Introduction

Sporadic cerebral amyloid angiopathy (CAA) is characterized by amyloid beta (Aβ) deposits in leptomeningeal vessels, cortical arteries, and capillaries in the brain. Loss of the integrity of cerebral blood vessels caused by intercalation of Aβ into cerebral blood vessels led to spontaneous lobar hemorrhages and dementia in patients with CAA [30]. Postmortem studies revealed that CAA incidences were more than 80 and 70% in patients with Alzheimer’s disease (AD) and elderly people, respectively [8, 16].

Several molecules have reportedly co-accumulated with tissue amyloid deposits in patients with amyloidosis. Apolipoprotein E (ApoE) commonly co-accumulated with Aβ deposits in senile plaques in AD. Also, the ε4 genotype of APOE was well documented to promote development of AD [12]. The ε2 and ε4 genotypes of APOE were both reportedly related to the development and severity of CAA [1, 4, 6]. In addition, serum amyloid P component (SAP) co-accumulated with tissue amyloid deposits and has attracted increasing attention as a target in amyloidosis diagnosis and therapy [23]. In CAA, however, the exact molecules that co-accumulate with cerebrovascular Aβ deposits are still not fully known.

Here, to identify the key molecules in CAA diagnosis and pathogenesis that may lead to therapy for CAA, we used laser capture microdissection (LCM) to perform proteomic analyses with cerebral blood vessels obtained from CAA cases. We investigated highly expressed proteins in these cerebral blood vessels. In the present study, we focused on sushi repeat-containing protein 1 (SRPX1) because of its potential and specific expression in blood vessels as suggested by the human gene expression database available at BioGPS (http://biogps.org), which provides the highest SRPX1 mRNA levels in smooth muscle among various human tissue sites. Also, because SRPX1, which is known as a tumor suppressor gene, reportedly induced apoptosis in tumor cells [26, 27], we hypothesized that SRPX1 may play an important role in Aβ-induced apoptosis in CAA. Furthermore, previous studies had not indicated that SRPX1 associated with the other form of Aβ deposits in senile plaques of AD brains [7, 15]. SRPX1, a transmembrane protein consisting of 464 amino acids, has three sushi domains, which are found in various kinds of complement and adhesion proteins [13]; it also has a short intracellular domain in the C-terminal region [22, 25,26,27]. SRPX1 was initially identified as a causative gene in patients with X-linked retinitis pigmentosa [17]. Expression of SRPX1 mRNA was reportedly down-regulated in tumor cells, and the SRPX1 gene was thought to be a tumor suppressor gene [14, 25,26,27]. SRPX2, a paralog of SRPX1, was reportedly involved in angiogenesis, cell adhesion, and synapse formation. Recent studies also suggested that the C-terminal regions of those SRPXs may be involved in redox-dependent regulation [22]. In our study here, we demonstrated that SRPX1 bound to Aβ in vitro and that SRPX1 enhanced the cytotoxic effects of Aβ in primary cultured cerebrovascular smooth muscle cells.

Materials and methods

Patients and sample collection

We studied 55 consecutive cases, autopsied at Kumamoto University Hospital from 1990 to 2012, for which brain tissue samples were available. Autopsies had been performed on cases aged 50 years or older (4 cases in their 50s, 18 in their 60s, 21 in their 70s, 11 in their 80s, and 1 in her 90s). To determine the occurrence of CAA, we studied cerebral neocortical tissues in each case (Supplementary Table S1). In addition, we used occipital cortex tissues obtained from five randomly selected cases with severe CAA and five control non-CAA cases autopsied at the Brain Research Institute, Niigata University, Niigata, Japan (Supplementary Table S1).

The pathological staging of neurofibrillary tangles was assessed according to the modified Braak neuropathological staging system of Alzheimer-related changes [3]. The burden of neuritic plaques in the cortical section was evaluated according to the criteria of the Consortium to Establish a Registry for Alzheimer’s disease (CERAD) [18]. Supplementary Table S1 provides clinicopathological information about the autopsied cases, and the control cases, used in this study. Supplementary Fig. S1 presents a flow chart illustrating sample collection and the inclusion criteria.

