Potential biofluid biomarkers for vascular dementia

Abstract

Vascular dementia (VaD) is the second most common causative disease of dementia, after Alzheimer’s disease (AD). Regarding the diagnosis of VaD, the presence of cerebrovascular impairments such as cerebral infarction and intracranial hemorrhage is confirmed by brain imaging and then followed by the temporal causality that cognitive dysfunction develops. As VaD has a wide range of pathologies and clinical symptoms, its diagnosis and the development of therapeutics specific to VaD could be difficult. The challenge is in searching for biomarkers that reflect the pathological conditions, prognosis, and severity of VaD and the detailed stratification of patients. Breakdown of the blood-brain barrier and extracellular matrix, along with axonal disorders, demyelinating disorders, and gliosis, plays a significant role in the pathogenesis of cerebrovascular impairment. Increased cerebrospinal fluid (CSF)/serum albumin quotient, decreased matrix metalloproteinase-2 index, and increased neurofilament light have been reported as biomarkers of these disorders. Measuring CSF amyloid-β 42 (Aβ42), tau, and phosphorylated tau (p-tau) as biomarkers of AD has proved useful, but the significance of these markers in VaD has not been investigated in detail. The complications of VaD and AD are common, so determining which is closer to the core of the pathological conditions is often difficult. Biomarkers specific to VaD are expected to be useful for the differential diagnosis and prognosis prediction of dementia.

 Significance of evaluating VaD using biomarkers

Vascular dementia (VaD) is a leading cause of dementia that has the second highest prevalence after Alzheimer’s disease (AD)¹⁾. The risk factors of VaD are lifestyle-related diseases such as hypertension, diabetes, and dyslipidemia, and aging; thus, the number of patients with VaD has been increasing owing to population aging. VaD can be diagnosed on the basis of its gradual progression, focal neurological signs/symptoms, cognitive impairment, patient history of multiple ischemic stroke, neuroimaging for cerebrovascular impairment, and the temporal relationship between cerebrovascular impairment and cognitive dysfunction²⁾. However, because clinical symptoms are often not specific, diagnostic accuracy based on these criteria is insufficient, often delaying the diagnosis³⁾. In recent years, new concepts of vascular cognitive impairment (VCI)⁴⁾ and vascular cognitive disorders⁵⁾, including mild cognitive impairment (MCI), have been proposed for early diagnosis and prevention by early intervention.

Traditionally, VaD has been considered to be related to the amount of neuronal loss due to multiple infarctions called multiple infarction dementia⁶⁾. With the widespread use of brain magnetic resonance imaging (MRI), opportunities for brain imaging have increased, revealing the presence of cerebrovascular impairments, which are not necessarily associated with the onset of clinical stroke. Thus, the concept of VaD disease has changed tremendously, and the fact that VaD shows a complexity of pathogenesis formed by multiple pathological factors and the heterogeneity of clinical symptoms has been revealed. On the basis of the diagnostic criteria of the National Institute of Neurological Disorders and Stroke and Association Internationale pour la Recherché et l’Enseignement en Neurosciences, VaD can be further divided into multi-infarct dementia (MID), strategic single-infarct dementia, small vessel disease dementia (SVD), hypoperfusion VaD, hemorrhagic VaD, and others. Hereditary VaD includes cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy (CADASIL), and other adult-onset hereditary leukoencephalopathies²⁾. SVD is the most prominent type⁷⁾, and the importance of slowly progressing changes in the brain associated with SVD has been recognized⁸⁾. This progression pattern of SVD is quite in contrast to VaD, which occurs after an acute stroke (cerebral infarction/hemorrhage). In fact, SDV is the most common neuropathological feature in elderly patients and is highly associated with an increased risk of dementia⁹⁾.

Brain MRI is an important imaging modality for diagnosing VaD and is useful for the morphological assessment of cerebrovascular damage and cerebral atrophy. Nevertheless, microscopic pathological changes are difficult to detect because of the limitations of the temporal resolution of MRI scanners⁵⁾. Evidence has shown that microinfarction is a major risk factor of VaD¹⁰⁾; however, microinfarctions are difficult to detect with conventional 1.5- and 3-Tesla MRI scanners¹¹⁾. Lately, microinfarctions with a diameter of 0.7 mm can be detected using 7-Tesla high-resolution MRI scanners, but the average microinfarction size is approximately 0.3 mm in diameter, which is far smaller than the standard temporal resolution of MRI scanners¹²⁾.

Cerebrospinal fluid (CSF) measurements reflect pathological changes in the central nervous system¹³⁾. Hence, CSF biomarkers are used to diagnose various neurological diseases, monitor disease progression, and determine the effects of therapeutic agents. The CSF biomarkers of VaD could accurately stratify patients with VaD who have clinically diverse symptoms and improve the accuracy of diagnosis^14,15). CSF biomarkers are useful for revealing the mechanism of the development of VaD¹⁶⁾. VaD is heterogeneous in its etiology and pathological characteristics, so patient stratification using biofluid biomarkers may help in the efficient development of specific therapeutic agents^17,18). As VaD varies greatly between patients in terms of severity and cognitive prognosis, it is crucial to develop biomarkers that reflect these^19,20).

