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Amyloid -proteins 1Ч40 and 1Ч42(43) in the soluble fraction of extra- and intracranial blood vessels.

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Amyloid P-Proteins 1-40 and 1-42(43)
in the Soluble Fraction of Extraand Intracranial Blood Vessels
Yasuhisa Shinkai, MD," Masahiro Yoshimura, MD,? Yuji Ito, MDJ Asano Odaka, MSc,$
Nobuhiro Suzuki, PhD,§ Katsuhiko Yanagisawa, MD,* and Yasuo Ihara, MD"
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To investigate the process of amyloid f3-protein (Ap) accumulation in cerebral amyloid angiopathy (CAA), the levels
of AP were determined in the soluble fraction of extra- and intracranial blood vessels and leptomeninges obtained at
autopsy. Two enzyme immunoassays were employed that are known to sensitively and specifically quantify two Ap
species, Apl-40 and 1-42(43). Ap was detectable in the intracranial blood vessels and leptomeninges with the latter
containing the highest levels, while it was undetectable in the extracranial blood vessels. Thus the levels of soluble
Af3 correlated well with the predilection sites for CAA. Among individuals aged 20 to 90, the Ap levels in the
leptomeninges increased sharply in those aged 50 to 70 and thereafter tended to decline. However, only slight degrees
of CAA were detected by immunocytochemistry, even when those leptomeninges contained high levels of Ap comparable with those in Alzheimer's disease. The level of Apt-42 was almost always severalfold that of Apl-40 in the soluble
fraction of leptomeninges. This is in good agreement with the immunocytochemical result showing the presence of
AP40-negative, Ap42(43)-positive meningeal vessels. These results indicate that Apl-42 is the initially deposited
species in CAA and that the disruption of AP homeostasis precedes AP deposition in the meningeal vessels.
Shinkai Y, Yoshimura M, Ito Y, Odaka A, Suzuki N , Yanagisawa K, Ihara Y. Amyloid P-proteins 1-40 and
1-42(43) in the soluble fraction of extra- and intracranial blood vessels. Ann Neurol 1995;38:42 1-428
Cerebral amyloid angiopathy (CAA), also known as
congophilic angiopathy, was originally defined by the
presence of amyloid-bearing vessels showing Congo
red birefringence under polarized light. CAA affects
most commonly meningeal arteries and veins, and less
frequently parenchymal arterioles and capillaries. In
the latter condition, amyloid-bearing vessels sometimes produce a perivascular amyloid mass radiating
into the parenchyma and known as dysphoric angiopathy (drusige Entartung) (for review, see [l, 21).
CAA is often accompanied by Alzheimer's disease
(AD), but there appears to be two extreme conditions,
one, where severe CAA is isolated from parenchymal
A D lesions and, the other, where abundant parenchymal senile plaques do not accompany CAA El, 23.
Many investigators consider that the pathogenesis of
CAA and senile plaque may be similar, if not identical.
In fact, both types of amyloid are composed of amyloid
P-protein (AP), a small protein of M, -4,000, proteolytically derived from P-amyloid precursor protein
(APP). However, vascular amyloid was repeatedly
claimed to be distinct from senile plaque core amyloid.
The most important difference is the abundance of the
AP37 or 40 species in CAA 13-51, in contrast to the
senile plaque core where AP42(43) is by far the predominant species [b, 73.
The introduction of specific AP immunostaining has
revolutionized the detection of CAA. It has been
shown that a much larger proportion of aged subjects
and A D patients are affected by CAA to varying extents. Based on sensitive immunochemical and immunocytochemical procedures, we previously showed
that (1) soluble fractions from A D cortices contain
varying amounts of Apl-40; (2) high levels of soluble
Apl-40 are associated with CAA; and (3) some leptomeninges containing high levels of Apl-40 do not
exhibit CAA as judged by immunocytochemistry, suggesting the presence of a nonfibrillar stage preceding
CAA [S}. To learn more about the postulated preamyloid stage in the cerebral blood vessel, the levels of
Apl-40 and 1-42(43) in the soluble fractions of extraand intracranial vessels, from autopsy cases ranging in
age from 22 to 94 years, were quantified by two enzyme immunoassays 18, 93.
