Neurochemistry International 146 (2021) 105015

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Neurochemistry International
journal homepage: www.elsevier.com/locate/neuint

Diagnostic and prognostic blood biomarkers in vascular dementia: From the

viewpoint of ischemic stroke
Satoshi Hosoki, Tomotaka Tanaka, Masafumi Ihara *
Department of Neurology, National Cerebral and Cardiovascular Center, Suita, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Reliable quantitative blood biomarkers are important in vascular dementia (VaD) because early diagnosis and
Vascular dementia therapeutic intervention are effective in preventing progression of dementia. Although many blood biomarkers
Acute ischemic stroke for acute ischemic stroke (AIS) or VaD have been reported, there are few reliable blood biomarkers. VaD and AIS
Biomarker
have similar pathological conditions that are associated with small vessel disease (SVD) such as oxidative stress,
Noncoding RNA
Multimarker
inflammation, endothelial dysfunction, and neuronal injury. Therefore, it may be possible to find superior blood
biomarkers of VaD among AIS blood biomarkers. Owing to recent developments, noncoding RNAs such as
microRNA and long noncoding RNA, which can be analyzed using a single drop of blood, are also particularly
reliable VaD markers because they stably reflect brain tissue damage. A multimarker combining several blood
biomarkers or artificial intelligence technology may also be beneficial to compensate for insufficiencies of a
single blood biomarker. This review describes the blood biomarkers of VaD and how they are related to blood
biomarkers of AIS.

1. Introduction population (Pendlebury and Rothwell, 2019). According to a

Vascular dementia (VaD) is the second most common cognitive
impairment following Alzheimer’s disease (AD) (Gorelick et al., 2011)
and often complicated by co-existent AD (Kalaria and Ballard, 1999).
Management of vascular risk factors is directly associated with a
decrease in incidence of VaD (Fotuhi et al., 2009). Therefore, an early
diagnosis of VaD by blood biomarkers can contribute to early inter­
vention and prevention of dementia. Blood biomarkers may be optimal
candidates for identifying VaD in terms of the accessibility, minimal
invasiveness, and cost-effectiveness. However, there are few reliable
blood biomarkers of VaD. Meanwhile, many blood biomarkers for acute
ischemic stroke (AIS) have been explored for a long time (Whiteley et al.,
2008). VaD and AIS have similar pathological conditions that are asso­
ciated with small vessel disease (SVD), such as oxidative stress,
inflammation, endothelial dysfunction, and neuronal injury. Approxi­
mately one-fourth of the patients with AIS have SVD, mainly in the form
of lacunar infarction (Pasi and Cordonnier, 2020). In addition, SVD is
the major pathological alteration of VaD (Iadecola, 2013). Thus, SVD is a
shared pathological mechanism between VaD and AIS. Moreover, AIS
itself is a risk factor of VaD. The incidence of dementia is nearly 50 times
higher in patients in the year after a major stroke than in the general
meta-analysis (Pendlebury and Rothwell, 2009), approximately 10% of
patients develop dementia within a few months to a year after stroke,
and >30% develop dementia after stroke recurrence. However, the
remaining stroke cases also have SVDs such as white matter hyper­
intensities (WMH), cerebral microbleeds (CMBs), and lacunar infarction.
These SVDs are expected to cause dementia (Béjot et al., 2011; Pend­
lebury and Rothwell, 2009; Yatawara et al., 2020). Therefore, it is
possible to find new blood biomarkers of VaD among candidate blood
biomarkers of AIS. In addition, owing to the recent developments,
noncoding RNAs such as microRNA and long noncoding RNA, which can
be analyzed using a single drop of blood, are also particularly reliable
VaD markers. They are caused by transcriptional changes induced in the
pathological changes in VaD and stably reflect brain tissue damage. This
review describes the blood biomarkers of VaD and how they are related
to AIS blood biomarkers.
2. Pathomechanism and diagnosis of VaD
VaD accounts for at least 20% of dementia cases (Gorelick et al.,
2011), and vascular factors are known to accelerate AD pathology
(Kalaria et al., 2012; Snowdon et al., 1997). Inducing cerebral

* Corresponding author. Department of Neurology, National Cerebral and Cardiovascular Center, 6-1 Kishibe-Shimmachi, Suita, Osaka, 564-8565, Japan.
E-mail address: ihara@ncvc.go.jp (M. Ihara).