For proteomic analyses, we used cerebral neocortical tissues obtained from cases with severe CAA (n = 8) or mild CAA (n = 12) and cerebral neocortical tissues from randomly selected age-matched control subjects without CAA (n = 10). To investigate SRPX1 mRNA levels, we used frozen occipital cortex tissues dissected from randomly selected control subjects (n = 5).

Congo red (CR) staining and immunohistochemical staining

We performed CR staining and immunohistochemical staining with formalin-fixed, paraffin-embedded consecutive brain sections as described previously [29]. Brain tissues were stained with an anti-human Aβ/amyloid precursor protein (APP) antibody (pretreated with formic acid) (1:100 dilution, clone 6F/3D; DAKO, Glostrup, Denmark), an anti-human SRPX1 antibody (pretreated by autoclaving at 120 °C for 20 min in citric acid buffer) (1:100 dilution; Atlas Antibodies, Stockholm, Sweden), or an anti-phospho tau antibody (AT8, 1:200 dilution; Thermo Fisher Scientific, Waltham, MA). Secondary antibodies conjugated with horseradish peroxidase (HRP) (1:100, DAKO) were used for visualization of immunoreactive lesions by means of the ImmPACT DAB peroxidase substrate (Vector Laboratories, Burlingame, CA), followed by counterstaining with hematoxylin. For selective staining of Aβ40, we used the Amyloid β-protein immunohistostain kit (WAKO Pure Chemical Industries Ltd., Osaka, Japan).

For immunofluorescence studies, sections were blocked with 10% goat serum in PBS buffer-containing 0.2% Triton-X-100 and were incubated overnight with following primary antibodies: anti-human collagen IV (1:100 dilution; Chemicon, Temecula, CA), monoclonal antibody against α-smooth muscle actin (1:200 dilution; Sigma-Aldrich, St. Louis, MO), or anti-CD31 (1:20 dilution; DAKO). For these immunofluorescence studies, Alexa Fluor 594 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit, and Alexa Fluor 594 donkey anti-mouse (1:100 dilution; Invitrogen, Carlsbad, CA) were used as secondary antibodies. The samples were mounted by applying Vectashield Mounting Medium (Vector Laboratories) and were visualized using a fluorescence microscope (BZ-X700; Keyence, Osaka, Japan).

Three trained observers who were blinded to the clinical information independently assessed the histopathological findings.

Protein extraction and proteome analysis

We performed protein extraction and proteome analysis using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS), as previously described [28, 31]. Briefly, leptomeningeal and cortical vessels were isolated via LCM from 8 cases with severe CAA with extensive circumferential deposition of Aβ, 12 cases with mild CAA with a small amount of Aβ deposition, and 10 control subjects without CAA. LCM (LMD7000; Leica Microsystems, Wetzel, Germany) was performed using the bright-field setting to confirm the presence of cerebrovascular lesions, which were placed into microcentrifuge tubes containing 10 mM Tris, 1 mM EDTA, and 0.002% Zwittergent 3–16. Collected tissue samples were heated at 98 °C for 90 min. Samples were sonicated for 90 min and digested with 1.5 μl of 1 mg/ml trypsin (Promega, Madison, WI) overnight at 37 °C. Digested peptide mixtures were analyzed using nano-flow reversed-phase LC–MS/MS (LTQ Velos Pro; Thermo Fisher Scientific). We obtained the relative abundances of the identified molecules using the normalized spectral abundance factor [21].

Binding of Aβ40 and Aβ42 to SRPX1 in vitro

To determine the binding ability of Aβ40 and Aβ42 to SRPX1 in vitro, we performed an enzyme-linked immunosorbent assay (ELISA) as described earlier [9]. Briefly, after coating the anti-human SRPX1 antibody (1:200 dilution) in bicarbonate/carbonate coating buffer (100 mM, pH 9.5) at 4 °C overnight, 1 μM recombinant SRPX1 (Proteintech, Chicago, IL) was added as the capture antigen; 1% bovine serum albumin (BSA) was used as the negative control. Various concentrations (0–1.0 μM) of native Aβ40 or Aβ42 were added and then incubation proceeded with the anti-human Aβ/APP antibody (1:10,000 dilution, clone 6E10; Biolegend, San Diego, CA). After incubation with an HRP-conjugated secondary antibody and color development, the absorbance at 450 nm was measured with a microplate spectrophotometer (Bio-Rad, Hercules, CA).