From the 'Department of Neuropatholonv, Institute for Brain Research, Faculty-of Medicine, Univ&ity ofTokyo, ?Tokyo Metropolitan Medical Examiner's Office, $Department of Clinical Pathology,
Tokyo Metropolitan Tuna Geriatric Hospital, and WIhcovery Research Division, Takeda Chemical Industries, Ltd, Tsukuba, Japan.
Received Ian 30. 1995. and in revised form Mav 1. AcceDted for
publication May 3 1, 1995.
Address correspondence to Dr Ihara Department of Neuropatholfor Brain Research,
of Medicine, University
of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 13,
ogy,
Ficulty
Copyright 0 1995 by the American Neurological Association
421
Materials and Methods
Autopsy
The present study is based on autopsy cases at the Tokyo
Metropolitan Tama Geriatric Hospital, Higashimurayama,
and those at the Tokyo Metropolitan Medical Examiner's
Office, Otsuka, Tokyo. Postmortem delay ranged from 4 to
12 hours in autopsy cases at the former facility and, from
postmortem signs, was estimated to be less than 12 hours
for those at the latter facility. Those cases at the Geriatric
Hospital (Patients 33, 34, 36, 39, 41, 42, 45, 46, 48, 51, 52,
55,60,61, and 62 in the Table) were completely investigated
by general pathologists and neuropathologists and none were
diagnosed as having had AD. All subjects who died of unknown causes in the Tokyo Metropolitan area were subjected to postmortem examination at the latter facility.
Eleven AD cases (68-87 yr; mean, 79.4 yr) were neuropathologically investigated, all of which were accompanied to
some degree by CAA showing Congo red birefringence. The
diagnosis of AD was made based on both clinical and pathological findings [lo].
Blood Vessels and Leptomeninges
Intracranial vessels including the middle cerebral artery and
basilar artery were carefully removed from the brain. About
50 mg of leptomeninges (containing meningeal vessels) were
gently peeled off the cortical surface (-2 x 2 cm) at the
occipital pole. In AD brains, similar portions in the occipital
cortex were chosen for sampling leptomeninges. As judged
from our experience, the occipital cortex is the predilection
site for CAA [2). The cortical blocks (attached by leptomen-
Summary of Patients arid Data on AP Level and Deposition
PatientlCase No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
422
Age (yr)/Sex
22lMl
231"
331M
421hI
46iM
471M
481M
49llM
501M
5OIM
50lM
52lM
54lM
54iM
55/M
56lM
57lF
58lM
58lF
59lM
6OIM
6OIM
6OlM
6lIF
6llM
bllM
03IM
641M
691F
691M
iS91F
70lM
71lM
71lM
Annals of Neurology
A(31-401x-40d
ApI-42(43)/~-42(43)~
(pmollgm)
(pmollgm)
0.4
0.2
0.3
0.1
0.4
0.4
0.4
0.3
0.3
5.3
0.4
11
0.2
0.3
0.7
0.7
0.2
0.1
0.6
6
0.9
0.3
1.3
2.5
0.3
0.74
14
0.2
110
1.3
90
8.4118
14
24127
Vol 38 No 3 September 1995
0.8
0.2
0.3
<o. 1
1.2
0.6
0.4
0.9
0.3
20
0.3
55
<o. 1
0.3
0.2
0.5
0.1
0.1
1.o
CAA
BA271BC05'
-!-
-I ND
-1-
ND
ND
-1ND
-I-i-1ND
ND
-1ND
ND
ND
-IND
31
-1-
<o. 1
ND
-1ND
-1-!ND
ND
-1ND
ND
ND
ND
+i+
-1 -
0.3
0.5
0.5
0.2
<o. 1
180
0.3
1,900
2.1
1,200
27/12>
110
8.8127
Summary of Patients and Data on AD Level and Deposition (Continued)
Age (yr)/Sex
Apl-401~-40"
(pmollgm)
62
63
71lM
72lM
72lM
721F
73iM
73lF
74lM
75lM
75lM
76lM
771F
77lM
77lF
78lM
781F
79lM
80lM
80lF
80iF
81lF
82lF
821M
83/M
85lF
91lF
93lF
94lF
95lF
971F
1.6
3118.8
0.611.2
0.7
5.9114
32145
9,10017,000
16
0.912
30142
1.5
1,150
7.0114
4501288
5.8
59
3.0
25
1,750
0.4
530
5.2
0.2
41
270
330
290
160
1.4
PatientlCase No.