https://doi.org/10.1016/j.neuint.2021.105015
Received 31 December 2020; Received in revised form 25 February 2021; Accepted 2 March 2021
Available online 26 March 2021
0197-0186/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Hosoki et al.
circulatory failure in AD model mice that overexpress human mutant
amyloid precursor protein increases the polymerized amyloid β protein
in the soluble fraction, causing synergistic cognitive deterioration
function (Viswanathan et al., 2009). Moreover, an 8-month brain
hypoperfusion of wild-type mice caused hippocampal atrophy and
decreased glucose metabolism, resulting in impairment of episodic
memory (Nishio et al., 2010). Inducing cerebral circulatory insufficiency
in a mouse model of cerebral amyloid angiopathy significantly increases
the amount of amyloid β protein deposited on the blood vessel wall and
exacerbates cognitive dysfunction (Okamoto et al., 2012). These explain
the significant effects of vascular factors on neurodegeneration (Ihara
and Tomimoto, 2011). In patients with AD, cerebral amyloid angiopathy
and cortical vascular abnormalities are frequently observed (Kitaguchi
et al., 2007), and peripheral vascular resistance correlates with amyloid
accumulation in the brain observed by amyloid imaging (Hughes et al.,
2013). Therefore, VaD and AD may be linked bidirectionally and syn­
ergistically to exacerbation of dementia (Kalaria and Ihara, 2013). In
fact, the pathological changes with VaD and AD frequently occur
together, which is termed as mixed dementia (Saito and Ihara, 2016;
Zekry et al., 2002). In retrospective and prospective autopsy studies, the
prevalence of mixed dementia ranges from 2% to 58%, with reasonable
means ranging from 6% to 12% (Jellinger and Attems, 2007). Clinically,
mixed dementia also includes AD with cerebrovascular disease, vascular
lesions, and vascular risk factors (Kalaria, 2000). In the Rush Memory
and Aging Project, the frequency of mixed dementia was 40% among the
total dementia cases (Schneider et al., 2007). Understanding the pa­
thology of vascular dementia can also aid in better management of AD.
The Framingham study found a 44% reduction in dementia in 30 years
(Satizabal et al., 2016). The most significant decrease was in VaD, which
suggests that control of vascular risk factors is associated with the
reduction of VaD. In addition, the incidence of AD also declined,
although not statistically significant, suggesting that control of vascular
risk factors is related to the decrease in incidence of AD (Satizabal et al.,
2016). Thus, in the treatment or prevention of dementia, even AD,
physicians should determine vascular contribution to dementia and
consider early management of vascular risk factors, if present, such as
hypertension, diabetes, hypertension, and obesity (Fotuhi et al., 2009).
The current diagnosis of VaD is mainly based on cognitive and neuro­
imaging evaluation, and the sensitivity and specificity of diagnosis are
inadequate. Diagnosis by neuroimaging is difficult because similar
neuroimaging findings are associated with dementia in some patients
but with normal cognition in others (van der Flier et al., 2018). There is
no consensus on dementia screening tools (Ghafar et al., 2019). There­
fore, the development of objective blood biomarkers for VaD has great
clinical significance.
3. Shared pathology of VaD and AIS
The vascular alterations that cause VaD are diverse and include
systemic conditions that influence global cerebral perfusion, or alter­
ations involving cerebral blood vessels, most commonly SVD (Iadecola,
2013). SVD includes various pathological changes that affect arterioles,
venules, and capillaries (Iadecola, 2013), which are associated with
imaging findings of SVD, including lacunar infarction, WMH, CMBs, and
enlarged perivascular spaces (Staals et al., 2014). Despite the diversity
of the underlying brain pathology, VaD and AIS have similar pathogenic
bases associated with SVD, resulting from hypoperfusion, oxidative
stress, and inflammation, which in turn lead to endothelial damage,
blood-brain barrier (BBB) breakdown, activation of innate immunity,
disruption of trophic coupling between vascular and brain cells, and
neuronal injury (Iadecola, 2013). Such markers in each pathological
mechanism may be candidates for blood biomarkers in VaD. AIS begins
with sudden brain hypoperfusion, resulting in cell energy deficiency,
neurotoxicity, oxidative stress within minutes, microangiopathy, acti­
