Identifying new biomarkers beyond the established risk factors that make it possible to predict and prevent ischemic stroke has great significance. Extracellular vesicles are powerful cell‒cell ...messengers, containing disease-specific biomolecules, which makes them powerful diagnostic candidates. Therefore, this study aimed to identify proteins derived from extracellular vesicles enriched serum related to future ischemic stroke events, using a proteomic method. Of Japanese subjects who voluntarily participated in health checkups at our institute a number of times, 10 subjects (6 males and 4 females, age: 64.2 ± 3.9 years) who developed symptomatic ischemic stroke (7.3 ± 4.4 years' follow-up) and 10 age‒sex matched controls without brain lesions (6.7 ± 2.8 years' follow-up) were investigated. Extracellular vesicles enriched fractions were derived from serum collected at the baseline visit. Differentially expressed proteins were evaluated using isobaric tagging for relative and absolute protein quantification (iTRAQ)-based proteomic analysis. Of the 29 proteins identified, alpha-2-macroglobulin, complement C1q subcomponent subunit B, complement C1r subcomponent, and histidine-rich glycoprotein were significantly upregulated (2.21-, 2.15-, 2.24-, and 2.16-fold, respectively) in subjects with future ischemic stroke, as compared with controls. Our study supports the concept of serum-derived extracellular vesicles enriched fractions as biomarkers for new-onset stroke. These proteins may be useful for prediction or for targeted therapy.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The purpose of the current study was to predict intraocular pressure (IOP) using color fundus photography with a deep learning (DL) model, or, systemic variables with a multivariate linear regression ...model (MLM), along with least absolute shrinkage and selection operator regression (LASSO), support vector machine (SVM), and Random Forest: (RF). Training dataset included 3883 examinations from 3883 eyes of 1945 subjects and testing dataset 289 examinations from 289 eyes from 146 subjects. With the training dataset, MLM was constructed to predict IOP using 35 systemic variables and 25 blood measurements. A DL model was developed to predict IOP from color fundus photographs. The prediction accuracy of each model was evaluated through the absolute error and the marginal R-squared (mR
), using the testing dataset. The mean absolute error with MLM was 2.29 mmHg, which was significantly smaller than that with DL (2.70 dB). The mR
with MLM was 0.15, whereas that with DL was 0.0066. The mean absolute error (between 2.24 and 2.30 mmHg) and mR
(between 0.11 and 0.15) with LASSO, SVM and RF were similar to or poorer than MLM. A DL model to predict IOP using color fundus photography proved far less accurate than MLM using systemic variables.
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Cystatin C (CST3) is a cysteine protease inhibitor abundant in the central nervous system, and demonstrated to have roles in several pathophysiological processes including vascular remodeling and ...inflammation. Previously, we showed a relation of CST3 gene polymorphisms with deep and subcortical white matter hyperintensity (DSWMH) in a small case-control study. In this study, we aimed to investigate the relation in a larger cross-sectional study. Participants of a brain health examination program were recruited (n = 1795) in the study, who underwent routine blood tests and cognitive function tests. Cerebral white matter changes were analyzed by MRI. Additionally, 7 single nucleotide polymorphisms (SNPs) (-82G/C, -78T/G, -5G/A, +4A/C, +87C/T, +148G/A and +213G/A) in the promoter and coding regions of CST3 gene were examined. Among them, carriers of the minor allele haplotype -82C/+4C/+148A were significantly associated with decreased CST3 concentration in the plasma. Unadjusted analysis did not show significant relation between carriers of the minor allele haplotype and periventricular hyperintensity (PVH), but DSWMH was marginally (p < 0.054) increased in this group. After adjusting the effects of other variables like age and kidney function, logistic regression analysis revealed that carriers of the minor allele haplotype were at a significantly increased risk of developing both PVH and DSWMH. Thus, our results suggest that carriers of the minor allele haplotype -82C/+4C/+148A of CST3 gene could be at an increased risk to develop cerebral white matter disturbance.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Increased angiogenesis, especially the pathological type, has been documented in Alzheimer's disease (AD) brains, and it is considered to be activated due to a vascular dysfunction-mediated hypoxic ...condition. To understand the role of the amyloid β (Aβ) peptide in angiogenesis, we analyzed its effects on the brains of young APP transgenic AD model mice. Immunostaining results revealed that Aβ was mainly localized intracellularly, with very few immunopositive vessels, and there was no extracellular deposition at this age. Solanum tuberosum lectin staining demonstrated that compared to their wild-type littermates, the vessel number was only increased in the cortex of J20 mice. CD105 staining also showed an increased number of new vessels in the cortex, some of which were partially positive for collagen4. Real-time PCR results demonstrated that placental growth factor (PlGF) and angiopoietin 2 (AngII) mRNA were increased in both the cortex and hippocampus of J20 mice compared to their wild-type littermates. However, vascular endothelial growth factor (VEGF) mRNA did not change. Immunofluorescence staining confirmed the increased expression of PlGF and AngII in the cortex of the J20 mice. Neuronal cells were positive for PlGF and AngII. Treatment of a neural stem cell line (NMW7) with synthetic Aβ
directly increased the expression of PlGF and AngII, at mRNA levels, and AngII at protein levels. Thus, these pilot data indicate that pathological angiogenesis exists in AD brains due to the direct effects of early Aβ accumulation, suggesting that the Aβ peptide regulates angiogenesis through PlGF and AngII expression.