Age (yr)lSex
AP 1-401x-40a
(pmollgm)
Apl-42(43)1~-42(43)~
(pmollgm)
CAA in Occipital
Cortexd
AD 1
2
3
4
5
68lM
74lF
75lF
76lF
76lM
81lF
82lF
841F
84lF
86lF
87lF
2 3,000l20,000
35,000/12,000
58012,800
1001380
67014,100
47013,300
1,800114,000
5,900111,000
4301520
2,90015,300
191270
291230
Moderate
Marked
Focal
Marked
Focal
Moderate
Moderate
Marked
Marked
Moderate
Focal
PatientlCase No.
Apl-42(43)/~-42(43)~
(pmollgm)
BA27lBC05'
CAA
~~~
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
6
7
8
9
10
11
2.2
1.214
3.215
0.8
1.713
7.6164
4,30018,500
380
1.411.5
3101910
210
7,750
2001390
26014,300
37
380
5.1
3.0
5,750
0.3
1,100
1.2
0.1
280
180
34
550
600
7.4
26,000/50,000
5301870
5,5 00110,000
24,000/47,000
8,900115,000
5,800110,000
21,000116,000
77,000174,000
69011,500
55011,300
ND
ND
ND
ND
ND
-I+
+I+
ND
ND
ND
t/+
_ti*
t l
?I+
ND
ND
?I+
+
-1%
-1%
-1+/+
ND
ND
-I+-+
ND
?I*
+I +
ND
ND
-t
".b4G8-basedenzyme immunoassay (EJA) values representing total AP40 and AP42(43) amounts.
'Rating of cerebral amyloid angiopathy (CAA): ( - ) = none; ( 2 ) = questionable; ( + ) = definite, a few; ( + + ) = definite, many;
ND = not determined. None of the CAA in this group as judged by AD immunostaining showed Congo red birefringence.
dThe extent of CAA was rated by Congo red birefringence under polarized light.
AP = amyloid P-protein; AD = Alzheimer's disease.
Shinkai et al: AP Quantitation in Meningeal Vessels 423
inges) located at the same site in the contralateral hemisphere
were fixed in 10% formalin and subjected to histological and
immunocytochemical examination (see below). Small pieces
were taken from extracranial vessels, including the internal
carotid artery and aorta (ascending portion), and from the
liver and kidney. The specimens for AP quantitation were
immediately rinsed in saline, weighed, and stored at - 80°C
until use.
Tissue Extraction
Sampled leptomeninges were cut into small pieces with a
sharp blade and homogenized in 19 volumes of Tris-saline
(50 mM Tris-HCI, p H 7.4, 0.15 M NaCI) containing 1 mM
EGTA, 0.5 mM diisopropyl fluorophosphate, 0.5 mM phenylmethylsulfonyl fluoride, 1 pglml N-a-p-tosyl-L-lysine
chloromethyl ketone, 1 pg/ml antipain, 0.1 p,g/rnl pepstatin,
and 1 pg/ml leupeptin, with a Teflon-glass motor-driven
homogenizer with 20 strokes. The homogenates were centrifuged at 100,000 g for 15 minutes on a TI.. 100 rotor in a
TLX centrifuge (Beckman). The supernatants were diluted
more than fivefold with 20 mM phosphate buffer (pH 7.0)
containing 0.4 M NaCI, 2 mM E'DTA, 10% Block Ace (Dainippon, Tokyo, Japan), 0.2% bovine serum albumin, and
0.05% NaN, (buffer EC) and applied to each two-site enzyme immunoassay (EIA) [8, 91.