vation of hemostatic mechanisms, inflammatory response, endothelial
damage, and dysfunction of the blood–brain barrier within hours,
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Neurochemistry International 146 (2021) 105015
resulting in neuronal injury (Brouns and De Deyn, 2009). Although VaD
is generally caused by slower response to more chronic cerebral hypo­
perfusion, oxidative stress, inflammation, endothelial dysfunction, and
neuronal injury are shared pathological mechanisms between VaD and
AIS. The next section will outline the blood biomarkers of VaD and how
they are related to the blood biomarkers of AIS (Fig. 1, Table 1).
4. Oxidative stress
Brain ischemia generates reactive oxygen species (ROS), such as
superoxide anion, hydroxyl radical, hydroperoxyl radical, hydrogen
peroxide, and singlet oxygen, and reactive nitrogen species (RNS), such
as nitric oxide and peroxinitrite anion. Oxidative stress refers to a situ­
ation in which cells’ antioxidant capacity is exceeded in the state where
ROS and RNS are excessively generated (Brouns and De Deyn, 2009).
Excessive ROS and RNS production causes DNA damage, protein
destruction, lipid peroxidation, and tissue damage, resulting in the death
of the nerve cell (Chan, 2001). ROS and RNS also trigger necrosis and
apoptosis of nerve cells (Rana and Singh, 2018) (Fig. 1A).
A product of membrane phospholipid peroxidation is oxidized low-
density lipoprotein (LDL). The pathological roles of oxidized LDL
(oxLDL) are endothelial cell injury, promotion of foam cell formation,
proliferation of vascular smooth muscle cells, and platelet adhesion
aggregation (Wang et al., 2017). Elevated oxLDL levels are associated
with lower MMSE scores in patients with AIS in a large cohort (VaD
group, 2711; control group, 1015) (Wang et al., 2018a). OxLDL is
reportedly related to AIS (Yokota et al., 2016) and the prognosis of AIS
(Uno et al., 2003).
Malondialdehyde (MDA) is produced as a reactive oxygen peroxide
and degrades membrane phospholipids in cell membranes (Cano et al.,
2003). MDA is a blood biomarker of AIS (Cano et al., 2003) and has been
associated with early AIS outcome (Polidori et al., 2002) and related to
VaD.
DJ-1, a translation product of DJ-1, which is the causative gene of
familial Parkinson’s disease PARK7, is implicated in oxidative stress
after ischemia and has an antioxidant effect by promoting self-oxidation
and transcription of antioxidant stress factors (Ariga et al., 2013).
Elevated blood DJ-1 level can distinguish patients with acute stroke
from controls (Allard et al., 2005).
Nicotinamide adenine dinucleotide phosphate oxidase (NOXs) is a
molecular source of ROS during aging, hypoperfusion, stroke, and hy­
pertension (Drummond et al., 2011). Several in vivo experiments
demonstrate that NOXs are involved in cognitive dysfunction (Choi and
Lee, 2017). Thus, NOXs can be a promising blood biomarker for VaD.
Several microRNAs associated with apoptosis are blood biomarkers
for VaD and AIS. miR-502-3P regulates SET, which is a potent and
specific inhibitor of protein phosphatase 2A, and is associated with
many cellular processes, such as cell cycle control, migration, and
apoptosis (Madeira et al., 2005), while miR-486-5p is associated with
proliferation, survival, apoptosis, and differentiation of endothelial
progenitor cells (Jiang et al., 2019). In a cohort of 204 patients with VaD
and 200 healthy and age- and sex-matched controls, elevated blood
miR-502-3p levels can distinguish patients with VaD with 75% sensi­
tivity and 89% specificity, while elevated blood miR-486-5p levels can
distinguish patients with VaD from 200 control patients with 75%
sensitivity and 83% specificity (Prabhakar et al., 2017). As for other
apoptosis-related microRNAs, miR-16 promotes ischemic brain damage
by negatively regulating anti-apoptotic Bcl-2 and Bcl-w (Yang et al.,
2017b), while miR-335 promotes stress granule formation and inhibits
apoptosis by reducing Rho-associated protein kinase 2 expression in AIS
(Si et al., 2019). Moreover, miR-30a-5p regulates the expression of RhoB
and beclin-1 genes and promotes apoptosis (Wang et al., 2018b). With
regard to the association between apoptosis-related microRNA and AIS,
blood levels of miR-16, miR-335, and miR-30a-5p, which are
apoptosis-related microRNAs, are diagnostic blood biomarkers of AIS
(Leung et al., 2014; Wang et al., 2018b; Zhao et al., 2016). High blood
S. Hosoki et al. Neurochemistry International 146 (2021) 105015