Knockdown of SRPX1 by siRNA

Primary murine cerebrovascular smooth muscle cells isolated from 3-month-old C57BL/6 J mice were cultured according to a previous report [10]. To knock down SRPX1 expression, we transfected primary cerebrovascular smooth muscle cells with SRPX1-specific siRNA (MISSION esiRNA; Sigma-Aldrich) complexed with Lipofectamine RNAi Max (Invitrogen) in Opti-MEM (Gibco, Grand Island, NY) for 48 h, according to the manufacturer’s protocol. Luciferase siRNA (MISSION esiRNA; Sigma-Aldrich) served as the negative control siRNA.

Treatment of murine cerebrovascular smooth muscle cells with Aβ

Primary cultured murine cerebrovascular smooth muscle cells were treated with freshly prepared Aβ40 or Aβ42, at a final concentration of 10 μM, for 24 h. The same volume of PBS was used as a negative control. Hydrogen peroxide, at final concentrations of 100–1000 nM, was also used as a control agent for 24 h. To detect Aβ deposits in cultured cells, we performed immunohistochemistry with anti-human Aβ antibody (1:100 dilution, clone 6F/3D; DAKO). Oligomerization of Aβ was analyzed via glutaraldehyde cross-linking and Western blotting with the anti-human Aβ/APP antibody (1:1000 dilution, clone 6E10; Biolegend).

Determination of caspase-3/7 activity

Caspase-3/7 activity was determined in cultured smooth muscle cells treated with Aβ40 at a final concentration of 10 μM for 24 h using the Apo-ONE Homogeneous Caspase-3/7 Assay (Promega), according to the manufacturer’s instructions. Fluorescence was measured with the FilterMax F5 Microplate Reader (Molecular Devices, Sunnyvale, CA).

Real-time quantitative reverse transcription polymerase chain reaction (PCR)

We extracted total RNA from primary cultures of murine cerebrovascular smooth muscle cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany). We also extracted total RNA from human and murine cerebral cortex, hippocampus, and purified cerebral blood vessels using the TRIzol reagent (Thermo Fisher Scientific). We isolated cerebral blood vessels according to the method of Boulay et al. while allowing for mechanical isolation [2]. Briefly, brain tissues obtained from human cases (see Supplementary Table S1) and from 3-month-old C57BL/6 J male mice were homogenized in Hanks’ balanced salt solution (HBSS) buffer supplemented with 10 mM HEPES, followed by centrifugation at 2000×g for 10 min at 4 °C. The precipitates were resuspended in 18% dextran in HBSS buffer and centrifuged at 4400×g for 15 min at 4 °C. The final pellets were resuspended in 2 ml of HBSS buffer and were passed through a 40-μm nylon mesh (BD Biosciences, San Jose, CA). We used cerebral blood vessels retained on the mesh for real-time PCR analyses.

Real-time PCR was performed as previously described [19]. Primers used were as follows: mouse SRPX1 forward: 5ʹ-GATCAGAGCAAAGATTATGCCTCCA-3ʹ; mouse SRPX1 reverse: 5ʹ-CTTTATCCATGCCATGCTTATCCA-3ʹ; mouse GAPDH forward: 5ʹ- TTAGCACCCCTGGCCAAGG-3ʹ; mouse GAPDH reverse: 5ʹ- CTTACTCCTTGGAGGCCATG-3ʹ; human SRPX1 forward: 5ʹ-GCCATGCCAGCAAATGGAG-3ʹ; human SRPX1 reverse: 5ʹ-ACACTTGGGCACTTGATTCTAGGAG-3ʹ; human GAPDH forward: 5ʹ- GAGTCAACGGATTTGGTCGT-3ʹ; human GAPDH reverse: 5ʹ- TTGATTTTGGAGGGATCTCG-3ʹ.