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Cerebral microbleeds (CMBs) are associated with focal hemosiderin deposits and represent a form of cerebral small vessel disease. To date, indefinite and inconsistent reports are available regarding ...the association between serum lipid fractions and CMBs. In addition, these previous studies did not include Asian populations, who may have a higher risk of cerebral hemorrhage. The purpose of this study was to examine the associations between serum lipid fractions and CMBs in healthy Japanese subjects.
We performed a cross-sectional study involving 4,024 neurologically normal Japanese subjects (mean age 61.6 years). All the participants underwent 1.5-Tesla magnetic resonance imaging scan, and CMBs were classified into 3 groups based on their locations. The concentrations of lipid fractions were categorized into quartiles and the association between the lipid fractions and CMBs were investigated using logistic regression analysis.
CMBs were observed in 164 (4.1%) of participants. Of these participants with CMBs, 33 (20.1%) had lobar CMBs and 91 (55.5%) had deep CMBs. Subjects with deep CMBs had lower total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) levels. After adjusting for confounding factors, lower TC and HDL-C levels were still associated with the presence of deep CMBs (OR for the highest vs. the lowest quartiles of TC and HDL-C was 2.28 95% CI 1.05-4.94, and 1.93 95% CI 1.02-3.65, respectively). The presence of subcortical infarcts and periventricular hyperintensities was more frequently observed in deep CMBs, whereas white matter hyperintensities were more frequently observed in lobar CMBs.
Our results suggest that low serum TC and HDL-C levels are closely associated with deep CMBs.
Growing evidence suggests that vascular risk factors, especially hypertension, relate not only to cardiovascular disease but also to cognitive impairment. However, the impact of pulse pressure on ...cognitive function remains controversial. In this study, we evaluated the associations between pulse pressure and cognitive function in a Japanese health examination cohort using propensity matching analysis.
We examined 2,546 individuals with a mean age of 60.8 ± 10.3 years who voluntarily participated in health examination. Clinical variables included pulse pressure, and brain magnetic resonance imaging (MRI). We divided the participants into the high and low pulse pressure groups with a pre-defined cut-off value of 65 mmHg and evaluated their physical examination data, cognitive functions including Okabe's test, Kohs' test, and silent brain lesions using propensity matching. To clarify whether pulse pressure and blood pressure have different implications for cognitive function, a mediating analysis was also conducted.
From the 2,546 subjects, 439 (17.2%) were in the high PP group. The propensity matching algorithm produced 433 pairs of patients with similar propensities. Higher pulse pressure corresponded to lower Okabe and Kohs' scores (44.3 ± 7.1 vs 42.7 ± 7.5; p = 0.002, 97.9 ± 18.0 vs 95.0 ± 18.1 p = 0.019, respectively). The relationship between pulse pressure and cognitive impairment was not significantly mediated by systolic blood pressure. We observed no significant associations between silent brain lesions and pulse pressure.
High pulse pressure was associated with lower cognitive performance without systolic blood pressure mediation in Japanese subjects without dementia.
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Cystatin C (CST3) is an endogenous cysteine protease inhibitor, which is implicated in cerebral amyloid angiopathy (CAA). In CAA, CST3 is found to be aggregated. The purpose of this study is to ...investigate whether this aggregation could alter the activity of the protein relevant to the molecular pathology of CAA. A system of CST3 protein aggregation was established, and the aggregated protein was characterized. The results showed that CST3 aggregated both at 80 °C without agitation, and at 37 °C with agitation in a time-dependent manner. However, the levels of aggregation were high and appeared earlier at 80 °C. Dot-blot immunoassay for oligomers revealed that CST3 could make oligomeric aggregates at the 37 °C condition. Electron microscopy showed that CST3 could make short fibrillary aggregates at 37 °C. Cathepsin B activity assay demonstrated that aggregated CST3 inhibited the enzyme activity less efficiently at pH 5.5. At 7.4 pH, it lost the inhibitory properties almost completely. In addition, aggregated CST3 did not inhibit Aβ1-40 fibril formation, rather, it slightly increased it. CST3 immunocytochemistry showed that the protein was positive both in monomeric and aggregated CST3-treated neuronal culture. However, His6 immunocytochemistry revealed that the internalization of exogenous recombinant CST3 by an astrocytoma cell culture was higher when the protein was aggregated compared to its monomeric form. Finally, MTT cell viability assay showed that the aggregated form of CST3 was more toxic than the monomeric form. Thus, our results suggest that aggregation may result in a loss-of-function phenotype of CST3, which is toxic and responsible for cellular degeneration.