Fig. 1. The association between the pathology of VaD and the biomarkers. Many biomarkers are associated with the pathology of VaD, oxidative stress (A),
inflammation (B), endothelial dysfunction (C), and neuronal injury (D). ADMA: asymmetric dimethylarginine, AM: adrenomedullin, BBB: blood–brain barrier, DAMP:
damage-associated molecular patterns, GFAP: Glial fibrillary acidic protein, H-FABP: heart-type fatty acid-binding protein, ICAM-1: intercellular adhesion molecule-
1, IL-6: interleukin-6, IL-10: interleukin-10, MDA: malondialdehyde, MIAT: myocardial infarction-associated transcript, MMP-9: matrix metalloproteinase-9, NO:
nitric oxide, NOS: nitric oxide synthase, NOX: nicotinamide adenine dinucleotide phosphate oxidase, NSE: neuron-specific enolase, ox LDL: oxidized low-density
lipoprotein, TM: thrombomodulin, TNF-α: tumor necrosis factor-α, ROS: reactive oxygen species, RNS: reactive nitrogen species, UCH-L1: ubiquitin C-terminal
hydrolase L1, VCAM-1: vascular cell adhesion protein, vWF: von Willebrand factor.

miR-16 level is also associated with poor outcomes (Tian et al., 2016).
These microRNAs are promising blood biomarkers of VaD.
5. Inflammation
Damage-associated molecular patterns, released during cell death,
activate macrophages and neutrophils and produce inflammatory cy­
tokines. T cells are further activated to prolong inflammation (Naka­
mura and Shichita, 2019). Inflammation exacerbates leukoaraiosis
through demyelination, loss of axons, and oligodendrocyte degenera­
tion, causing neurodegeneration and cell death, and promoting the
inflammation of glial cells (Venkat et al., 2015) (Fig. 1B).
Interleukin-6 (IL-6) is a potent pro-inflammatory factor secreted by T
cells, macrophages, and neutrophils. A positive correlation has been
detected between IL-6 and WMH. IL-6 is significantly related to WMH
severity independent of age and systolic blood pressure (Nagai et al.,
2011).
Matrix metalloproteinase-9 (MMP-9) activated by ROS attacks the
basal lamina and tight junctions of the cerebral endothelial cells, causing
BBB dysfunction. MMP-9 levels are significantly increased in the cere­
brospinal fluid of patients with VaD compared to those with AD (Adair
et al., 2004). With regard to the blood biomarker of AIS, high plasma
MMP-9 level in AIS is associated with hemorrhagic transformation
(Castellanos et al., 2003).
Several microRNAs and long noncoding RNA associated with
inflammation are blood biomarkers for VaD and AIS. miR-126 inhibits
the secretion of tumor necrosis factor-α (TNF-α) and IL-6 in human
gingival fibroblasts under high-glucose conditions by targeting TNF
receptor-associated factor 6; and protects human cardiac microvascular
endothelial cells from hypoxia-reoxygenation-induced injury and in­
flammatory response (IL-6, IL-10, and TNF-α) by activating the PI3K-
Akt-eNOS signaling pathway (Yang et al., 2017a). miR-126 can be a
diagnostic marker of AIS (Long et al., 2013) and is also associated with
the National Institutes of Health Stroke Scale (NIHSS) score on admis­
sion and modified Rankin Scale (mRS) score at 3 months (Qi et al.,
2020). Myocardial infarction-associated transcript (MIAT), a long non­
coding RNA, is related to vascular endothelial cell inflammation. The
function of MIAT is to prolong inflammation by suppressing effer­
ocytosis and inhibiting clearance of apoptotic cells (Ye et al., 2019).
MIAT is a diagnostic marker for AIS (Zhu et al., 2018) and also associ­
ated with NIHSS at admission and mRS at 3 months (Zhu et al., 2018).
miR-126 and MIAT are candidate blood biomarkers of VaD.
6. Endothelial dysfunction
In the inflammatory response, leukocytes are recruited from the
circulation into the extravascular space. Activation of endothelial cells
allows them to express adhesion molecules on their surface and bind to
interacting molecules on the surface of circulating white blood cells
(Fig. 1C).
The cell adhesion molecules include intercellular adhesion molecule-
1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and selectins
(P- and E-selectin) (Cipollini et al., 2019). Upregulated expression of
ICAM-1 is associated with progression of WMH. After adjusting for age,
sex, cardiovascular risk factors, and C-reactive protein, ICAM-1 is
associated with WMH lesion progression at both 3 and 6 years (Markus
et al., 2005). As for the blood biomarker of AIS, increased ICAM-1 levels
are associated with an increased risk of AIS in patients with coronary
heart disease (Tanne et al., 2002) and with early death in middle-aged
patients with AIS (Rallidis et al., 2009).
VCAM-1 is a ligand expressed on the vascular endothelium that binds
to α4 integrin, which plays an essential role in the infiltration of T cells

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Table 1
Association between VaD and AIS biomarkers.
VaD AIS

Assoc VaD Ctrl Sn Sp ref Assoc AIS Ctrl OTMT Sn Sp ref

(n) (n) (%) (%) (n) (n) (%) (%)