Western blot analysis

Primary cultured murine cerebrovascular smooth muscle cells were washed twice in ice-cold PBS and then lysed by the addition of RIPA buffer (Thermo Fisher Scientific) containing freshly added protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Protein concentrations were determined using the BCA Protein Assay kit (Pierce Chemical, Rockford, IL), and equal amounts of protein from each sample were resolved by means of 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad). We performed Western blot analysis as previously described [19]. Briefly, the PVDF membranes containing the proteins were first incubated with a blocking buffer (Blocking One; Nacalai Tesque) and were then incubated with an anti-mouse SRPX1 antibody (1:4000 dilution; ProSci, Poway, CA) or an anti-mouse GAPDH antibody (1:1000 dilution; MBL, Nagoya, Japan) followed by incubation with an HRP-conjugated secondary antibody. We used ImageJ 1.47 software (NIH, Bethesda, MD) to analyze the intensity of specific bands.

APOE genotyping

Genomic DNA was extracted from the formaldehyde-fixed paraffin-embedded brain tissues according to the manufacturer’s instructions (QIAamp DNA FFPE Tissue Kit; Qiagen, Düsseldorf, Germany) [24]. APOE genotyping was performed using a semi-nested PCR assay followed by digestion with the restriction enzyme HhaI as described previously [11].

Statistical analysis

We analyzed data via Student’s t test and one-way ANOVA followed by Tukey’s post hoc test when the ANOVA reached significance. A non-parametric test (Mann–Whitney U test) was used for mass spectrometric analyses. The level of statistical significance was set at p < 0.05. Statistical analyses were performed using JMP 9.0 statistical software (SAS Institute, Cary, NC).

Ethics

The Human Ethics Review Committee of Kumamoto University approved the study protocol. Families of subjects provided signed consent forms. All patients’ family members gave informed consent for performance of an autopsy. The Animal Care and Use Committee of Kumamoto University School of Medicine approved the protocols for animal experiments.

Results

Up-regulated molecules in autopsied CAA cases

In our postmortem analyses, the occurrence of CAA was 27.3% (n = 15), with the value increasing with age (the trend being 0, 5.6, 38.1, and 50.0% for patients who had had autopsies in their 50s, 60s, 70s, and 80s and older, respectively, p = 0.017) (Supplementary Fig. S2). The occurrence of senile plaques was 60.0% (n = 33) in those autopsied cases. Genotype frequencies for cases with CAA differed from frequencies for cases without CAA (Table 1).

Table 1 Clinicopathological characteristics of autopsied cases with and without CAA
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We performed proteomic analyses with microdissected cerebrovascular tissue samples and identified the up-regulated molecules in the cerebral blood vessels of severe CAA cases (Table 1). Six molecules, Aβ, ApoE, glial fibrillary acidic protein (GFAP), two isozymes (α and β) of enolase, and SRPX1, were significantly up-regulated in severe CAA cases, and those molecules were detected in all eight cases with severe CAA (Table 2). In the present study, we focused on SRPX1 because of its potential specific expression in blood vessels. In addition, because SRPX1, which is known as a tumor suppressor gene, reportedly induced apoptosis in tumor cells [26, 27], we hypothesized that SRPX1 may play an important role in Aβ-induced apoptosis in CAA. Our proteomic analyses also revealed that SRPX1 levels in cerebral blood vessels of severe CAA cases were higher than those of mild CAA cases (Supplementary Table S2). Although we did not find SRPX1 up-regulation in mild CAA cases compared with control non-CAA cases by means of proteomic analyses (Supplementary Table S3), immunohistochemical staining, which is thought to be a more sensitive method for detecting SRPX1 in cerebral blood vessels, revealed that SRPX1 was indeed up-regulated in mild CAA cases compared with control non-CAA cases (Fig. 1).

Table 2 Significantly up-regulated molecules in cerebral blood vessels of cases with severe CAA
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Fig. 1
figure 1

Representative histopathological images from a case with severe CAA (case no. SC-4). Serial sections show CR-positive leptomeningeal arteries surrounding the occipital cortex (a) and those same arteries viewed under polarized light (CRPL) (b). These arteries stained positive for Aβ/APP (c), Aβ40 (d), and SRPX1 (e) (arrows). An Aβ-stained brain parenchymal lesion revealed that senile plaques were negative for SRPX1 (a–f). Arrowheads point to senile plaques. Scale bars 200 μm in a–e. f–h Percentages of SRPX1-positive cerebral blood vessels in autopsied cases with mild or severe CAA and in autopsied cases without CAA (control). Leptomeningeal arteries (≥100 μm in diameter) (f), leptomeningeal arterioles (<100 μm in diameter) (g), and cortical arteries (h). *p < 0.05, **p < 0.01, ***p < 0.001. NS not significant. i–k Representative cerebrovascular SRPX1-positive images from a control non-CAA case (case no. C-17) (i), a mild CAA case (case no. MC-11) (j), and a severe CAA case (case no. SC-3) (k). Scale bars 100 μm in i–k. l–n Early accumulation of SRPX1 (l) in the collagen IV-positive (m) basement membrane of cerebral blood vessels in a mild CAA case (case no. MC-6). n Merged image for SRPX1 (green) and collagen IV (red). Scale bars 100 μm in l–n