Immunocytochemistly
Antibodies
AP monoclonal antibodies used in the present study were
BAN50 (raised against AP1-16), BA27 (specific for AP40),
BCOS (specific for AP42 and A[343), and 4G8 (raised against
AP17-24), whose characteristics were described in detail
elsewhere [8, 9, 11). In EIA, ]BAN50 or 4 G 8 was used as
the capture antibody by coating a multiwell plate (Immunoplate I, Nunc, Roskilde, Denmark), while BA27 o r BC05
was used as detection antibody following conjugation with
horseradish peroxidase [S, 91.
Enzyme lmmunoassay
The EIAs for AP were carried out in the same way as described previously {8, 9f. In brief, 100 pl of the diluted
Tris-saline supernatants of tissue homogenate, as well as synthetic Apl-40 or Apl-42 (Bachem, Torrance, CA, USA)
were applied to a BANSO- or 4G8-coated multiwell plate
and incubated at 4°C overnight. After rinsing with phosphatebuffered saline, loaded wells were reacted with appropriately
diluted horseradish peroxidase-con jugated BA27 or BCO5
at room temperature for 6 hours. Bound enzyme activity
was measured by the T M B Microwell Peroxidase Substrate
System (Kirekegaard and Perry Laboratories, Gaithersburp
MD, USA).
The BANSO-based EIAs probably quantify intact AP 1-40
and APl-42(43). This is because (1) the BAN50 epitope
appears to be located in the amino-terminal-most portion
of AP (Suzuki N , et al, unpublished observation); and
(2) isomerization at positions 1 and 7 [GI greatly reduces its
affinity for BAN50 (Suzuki N , et al, unpublished observation). O n the other hand, the 4G8-based EIAs are assumed
to measure all AP species including intact, isomerized, and
many amino-terminally ragged species with truncation up to
position 10 1121.
424 Annals of Neurology
The leptomeninges from 11 subjects in their 70s were
quantified for AP40 and AP42(43) by both BANSO- and
4G8-based EIAs, and results obtained were compared (see
the Table). The BANSO-based EIA values were found to be
strictly proportional to the 4G8 values {y = 0.29913 +
0.85343x, Y' = 0.950 for AP40, and y = 0.39133 +
1.0690x, r2 = 0.934 for AP42, where x = log(BAN50
value) and y = log(4G8 value)]. In the A D group the
BANSO- and 4G8-based EIA values were also in direct proportion iy = 0.70245 + 0.86472x, r2 = 0.960 for AP40,
and y = 1.2639 + 0.76699x, r2 = 0.853 for AP42(43)].
Thus, we conclude that the BANSO-based EIA values faithfully reflect the total amount of AP in the soluble fraction of
leptomeninges.
Augmentation factors (the 4G8/BAN50 ratios) were 1.48
2 0.71 for AP40 and 4.29 2 4.54 for AP42(43) in the
subjects in their 70s, and 1.62 k 0.161 and 5.46 % 3.77 in
A D patients, respectively. The differences between the ratios
for AP40 and AP42(43) were statistically significant only in
the A D group ( p < 0.05, t test). To examine the effect of
postmortem delay, leptomeninges from 1 autopsy case were
kept at room temperature and serially sampled for AP quantitation. The levels of Apl-40 and 1-42 in the soluble fractions were not significantly altered up to 24 hours at room
temperature (data not shown).