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•PC was decreased and LPC was increased in the hippocampus of J20 mice at 6 months.•PC, PE, and LPC were Increased at 3 months in the cortex of J20 mice.•PC, PE, and LPC were ...decreased at 6 months in the cortex of J20 mice.•LPC 16:0, LPC 18:1, and LPC 20:4 were increased in J20 hippocampus at 6 months.•Compared to 3 months, ROS levels were higher at 6 months in J20 mice.
Phospholipid levels are reported to be decreased in Alzheimer’s disease (AD). For a better understanding, we investigated the time-dependent changes of phospholipids species in a mouse model of AD. The levels of phospholipids in the hippocampus and prefrontal cortex of wild-type and APP-Tg (J20) mice were measured by LC-ESI-MS/MS. Compared to wild-type, total phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lysophosphatidylcholine (LPC) were Increased at 3 months but decreased at 6 months in the cortex of J20 mice. Total lysophosphatidylethanolamine (LPE) was decreased both at 3 and 6 months. PC was decreased and LPC was increased at 6 months, resulting in an increased LPC/PC ratio in the hippocampus of J20 mice. At species levels, PCA analysis could discriminate wild-type and J20 based on PC and LPC distribution at 6 months. At 6 months, several highly abundant PC including PC (16:0/16:0), PC (16:0/18:0), PC (16:0/18:1), and PC (18:0/18:1) were decreased in the cortex and hippocampus of J20. Conversely, LPC species including LPC 16:0, LPC 18:1, and LPC 20:4 were increased especially in the hippocampal area. Increased activation of phospholipid-metabolizing enzyme cPLA2 was seen in the hippocampus and cortex of J20 mice at 9 months. On the other hand, ROS levels started to increase as early as 3 months. Compared to 3 months, ROS levels were higher at 6 months in J20 mice. Thus, we demonstrated here a time- and area-dependent alteration of phospholipid composition during the early stage of AD, which could be important in understanding the pathological process.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Metabolic syndrome (MetS) has been associated with silent brain lesions; however, there are no data on the relationship between MetS and the incidence of cerebral microbleeds (CMBs) in Asian ...populations. The aim of this study was to evaluate the longitudinal association between MetS and incidence of CMBs in the Japanese population. We performed a prospective cohort study involving 684 Japanese participants (mean age, 61.7 years) with a mean 6.5 ± 3.4 years follow-up. All participants underwent 1.5 T magnetic resonance imaging, and CMBs were classified by their locations. Logistic regression analyses were performed to examine the relationship of MetS and its components with the incidence of CMBs. MetS was observed in 7.5% of the study population. Forty-nine (7.2%) subjects (36 had new deep or infratentorial CMBs, 13 had new strictly lobar CMBs) developed new CMBs during the follow-up period. In multivariable analysis, MetS was significantly associated with the incidence of deep or infratentorial CMBs (odds ratio, 4.03; 95% confidence interval, 1.72-9.41), and the elevated blood pressure component was most robustly associated with the incidence of deep or infratentorial CMBs (odds ratio, 5.16; 95% confidence interval, 2.02-13.2). Increased body mass index was also associated with incidence of deep or infratentorial CMBs (odds ratio, 2.45; 95% confidence interval, 1.06-5.67). The present study showed that MetS predicts incidence of CMBs in the deep brain regions and high blood pressure is the most important among the MetS components.
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Background
Rho-kinase inhibition in a rat middle cerebral artery occlusion (MCAO) model is reported to improve neurological functions and decrease infarction size.
Objective
The objective of this ...study is to investigate the underlying mechanisms of such improvement by evaluating the effects of Rho-kinase inhibition on astrocytes and microglial accumulation and activation in this condition.
Methods
Adult male Sprague-Dawley (SD) rats were used to generate the MCAO model, which received an I.P injection of a chemical Rho-kinase inhibitor (Fasudil- 5 mg/kg/day) or vehicle (PBS) for 2 and 4 days.
Results
Fasudil treatment significantly decreased the stroke volumes and water content in the lesion areas, as revealed by MRI. Immunostaining and Western blotting results demonstrated that Fasudil significantly decreased the levels of Aquaporin-4, a water channel protein. The number of GFAP+ astrocytes and Iba-1+ macrophage/microglia was decreased in the lesion areas. Proinflammatory transcription factor NF-κB protein levels were decreased in the Fasudil group 2 days after MCAO. Also, proinflammatory mediators including TNF-α, IL-1β, and iNOS levels were decreased. In vitro migration study using a human microglial cell line (HMO6) confirmed the inhibitory effects of Fasudil on the process. Fasudil also decreased combined IL-1β and IFNγ-induced NF-κB nuclear translocation in HMO6. Moreover, Fasudil transiently decreased combined IL-1β and IFNγ-induced iNOS, TNFα, and IL-1β mRNA levels in HMO6.
Conclusion
Our study demonstrates the inhibitory effects of Rho-kinase on NF-κB-mediated glial activation and cerebral edema, which might be a promising therapeutic target in acute cerebral ischemia conditions.
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