Oxidative oxLDL Higher 2711 1015 – – Wang et al. AIS 250 250 <72 h N/ N/ Yokota et al. (2016)
Stress prevalence of (2018a) Dx A A
lower MMSE Associated with the prognosis of AIS Uno et al. (2003)
score
MDA not reported AIS 15 15 <24 h N/ N/ Cano et al. (2003)
Dx A A
Associated with the early outcomes of AIS Polidori et al. (2002)
DJ-1 AIS 622 31 <3 h N/ N/ Allard et al. (2005)
Dx A A
miR-502- VaD Dx 204 200 75 89 Prabhakar not reported
3p et al. (2017)
miR-486- 75 83
5p
miR-16 not reported AIS 74 19 <24 h 95 35 Leung et al. (2014)
Dx
Associated with the early prediction of mortality Tian et al. (2016)
and mRS
miR-335 AIS 168 104 <24 h 98 69 Zhao et al. (2016)
miR-30a- Dx 15 24 <6 h N/ N/ Wang et al. (2018b)
5p A A
Inflammation IL-6 WMH severity 137 – – – Nagai et al. not reported
(2011)
MMP-9 not reported Associated with the hemorrhagic transformation Castellanos et al.
in acute ischemic stroke (2003)
miR-126 AIS 197 50 <24 h 92 84 Long et al. (2013)
Dx
Associated with mRS at 3 months Qi et al. (2020)
MIAT AIS 189 189 <24 h 74 80 Zhu et al. (2018)
Dx
Associated with mRS at 3 months
Endothelial ICAM-1 Progression of 296 – – – Markus AIS 134 134 – – – Tanne et al. (2002)
Dysfunction WMH et al. (2005) Dx
Associated with early death in middle-aged Rallidis et al. (2009)
patients with AIS
VCAM-1 Severe WMH 11 18 – – de Leeuw Associated with outcome at 3 months in patients Richard et al. (2015)
et al. (2002) with CVD
P-selectin – – Associated with the atherothrombotic properties Pawelczyk et al.
of hyperglycemia and hyperlipidemia in stroke (2018)
patients
vWF WMH severity 198 – – – Nagai et al. Associated with the severity, outcome, and infarct Sato et al. (2006)
(2012) size in ischemic stroke
TM Severe WMH 110 50 – – Hassan Associated with fatal outcomes in patients with Olivot et al. (2004)
and the number et al. (2003) AIS
of lacunar
infarction on
MRI
ADMA Prevalence of 2013 – – – Pikula et al. AIS 52 48 <24 h – – Ercan et al. (2019)
silent brain (2009) Dx
infarcts
Fibrinogen Increased risk of 185 – – – Xu et al. Associated with the prognosis of AIS Kernagis and
both AD and (2008) Laskowitz (2012)
VaD
mid- WMH severity 288 – – – Kuriyama not reported
regional and cognitive et al. (2017)
proAM function test
scores
Neuronal NSE VaD Dx 42 38 – – Shen and AIS 66 N/A <3 h N/ N/ Wunderlich et al.
injury Gao (2015) Dx A A (2006)
Associated with the prognosis of LAA and IS after Wang et al. (2018c)
IVT
S100β Cognitive 210 207 – – Gao et al. Distinguishing between AIS and ICH (Wunderlich et al.,
impairment in (2015) Associated with the prognosis of AIS 2004; Zhou et al.,
patients with 2016)
WMH
GFAP not reported Distinguishing between AIS and ICH in 4.5 h Ren et al. (2016)
H-FABP AIS 22 22 <24 h 68 100 Zimmermann-Ivol
Dx et al. (2004)
Associated with poor outcomes Lyang et al. (2019)
miR-134 AIS 50 50 <24 h 75 73 Zhou et al. (2018)
Dx
Associated with the poor outcomes of AIS
miR-451a VaD Dx 204 200 70 75 not reported
(continued on next page)

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Table 1 (continued )
VaD AIS

Assoc VaD Ctrl Sn Sp ref Assoc AIS Ctrl OTMT Sn Sp ref

(n) (n) (%) (%) (n) (n) (%) (%)

Miscellaneous miR-409- 76 89 Prabhakar

markers 3p et al. (2017)
Multimarker 4-protein not reported AIS 65 157 <6 h 90 90 Lynch et al. (2004)
panel Dx