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Accumulation and Aβ binding of SRPX1 in cerebral blood vessels in autopsied cases with and without CAA and in mouse brain

Immunohistochemical studies revealed that SRPX1 co-accumulated with Aβ amyloid deposits in cerebral blood vessels of autopsied cases with CAA (Fig. 1a–e). In contrast, SRPX1 did not co-accumulate with Aβ deposits in senile plaques (Fig. 1e). SRPX1 accumulated in cerebral blood vessels in cases with CAA significantly more often than in those without CAA (Fig. 1f–k, Table 1). The percentages of SRPX1-positive vessels were significantly higher for both leptomeningeal arteries (≥100 μm in diameter) (Fig. 1f) and leptomeningeal arterioles (<100 μm in diameter) (Fig. 1g) in CAA cases compared with control non-CAA cases. In cortical arteries, the percentages of SRPX1-positive vessels were not different in both CAA and non-CAA cases (Fig. 1h). SRPX1 accumulation was not found in white matter vessels (Supplementary Fig. S3). SRPX1 was frequently found in the basement membrane of cerebral blood vessels in both mild and severe CAA cases (Fig. 1l–n, Supplementary Table S4). The percentages of SRPX1-positive endothelial cells tended to be higher in severe CAA cases than in mild CAA cases (Supplementary Table S4, Supplementary Fig. S4).

Real-time PCR analyses revealed significantly higher SRPX1 mRNA expression in isolated cerebral blood vessels than in the cerebral cortex and hippocampus in mice (Fig. 2a). In human brain samples, SRPX1 mRNA levels in cerebral blood vessels isolated from the cortex region were also significantly higher than tissue levels in the cortex obtained from the same cases (Fig. 2b).

Fig. 2
figure 2

SRPX1 mRNA levels in cerebral cortex tissues and blood vessels. a SRPX1 mRNA levels in the cerebral cortex, hippocampus, and cerebral blood vessels from 3-month-old C57BL/6 J male mice (n = 3). b SRPX1 mRNA levels in human cortex tissues and isolated cerebral blood vessels obtained from autopsied cases (n = 5). SRPX1 mRNA levels were normalized to GAPDH mRNA. Data represent mean ± SEM. NS not significant. *p < 0.05, **p < 0.01

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We investigated the binding of Aβ40 and Aβ42 to SRPX1 using an ELISA with surfaces coated with SRPX1. Both Aβ40 and Aβ42 bound to SRPX1 in a concentration-dependent manner (ranging from 0.1 to 1.0 μM) but did not bind to the control protein 1% BSA (Fig. 3a, b).

Fig. 3
figure 3

Aβ binding of SRPX1. ELISA results for the binding ability of Aβ40 (a) and Aβ42 (b) to SRPX1 in vitro. White circles indicate the amount of Aβ on antigen-free surfaces, and black circles indicate the amount of Aβ40 on surfaces coated with SRPX1 (1 μM). *p < 0.005, **p < 0.001, surfaces coated with SRPX1 versus control surfaces. c Representative Western blots of cell lysates of cultured cerebrovascular smooth muscle cells, which were treated with control siRNA or SRPX1 siRNA for 48 h and analyzed using anti-SRPX1 and anti-GAPDH antibodies. d Quantified SRPX1 protein levels in the Western blots of cell lysates of cultured cerebrovascular smooth muscle cells treated with control siRNA or SRPX1 siRNA. Data were normalized to GAPDH. **p < 0.05 versus control. e Formation of Aβ accumulations in cultured murine cerebrovascular smooth muscle cells treated with Aβ40 at a final concentration of 10 μM for 24 h with control siRNA or SRPX1 siRNA. Immunofluorescence studies with anti-human Aβ/APP antibody (1:100 dilution, clone 6F/3D; DAKO) in cultured cells treated with control siRNA or SRPX1 siRNA. Scale bars 100 μm. f Percentages of Aβ40-positive lesions were quantified by averaging ten random fields for cultured cells treated with control siRNA or SRPX1 siRNA. Data represent mean ± SEM from three independent experiments. **p < 0.01