The formalin-fixed specimens were dehydrated and embedded in paraffin and cut into 6-pm-thick sections. Those
sections were immunostained with BA27 or BCO5 by
the avidin-biotin method (Vectastain, Vector Laboratories,
Burlingame, CA, USA), as described previously [71. For enhancement of AP immunostaining, tissue sections, after deparaffinization, were immersed in concentrated formic acid
for 5 minutes.
Results
Tissue-soltcble APl-40 and 1-42(43) Are Present in
lntracranial Vessels Bat Not in Extracranial Vessels
The two EIAs can reliably detect as little as 0.1 to 1.0
fmol Apl-40 or 1-42(43) per well and their specificities were repeatedly confirmed 17-91. We first examined whether vascular-rich organs, for example, liver
and kidney, contained detectable levels of AP. The
specimens from these two organs showed no detectable levels of Apl-40 and 1-42(43) (data not shown).
This was also the case with extracranial arteries including the aorta and internal carotid artery (Fig 1). In contrast, intracranial vessels including the basilar artery
and middle cerebral artery showed detectable levels of
tissue-soluble AP (see Fig 1).In particular, leptomeninges contained the highest levels of AP among tissues
from a given individual (see Fig 1).The leptomeninges
consist of arachnoid mater, pia mater, and meningeal
arteries and veins of various sizes in the subarachnoid
space. To investigate the source of AP, visible meningeal vessels were carefully dissected from leptomeninges. Vessels were found to contain high levels of AP
Vol 38 N o 3 September 1095
I I
3.0
0
-
E
w
-t
.
2.0
I
L
w
-E
0
e
1.0
0.16
U
0
$
AP level as a function of age was most remarkable;
both A@ levels increased sharply in subjects in their
50s to 70s (see Fig 2). Those levels started to increase
at age 50, peaked at age 70 to 80, and thereafter declined somewhat (see Fig 2). At less than 70 years,
more than 60% of subjects had less than 1.0 pmol of
AP per gm wet weight of leptomeninges; while among
19 subjects 70 to 79 years old only 1 showed this level
of AP (see Table).
Eleven A D cases were similarly analyzed. In contrast
to controls, the level of Apl-40 was significantly
higher than that of Apl-42(43) in AD patients ( p <
0.05, t test). In addition, the levels of Awl-40 in AD
specimens were significantly higher ( p < 0.05, t test)
than those in the specimens from subjects 70 years
old and older, selected from consecutive autopsy cases;
while the difference between the levels of Apl-42 in
subjects 70 years old and older and those levels in AD
patients were not statistically significant (see Fig 2).
a
AP42(43)-positive, AP40-negative Meningeal Vessels
Fi g 1 . Quantitation of amyloid p-protein (A01 in the soluble
fraction of intra- and extracranial vessels and leptomeninges.
Apl-40 (open circles) and Apl-42 (closed circles) in the
soluble fraction were quantified by two enzyme immunoassays.
The detection limit in the present system was 0.1 pmollgm wet
weight. ICA = internal carotid artery; M C A = middle cerebral artery.
(data not shown), suggesting that soluble AP in leptomeninges is derived mainly from meningeal vessels.
This is supported by the immunocytochemical observation that arachnoid or pia mater did not exhibit dense
AP accumulation. However, we cannot completely exclude the possibility that a significant fraction of tissuesoluble AP also comes from arachnoid or pia mater.
APl-40 and 1 -42(43) Levels Increase Sharply in the
Leptomeninges from Subjects in their JOs to 70s
The levels of Apl-40 and A@l-42(43) behaved similarly in the leptomeninges. A low or high Apl-40
level invariably accompanied a low o r high AP142(43) level, respectively (see Table). This suggests
that the levels of the two Af3 species are coordinately
regulated in the leptomeninges. Usually, the level of
soluble AP 1-42(43) was severalfold higher than that
of Apl-40 in the leptomeninges (see Table). In some
cases Apl-42(43) levels were higher by one order of
magnitude than AP 1-40 levels (see Table). The difference between Apl-42(43) and 1-40 in an individual
60 to 79 years old was significant ( p < 0.05, Student’s
t test) but not significant in an individual 80 years old
or older (Fig 2).