ADMA: asymmetric dimethylarginine, AIS: acute ischemic stroke, Assoc: association, AM: adrenomedullin, Ctrl: control, Dx: diagnosis, GFAP: Glial fibrillary acidic
protein, H-FABP: heart-type fatty acid-binding protein, ICAM-1: intercellular adhesion molecule-1, ICH: intracerebral hemorrhage, IL-6: interleukin-6, IVT: intrave­
nous thrombolysis, LAA: large-artery atherosclerosis, MDA: malondialdehyde, mid-regional proAM: mid-regional pro-adrenomedullin, MIAT: myocardial infarction-
associated transcript, MMP-9: matrix metalloproteinase-9, MMSE: Mini Mental State Examination, MRI: magnetic resonance imaging, mRS: modified Rankin Scale,
NO: nitric oxide, NOS: nitric oxide synthase, NOX: nicotinamide adenine dinucleotide phosphate oxidase, NSE: neuron-specific enolase, OTMT: onset to the mea­
surement time, ox LDL: oxidized low-density lipoprotein, Sn: Sensitivity, Sp: Specificity, ref: references, TM: thrombomodulin, VaD: vascular cognitive impairment,
VCAM-1: vascular cell adhesion protein, vWF: von Willebrand factor, WMH: white matter hyperintensity.

to the brain. The plasma P-selectin and VCAM-1 levels were associated
with severe WMH. Those with severe periventricular WMH and symp­
tomatic carotid artery stenosis had higher VCAM-1 and P-selectin levels
than those without (de Leeuw et al., 2002). With regard to the blood
biomarker of AIS, the blood VCAM-1 level is associated with 3-month
outcomes in patients with cerebrovascular disease (Richard et al.,
2015), and the blood P-selectin level can be useful in assessment of
atherothrombotic properties of hyperglycemia and hyperlipidemia in
AIS (Pawelczyk et al., 2018).
When subcutaneous matrix proteins are exposed to the bloodstream,
platelets attach to the vessel wall by binding platelet surface receptors to
the endothelial von Willebrand factor (vWF). High plasma vWF levels
expressed by endothelial cells after tissue damage are associated with
WMH severity (Nagai et al., 2012). Likewise, the vWF level correlated
with severity, outcome, and infarct size of AIS and is significantly higher
in patients with stroke than in those without atrial fibrillation (Sato
et al., 2006).
Thrombomodulin (TM) is present on the surface of vascular endo­
thelial cells and forms a complex with thrombin that appears in the
blood to activate protein C, a coagulation inhibitor. TM is induced by
endothelial damage and degraded by intracellular proteases and
released into the blood, mainly due to damage or destruction of
microvessel walls. Therefore, blood TM level measurement estimates
endothelial cell production function and degree of damage, so it is useful
as a marker for damage to vascular endothelial cells. Blood TM level is
associated with severe WMH and the number of lacunar infarctions on
magnetic resonance imaging (Hassan et al., 2003). Increased TM level
also predicts a fatal outcome in patients with AIS (Olivot et al., 2004).
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of
nitric oxide synthase (NOS). L-arginine is the substrate of NOS to pro­
duce NO (nitric oxide). As the structure of ADMA is similar to that of L-
arginine, ADMA competes with L-arginine for NOS binding, thereby
inhibiting NOS function and impairing NO formation, which impairs
normal vascular endothelial cell function, and is associated with endo­
thelial dysfunction (Vallance et al., 1992). High plasma ADMA levels are
associated with silent brain infarcts (SBIs). The presence of SBIs is a risk
factor for developing symptomatic stroke (Vermeer et al., 2003a) and
cognitive dysfunction (Vermeer et al., 2003b, 2007). In multivariable
analyses adjusting for age, sex, and traditional stroke risk factors, higher
ADMA levels are associated with an increased risk of prevalent SBIs
(Pikula et al., 2009). As for the blood biomarker of AIS, ADMA levels in