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Reduction of Aβ40 accumulations by SRPX1 knockdown in primary cultures of cerebrovascular smooth muscle cells

siRNA targeting SRPX1 reduced SRPX1 mRNA and protein levels to 30.3 and 37.4%, respectively, in primary cultures of murine cerebrovascular smooth muscle cells (Fig. 3c, d, Supplementary Fig. S5). We added freshly prepared Aβ peptides to the culture medium of cultured cells treated with siRNAs at a final concentration of 10 μM for 24 h and found Aβ accumulations in the cells using immunohistochemical analysis with anti-human Aβ/APP antibody. Aβ accumulations were reduced in cultured cerebrovascular smooth muscle cells treated with SRPX1 siRNA compared with cultured cells treated with control siRNA (Fig. 3e, f). The Aβ that was added to the cultured cells formed oligomeric aggregates in the cultured cells after 24 h (Supplementary Fig. S6).

Induction of SRPX1 expression by Aβ40 and Aβ42 in primary cultures of cerebrovascular smooth muscle cells

To investigate whether Aβ40 and Aβ42 could induce SRPX1 expression in primary cultures of cerebrovascular smooth muscle cells, we examined SRPX1 mRNA and protein levels in cultured smooth muscle cells treated with Aβ at a final concentration of 10 μM for 24 h and in cells that were not treated. Both Aβ40 and Aβ42 significantly increased the SRPX1 mRNA level and protein expression (Fig. 4a, b). We used hydrogen peroxide as a control toxic agent in the cell culture studies. A low concentration (100 nM) of hydrogen peroxide did not affect SRPX1 expression, but a high concentration (500 nM) reduced SRPX1 expression in cultured cerebrovascular smooth muscle cells (Supplementary Fig. S7a).

Fig. 4
figure 4

SRPX1 mRNA levels in primary cultured murine cerebrovascular smooth muscle cells treated with Aβ40 (a) or Aβ42 (b) at a final concentration of 10 μM for 24 h and in cells without such treatment. SRPX1 mRNA levels were normalized to GAPDH mRNA levels. *p < 0.05, **p < 0.01 versus control. c, d Representative Western blots of cell lysates of cultured cerebrovascular smooth muscle cells, which were treated with Aβ40 (c) or Aβ42 (d) at a final concentration of 10 μM for 24 h, or were not treated (Control), as analyzed using anti-SRPX1 and anti-GAPDH antibodies. e, f Quantified SRPX1 protein levels in the Western blots of cell lysates of cultured cerebrovascular smooth muscle cells treated with Aβ40 (e) or Aβ42 (f) or not treated (Control). Data were normalized to GAPDH. *p < 0.05, **p < 0.01 versus control

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Augmentation of caspase 3/7 activity by concomitant administration of SRPX1 and Aβ40

Aβ40 induced caspase-3/7 activity in primary cultures of murine cerebrovascular smooth muscle cells. Although SRPX1 alone did not induce caspase-3/7 activity in these cells, concomitant administration of SRPX1 and Aβ40 enhanced caspase-3/7 activity, which was significantly greater than that induced by a single administration of Aβ40 in primary cultures of cerebrovascular smooth muscle cells (Fig. 5a). Knockdown of SRPX1 significantly reduced caspase-3/7 activity induced by Aβ40 (Fig. 5b).

Fig. 5
figure 5

a Caspase-3/7 activity in cerebrovascular smooth muscle cells. Enhancement of caspase-3/7 activity by concomitant administration of SRPX1 and Aβ40. b Reduction of Aβ40-induced caspase-3/7 activity by SRPX1 knockdown. Data represent mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01. NS not significant

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We used hydrogen peroxide as a control toxic agent in the cell culture studies. A low concentration (100 nM) of hydrogen peroxide did not affect caspase-3/7 activity, whereas a high concentration (500 nM) induced increased caspase-3/7 activity in cultured cerebrovascular smooth muscle cells (Supplementary Fig. S7b).

