solution (37°C; pH 7.3–7.4)32.CBF was monitored in the exposed cortex with a laser-Doppler probepositioned stereotaxically. Because CBF measurements by laser-Dopplercan vary depending on the monitoring site45, the probe was placed atthe same stereotaxic coordinates in all mice. Acetylcholine (10 µM),bradykinin (50 µM), A23187 (3 µM), U46619 (1 µM) or SNAP (100–500µM) were superfused on the cerebral cortex for 3–5 min. Agents wereapplied at concentrations that produce 50% of maximal responses (datanot shown). Hypercapnia (pCO2 = 50–60 mmHg) was produced byintroducing CO2 through the circuit of the ventilator. Arterial pressure,monitored via a femoral arterial catheter, was 85 ± 3 mmHg inAPP–/SOD1– mice, 84 ± 3 in APP+/SOD1– mice, 88 ± 3 in APP+/SOD1+

mice, 86 ± 3 in APP–/SOD1+ mice, 86 ± 3 in Tg6209 APP+ mice and 84± 4 in Tg6209 APP– mice. Topical superfusion with vasoactive agentsor hypercapnia did not affect arterial pressure or blood gases (data notshown). In experiments in which effects of topical SOD were studied,CBF responses to acetylcholine (10 µM) and hypercapnia were testedduring Ringer superfusion and 30 min after superfusion with bovineliver SOD (1000U /ml; Sigma)46.

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Fig. 5. Effect of topical application of SOD to the cerebral cortex on CBF responses in Tg6209 APP+ and APP– littermates. (a) Response to acetyl-choline. (b) Response to hypercapnia. Data were collected from 5–6 mice per group. * p < 0.001, t-test.

a bAcetylcholine Hypercapnia

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Determination of Aß. Techniques for Aß measurement have beendescribed in detail11,23. Brain tissue was homogenized in 1 ml of 70%formic acid and centrifuged at >100,000 g for 1 hour. Samples were dilut-ed and analyzed directly using either the BNT77/BA27 or BNT77/BC05sandwich ELISA system. Aß values were obtained by comparing theabsorbance obtained from duplicate samples to standard curves of eitherAß1–40 (BNT77/BA27) or Aß1–42 (BNT77/BC05) with a standard curve(Bachem, Torrance, California).

Electron microscopy. After transcardial perfusion of anesthetized micewith 2% paraformaldehyde in phosphate-buffered saline, a sample of pari-etal cortex was removed and fixed overnight in 2.5% glutaraldehyde and4% paraformaldehyde in 0.1 M sodium cacodylate buffer, 0.05 M sucroseat 4ºC. Samples were then postfixed, stained en bloc in 1% uranyl acetate,dehydrated with ethanol and embedded in Spurr’s resin. Thin sectionswere cut with an RMC MT-7000 ultramicrotome (Research and Manu-facturing Company, Tucson, AZ), stained with 1% uranyl acetate andReynold’s lead citrate and observed at 80 kV on a Philips CM-10 trans-mission electron microscope.

Data analysis. Data in text and figures are expressed as mean ± standarderror. Two-group comparisons were analyzed by the two-tailed t-test forindependent samples. Multiple comparisons were evaluated by the analy-sis of variance and Tukey’s test. Survival data were analyzed by the Fish-er’s exact test in a 2 × 2 contingency table. Probability values of less than0.05 were considered statistically significant.

ACKNOWLEDGEMENTSThis work was supported by grants from the National Institutes of Health (NS34179,

NS37853, NS35806 to C.I.; NS33249 to K.K.H; AG10681 to G.A.C.) and the

Fraternal Order of Eagles (G.A.C.). We thank G. Perry for the oxidative stress data

and Karen MacEwan for editorial assistance.

RECEIVED 24 JUNE; ACCEPTED 23 DECEMBER 1998

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