patients with AIS are significantly higher than those in the control group
(Ercan et al., 2019).
Fibrinogen converts to fibrin at the site of vascular endothelial cell
deficiency in the process of coagulation. An abnormal fibrinogen level is
a parameter of an abnormality in this hemostatic mechanism. It is also
involved in wound healing as a substrate for factor XIII and increases
during inflammation as an acute phase reactant. High fibrinogen levels
are correlated with an increased risk of AD and VaD (Xu et al., 2008). As
for the blood biomarker of AIS, fibrinogen level is associated with the
prognosis of AIS (Tombul et al., 2005).
Adrenomedullin (AM) is a potent vasodilator and bioactive natri­
uretic peptide that is secreted by endothelial cells (Shindo et al., 2000)
in response to hypoxic invasion (Nakayama et al., 1998), shear stress
(Chun et al., 1997), and inflammation (Ueda et al., 1999). Cerebral
endothelial cells are a significant source of AM (Kis et al., 2002). AM
suppresses leukocyte infiltration, oxidative stress, and apoptosis, and
simultaneously mobilizes CD34+ mononuclear cells to promote vascular
regeneration and compensate for brain protection in AIS (Koyama et al.,
2015; Miyashita et al., 2006). The levels of mid-regional proAM, which
is a stable fragment of the AM precursor, are significantly increased with
the severity of deep white matter lesions and inversely correlated with
cognitive function test scores (Kuriyama et al., 2017), which suggests
that AM can be an effective marker of AIS and cognitive decline.
7. Neuronal injury
Endothelial dysfunction leads to increased BBB permeability, sub­
sequent exposure of neurons to inflammatory factors, white matter
damage (demyelination, axonal loss, and oligodendrocyte degenera­
tion), neurodegeneration and cell death, increased glial inflammation,
glial cell activation, and ultimately neuronal injury (Cipollini et al.,
2019) (Fig. 1D).
Neuron-specific enolase (NSE) is an intracellular dimer enzyme of
glucose metabolism localized predominantly in neuronal cytoplasm
(Påhlman et al., 1984). Serum NSE levels in patients with VaD are
significantly higher than those in patients without VaD (Shen and Gao,
2015). Moreover, serum NSE levels are associated with the prognosis of
large-artery atherosclerotic strokes (Wang et al., 2018c) and AIS after
intravenous thrombolysis (Lu et al., 2015). With regard to the blood
biomarker of AIS, serum NSE levels initially increase in 2–3 h after AIS
(Wunderlich et al., 2006).
Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a cytoplasmic deu­
biquitinating enzyme of neurons that have been associated with synaptic
plasticity and homeostasis and the brain’s self-repair mechanisms after
injury (Papa et al., 2010) (Rojo et al., 2011). The blood UCH-L1 level
cannot distinguish AIS from intracerebral hemorrhage (Ren et al., 2016).
Protein S100 beta (S100β) is a calcium-binding protein expressed
mainly in human astroglial cells. S100β levels are elevated in patients
with SVD and associated with cognitive impairment in patients with
WMH (Gao et al., 2015). Moreover, S100β discriminates AIS from
intracranial hemorrhage with high sensitivity (Zhou et al., 2016) and is
related to prognosis of AIS (Wunderlich et al., 2004).
Glial fibrillary acidic protein (GFAP) is a brain-specific astrocytic
intermediate filament protein found almost exclusively in the central
nervous system (Eng et al., 2000). Higher GFAP levels distinguish be­
tween AIS and intracerebral hemorrhage (Ren et al., 2016).
Heart-type fatty acid-binding protein (H-FABP), which is a fatty acid-
binding protein in the heart, is located in the brain. Serum H-FABP levels
could help distinguish AIS from the control group with 68% sensitivity