Discussion

In the present study, we identified up-regulated molecules, including SRPX1, in cerebral blood vessels in CAA cases by means of proteomic analyses. Because SRPX1, which is known as a tumor suppressor gene, reportedly induced apoptosis in tumor cells [26, 27], we hypothesized that SRPX1 may play an important role in Aβ-induced apoptosis in CAA. We found significant up-regulation of SRPX1 in cerebral blood vessels in CAA cases. Furthermore, our in vitro analyses revealed that SRPX1 bound to Aβ and enhanced Aβ cytotoxicity in cultured cerebrovascular smooth muscle cells.

We first found that SRPX1 co-accumulated with cerebrovascular Aβ amyloid deposits in CAA and bound to Aβ in vitro but did not co-accumulate with Aβ in senile plaques, which is another important form of Aβ amyloid deposits in the cerebral cortex and hippocampus. We also found significant expression of SRPX1 in cerebral blood vessels, more than in the cerebral cortex. Higher expression of SRPX1 in cerebral blood vessels may contribute to specific accumulation of SRPX1 in cerebrovascular Aβ amyloid deposits in CAA. Our immunohistological studies revealed that SRPX1 frequently accumulated in the basement membrane of cerebral blood vessels in mild CAA cases, similar to the formation of Aβ deposits. This result suggests that the accumulation of SRPX1 may be an early event in CAA. In addition, the percentages of SRPX1-positive vessels were significantly higher in severe CAA cases compared with mild CAA cases in both proteomic and immunohistological analyses. SRPX1 may thus contribute to enhancing both early and late phases of CAA.

Second, we determined that Aβ up-regulated SRPX1 expression in cerebrovascular smooth muscle cells. Expression of SRPX1 mRNA was reportedly down-regulated in tumor cells, and the SRPX1 gene was thought to be a tumor suppressor gene [14, 25,26,27]. SRPX1 also induced apoptosis in human tumor cells [14, 25,26,27]. In the present study, we found that SRPX1 significantly enhanced apoptosis induced by Aβ40 in cerebrovascular smooth muscle cells. In contrast, knockdown of SRPX1 reduced Aβ40-induced apoptosis. Because we found that SRPX1 bound to Aβ, to support the binding interaction we used siRNA to knock down SRPX1 expression in murine cerebrovascular smooth muscle cells. We found reduced Aβ accumulations compared with the result with control siRNA. SRPX1 may play a role in the deposition of Aβ on the cell surface through its binding ability with Aβ and thus enhance Aβ-induced cerebrovascular degeneration in CAA. A potential limitation of this study is that the role of SRPX1 remains to be fully elucidated in cerebrovascular endothelial cells in CAA. Because our immunohistological analyses revealed that SRPX1 was also expressed in cerebrovascular endothelial cells, especially in severe CAA cases, SRPX1 may also play important roles in cerebrovascular endothelial cells in CAA. Further studies are needed to clarify this issue.

Our proteomic analyses also determined that other molecules, including ApoE, enolase, GFAP, vitronectin, clusterin, and midkine, were significantly up-regulated in cerebral blood vessels in CAA cases. Most of those up-regulated molecules were also found in AD brains [5, 7, 15, 20]. Those molecules may play roles in the formation of both vascular Aβ deposits in CAA and senile plaques in AD, and may therefore serve some functions in pathogenic pathways in CAA and AD.

On the basis of the findings just detailed, SRPX1 may be a novel molecular target in CAA diagnosis and therapy. Scintigraphy detecting another well-known co-accumulating molecule in amyloid deposits, SAP, is currently used to diagnose amyloidosis [23]. Scintigraphy with a novel co-accumulating molecule in cerebrovascular Aβ amyloid deposits, SRPX1, may therefore be a new diagnostic tool for CAA. In addition, inhibition of SRPX1 may be a novel therapeutic target to reduce Aβ-induced cerebrovascular degeneration in CAA. To pursue this issue, we must conduct additional in vivo studies.

In conclusion, SRPX1 may be a novel molecule that is associated with CAA and may enhance Aβ-induced cerebrovascular degeneration in CAA.