In one-half of the cases the occipital cortices contralateral to the sampling site for leptomeninges were examined for the presence of CAA by immunostaining with
BA27 and BC05.
Definite AP deposition in the meningeal vessels, as
judged by immunocytochemistry, was noted in 3 subjects in their 70s, 3 subjects in their 8Os, and 1 subject
in his 90s (see Table). Most important, in 3 cases, some
AP42-positive vessels were found to be Ap40 negative
in the adjacent section (Fig 3). Further, we were unable
to find AP40-positive, AP42(43)-negative vessels.
100000
-.-g
.pt
low0 :
low
:o
1
O . N S I
m
2
O
E
100
:
10
1
Y
9
20
30
40
50
60
70
80
90
AD
Age (years)
Fig 2. Amyloid p-protein (A@)level1 in the leptomeninges versus age. APl-40 (open circles) and APl-42 (closed circles)
were quantzj5ed by two enzyme immunoassays. The right column
represents AP levels in Alzheimer’s disease leptomeninges. Note
that the ordinate ic a logarithmic scale. Asterisk indicates that
the diyerence is statistically significant. NS = not significant.
Shinkai et al: Af3 Quantitation in Meningeal Vessels
425
Fig 3. Immunocytochemistry of amyloid /3-protein (Ap)-bearing
veuels in 2 cases. A set of serial sertions from Patients 45
(A and B ) and 47 (C and 0)were immunostained for AP40
(A and C) and AP42(43) (B and 0).Many meningeal vessels
in both cases were AP42(43) positive but AP40 negative.
Bar = 50 p n .
In general, there existed iz correlation between AP
levels and the presence of CAA. CAA, as judged by
AP immunostaining, was detected only in those cases
showing high levels of AP (see Table). Those brains
containing low levels of A@in the leptomeninges never
had immunocytochemically detectable CAA.
Discussion
The present work on AP qumtitation in leptomeninges
and other tissues, together with immunocytochemistry,
has clarified the following: ( 1) Tissue-soluble AP1-40
and AP 1-42(43) are detected only in intracranial vessels, not in extracranial vessels or organs, and their
highest levels were found in leptomeninges, most
likely in meningeal vessels; (2) the level of AP142(43) is almost always severalfold higher than that of
Apl-40, but in AD brains containing advanced CAA
this relationship is reversed; (3) the AP levels in leptomeninges remain very low until age 50 but increase
sharply thereafter; (4) even those brains containing
high levels of AP in leptomeninges show very low degrees of CAA; and (5) in some cases, Ap42(43)positive vessels are found to be negative for AP40.
Obviously, the tissue-soluble AP levels determine
the predisposition to CAA; the highest levels were
found in the leptomeninges containing meningeal vessels, predilection sites for CAA. Furthermore, only
cases showing high AP levels developed CAA to some
extent. This implies that soluble AP levels are markers
for potential CAA, which is consistent with our previous observation CS}. At present we cannot offer a
proper explanation as to why only meningeal vessels in
aged people contain such high levels of A@.As several
investigators have suggested, it is most likely that vascular smooth muscle cells themselves produce pamyloid, because (1) vascular smooth muscle cells are
rich in APP { 131; (2) the earliest Ap deposition occurs
in the outermost layer of the tunica media, most likely
in the ground substance between smooth muscle cells
[14-161; and finally (3) smooth muscle cells isolated
from CAA-affected vessels produce AP deposits in cytoplasmic vacuoles and in the vicinity of sarcolemma
in vitro [17). Since APP is abundant in all vasculature,
high tissue-soluble AP content should be characteristic
of intracranial vessels, in particular, meningeal vessels.