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S. Hosoki et al.
and 100% specificity in a study that examined 22 cases within 3 h after
stroke onset (Zimmermann-Ivol et al., 2004) and are also associated
with poor outcomes in patients with AIS (Lyang et al., 2019).
Several microRNAs associated with neuronal injury are blood bio­
markers for VaD and AIS. A brain-specific microRNA, miR-134, is
associated with dendrite spines. In a rodent model with VaD, histo­
pathological analysis reveals that miR-134-5p aggravates cognitive
impairment in rats with VaD through damage to cortical neurons and
loss of synaptic proteins (Liu et al., 2019). As for the blood biomarker of
AIS, a study that assessed stroke within 24 h of onset showed that
increased expression levels of miR-134 differentiated stroke from the
control group, with 75% sensitivity and 73% specificity (Zhou et al.,
2018). Serum miR-134 levels are also associated with NIHSS, cerebral
infarction volume, and poor prognosis (Zhou et al., 2018).
8. Miscellaneous markers
miR-451-a is associated with reduced natural killer cells in AIS (Kong
et al., 2020), while miR-409-3p is associated with a tumor suppressor
(Song et al., 2018). In a cohort of 204 patients with VaD and 200 healthy
and age- and sex-matched controls, elevated blood miR-451-a levels can
distinguish patients with VaD with 70% sensitivity and 75% specificity
with VaD, while decreased blood miR-409-3p levels can distinguish
patients with VaD with 76% sensitivity and 89% specificity (Prabhakar
et al., 2017).
9. Multimarkers
The use of multiple markers has been investigated since there is a
limit to a single marker’s diagnostic power. As a diagnostic blood
biomarker of AIS, four-protein panel (combination of S100β, vWF,
MMP-9, and VCAM) (Lynch et al., 2004) has been reported. This mul­
timarker is useful in the diagnosis of acute stroke with high sensitivity
and specificity of >90%. The multimarker of VaD is expected to achieve
a high diagnostic accuracy. VaD is attributed to various causes, and
different pathological pathways underlie the development of VaD
(Iadecola, 2013). Thus, a single marker is unlikely useful for the diag­
nosis or prediction of a disease with such heterogeneity; instead, mul­
tiple markers that encompass the different aspects of the disease
physiopathology may be more useful.
10. Perspective
Many blood biomarkers were found in both VaD and AIS. However,
some blood biomarkers have been reported in only AIS, and these are
candidates for new blood biomarkers for VaD. Among the VaD blood
biomarkers, oxLDL and ADMA are promising biomarkers because they
were reported in a prospective study with a large sample size. Some
noncoding RNAs were able to diagnose VaD with relatively high sensi­
tivity and specificity. Noncoding RNAs are present in human plasma in a
stable form, can be analyzed using a single drop of blood, regulate gene
expression at the post-translational level, and play an important role in
the pathogenesis of VaD and thus have the potential to be diagnostic
blood biomarkers for VaD (Mitchell et al., 2008; Yuan and Bi, 2020).
Currently, no blood biomarkers have yet to be proven useful in
clinical practice (Mijajlović et al., 2017). Factors that make clinical
application of blood biomarkers difficult are the BBB, limiting the
leakage of proteins from the brain parenchyma into the blood and
presence of these markers in other pathological conditions, such as ep­
ilepsy, encephalitis, and myocardial infarction (Whiteley et al., 2008).
By contrast, microRNAs have potential limitations related to reproduc­
ibility. One such limitation is that various methods can be used for
measuring microRNAs, and no gold standard has been established
(Witwer and Halushka, 2016).
Multimarkers of VaD are expected to encompass the different aspects
of the disease physiopathology and to compensate for the demerits of
6
Neurochemistry International 146 (2021) 105015
single markers.
The clinical application of artificial intelligence (AI) is also expected
to be appropriate. In 2017, a diagnostic accuracy comparison was made
between pathologists and machines in detecting lymph node metastases
in breast cancer. It was reported that machines performed better than
pathologists under certain conditions (Ehteshami Bejnordi et al., 2017).
In addition, the clinical application of deep learning is also expected, in
which AI independently extracts features from learning data and ac­
quires rules and knowledge. The expected areas where deep learning
may be imminently applied to stroke management are image segmen­
tation, automated featurization, and multimodal prognostication (Feng
et al., 2018). One of the largest barriers is said to be the complexity of
the underlying deep learning frameworks, which are largely inaccessible
to individuals without advanced training (Feng et al., 2018). AI will also
be applied to evaluations of biomarkers by combining such technology
with biomarkers and background factors, which will make VaD diag­
nosis more precise.
VaD is potentially preventable and treatable by managing the
vascular risk factors in the preclinical or early stages. Hypertension is a
strong risk factor of VaD (Novak and Hajjar, 2010). Antihypertensive
therapy for middle-aged individuals with hypertension is likely to sub­
sequently suppress the onset of dementia (Livingston et al., 2017).
However, its effectiveness has not been proven in intervention studies
owing to the difficulty of conducting long-term trials. Furthermore,
microRNAs have the potential to be considered as therapeutic targets
and may be important in VaD treatment (Yuan and Bi, 2020). In a rodent
study, post-ischemic treatment with intravenous miR-7 mimic admin­
istration decreased the ischemic volume and improved motor and
cognitive impairments with minimal peripheral toxicity, and minimized
the apoptosis and autophagic death of neurons (Kim et al., 2018). Thus,
miR-7 could be used as a potential therapeutic target for VaD. Diagnostic
biomarkers of VaD that are secreted as a defense mechanism can also be
considered as a therapeutic approach. The administration of AM as a
possible therapeutic agent for VaD and AIS can be evaluated owing to
the ability of AM to promote arteriogenesis and angiogenesis, inhibit
oxidative stress, preserve white matter integrity, and prevent cognitive
decline after chronic cerebral hypoperfusion in a rodent model (Maki
et al., 2011). Indeed, AM is being tested for its potential application in
patients with AIS in a Phase II clinical trial (jRCT ID: jRCT2051190092)
(Yoshimoto et al., 2021).
11. Conclusion
This review has outlined the increasing lines of evidence and possi­
bilities of blood biomarkers in VaD from the viewpoint of AIS pathol­
ogies and noncoding RNA multimarkers combined with multiple
biomarkers. Future studies are warranted to validate these promising
biomarkers.
Funding
This study is funded by the Japan Agency for Medical Research and
Development (19lk0201096h0001 and 20lk0201096h0002).
Authors’ Contributions
SH, TT, and MI were involved in writing of the manuscript, and made
critical revisions of the manuscript for important intellectual content.
All authors approved the final version of the manuscript.
Declaration of competing interest
The authors declare that they have no conflict of interest.
S. Hosoki et al.
Acknowledgments
We would like to thank Dr. Ahmad Khundakar for critical editing of
this manuscript.
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