In this respect it is noteworthy that smooth muscle cells are derived locally from individual organ parenchyma during embryogenesis { 181. Thus vascular
smooth muscle cells in different organs may respond in
different ways to aging. It is quite possible that smooth
muscle cells in intracranial vessels are distinct from others in terms of AP metabolism. In fact, cultured lepto-
426 Annals of Neurology Vol 38 No 3 September 1995
meningeal smooth muscle cells are distinct, with respect to APP metabolism, from aorta smooth muscle
cells; the latter secrete more truncated APP into medium, while the former retain more cell-associated
(full-length) APP [ 191.
The levels of tissue-soluble Apl--42(43) were always severalfold higher than those of Apl-40 among
individuals selected from consecutive autopsies. This
may complement the result recently obtained by Roher
and his colleagues { 51: In detergent-insoluble vascular
amyloid, the level of AP42 is higher than that of AP40.
These data suggest that Apl-42 plays a major role in
the initial stage of vascular, as well as parenchymal,
P-amyloidogenesis {6]. In contrast, in A D patients the
levels of tissue-soluble Apl-40 were much higher
than those of Apl-42 (see Table, Fig 2). This is a
remarkable characteristic of CAA in A D brain, which
may explain why CAA in AD is rich in Apl-40 [4,
81. CAA that shows Congo red birefringence was
abundant in all A D brains examined here but was undetectable in other brains in this series (see Table).
This suggests that massive accumulation of AP40 is
involved in the development of congophi!ic angiopathy, the final stage of CAA. Since Ap40 is less modified than Ap42(43) at its -mino-terminal portion (see
Materials and Methods), its accumulation can be assumed to be a k e r event.
Paraile: irnmunocytochemistry clearly showed in
some cases the presence of AP42(43)-positive, AP40negative meningeal vessels but the absence of
Af342(43)-negative, AP40-positive vessels. Together
with the above-mentioned data, these results strongly
suggest that AP42(43) is the initially deposited species
in CAA. It should be noted that these do not contradict our and others’ recent observations that AP40
staining was usually stronger than Ap42(43) staining in
CAA in A D { 7 , 2 0 ] and Down’s syndrome brains [21).
AP42(43) may be involved in the initiation of CAA,
while AP40 may be important for the subsequent development of CAA (see above). As AP40 accumulates
preferentially and progressively in the meningeal vessel
wall, AP42 that is deposited earlier becomes less accessible by end-specific antibodies. This may lead to seemingly decreased Af342(43) staining in advanced CAA
in A D brains, whereas the level of AP42(43) is not
significantly altered (see Fig 2).
The most remarkable finding in this study is that in
the leptomeninges, soluble AP levels increase sharply
between ages 50 and 70. Apparently, AP homeostasis
is acutely disrupted during this period. This disruption
appears to start at age 50, peak at 70 to 80 years, and
may precede the appearance of immunocytochemically
detectable CAA by more than several years. It is probably followed much later by congophilic angiopathy,
the final consequence of this disruption of AP homeostasis. Taking all these data into consideration, there
emerges one possible scenario: The smooth muscle
cells in the meningeal vessels encounter disruption of
AP homeostasis when their life span is complete;
thereafter, for unknown reasons, they fail to regenerate. Thus those affected vascular smooth muscles enter
the degeneration process between ages 50 and 70 and
the degeneration very slowly, but constantly, progresses; their regenerative attempts may be uncoordinated and unsuccessful {22, 231.
This study was supported by a Grant-in-Aid for Scientific Research
on Priority Areas (no. 06254203) from the Ministry of Education,
Science and Culture, and by a Grant-in-Aid for Scientific Research
from the Ministry of Health and Welfare, Japan.
We thank D. J. Selkoe for providing A D brains used in the present study, H. M. Wisniewski and K. S. Kim for providing 4G8,
J. Saishoji for technical assistance, C. Hamada and R. Oyama for
statistical analysis, and M. Anzai for typing the manuscript.
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vessels, extra, protein, amyloid, soluble, 1ч40, 1ч42, fractional, intracranial, blood
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