 CSF biomarkers of VaD

Some studies have reported CSF biomarkers that reflect the pathological processes of VaD^20,21); however, their clinical usefulness remains controversial. White matter lesion (WML) is a characteristic of SVD that is caused by chronic hypoperfusion and hypoxia in vascular beds supplied by long arterioles²²⁾. It is also thought to result from the destruction of the blood-brain barrier (BBB) accompanied by the activation of proteases such as matrix metalloproteinases (MMPs)^14,23). WML is associated with multiple processes such as axonal degeneration, gliosis, and demyelination^24–26).

The blood-CSF barrier (BCB) and BBB are vital for isolating the central nervous system from systemic circulation and maintaining an optimal microenvironment for the central nervous system. The barrier function is maintained by the interaction of endothelial cells, pericytes, and astrocytes²⁷⁾. The disruption of the BBB allows the entry of toxic inflammatory cytokines from the peripheral circulation into the brain parenchyma, which consequently damages neurons⁷⁾. An increasing number of studies have shown that BCB/BBB dysfunction may play an important role in the pathogenesis of VaD^28–30). The CSF/serum albumin quotient is a standard indicator of BBB and choroid plexus function. An increase in this ratio indicates enhanced permeability of the barrier²⁰⁾. Elevated CSF/serum albumin quotient in patients with VCI has been reported in many clinical studies^31–34). Table 1 shows the VaD biomarkers reported thus far.

Table 1 

Category	Reference
Blood-CSF or BCB/BBB dysfunction
CSF/serum albumin quotient (QA) ↑→	31–34
Inflammatory and glial activation
TNF-α ↑	37
TGF-β ↑	38
VEGF ↑	38
α1-Antichymotrypsin ↑	39
YKL-40 ↑	40
LCN2 ↑	42
Extracellular matrix breakdown
MMP-2 index ↓	45
MMP-3 activity ↑	45
MMP-9 ↑	33,44
MMP-10 ↑	33
TIMP-1 ↑	33,39
Axonal and myelin damage
NFL ↑	32–34,48
MBP ↑	33
Sulfatide ↑	52

Both the innate immune system and inflammation play a central role in the pathophysiology of cognitive impairment^35,36). CSF levels of tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF)^37,38); the acute inflammatory protein α 1-antichymotrypsin³⁹⁾; and the glial cell activation marker YKL-40 were elevated in patients with VCI⁴⁰⁾. Lipocalin 2 (LCN2) is a secretory glycoprotein involved in innate immunity that goes up to the central nervous system in response to injuries and inflammatory stimulation⁴¹⁾. The CSF level of LCN2 was reported to increase in patients with VaD⁴²⁾.

MMPs are members of a family of endopeptidases that are active in the extracellular matrix, cell surface, and intracellular environment. MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, and MMP-12 mainly show activities in the brain²⁰⁾. Whereas MMP-2 is detected in CSF in patients with physiological conditions, other MMPs (mainly MMP-3 and MMP-9) have extremely low CSF concentrations, except when accompanied by inflammation⁴³⁾. Changes in the CSF concentration of many MMPs have been reported in SVD^33,44,45). In addition, the CSF level of the tissue inhibitor of metalloproteinases-1 (TIMP-1), an inhibitor of MMP, has been reported to increase in patients with VCI^33,39).

The neurofilament (NF) is a major structural protein in neurons that consists of three subunits, namely low (NF-L), medium (NF-M), and heavy (NF-H), with different degrees of phosphorylation⁴⁶⁾. NF is expected to be a sensitive marker of nerve cell death and axonal damage⁴⁷⁾. Significant increases in NF-L levels in CSF have been reported in patients with severe WML^32–34,48). Other reports have also shown a positive correlation between the severity of WMLs and NF-L levels in CSF⁴⁹⁾. The myelin basic protein (MBP) is a major structural component of the myelin sheath⁵⁰⁾, and patients who have had a subcortical infarction stroke have been reported to have significantly higher MBP levels in CSF⁵¹⁾. Sulfatide is an acidic glycolipid in the myelin sheath produced by oligodendrocytes. It is regarded as a marker of white matter damage. Increased sulfatide levels in CSF have been reported in patients with SVD compared with healthy subjects and patients with AD⁵²⁾. The CSF level of sulfatide has also been shown to be a potential predictor of WML progression⁵³⁾.

Among all these biomarkers, increased CSF/serum albumin quotient, decreased MMP-2 index, and elevated CSF level of NF-L without alteration of AD-related core biomarkers are considered the most clinically useful biomarkers of VaD^14,54). However, a single marker is inadequate for diagnosing or differentiating VaD from AD. Thus, a combination of biofluid biomarkers and diagnostic imaging markers is considered essential for improving diagnostic accuracy²⁰⁾. Blood biomarkers of VaD remain an unexplored field. Nonetheless, as samples can be collected less invasively than CSF samples, we could expect better results from future studies²⁰⁾.

 Application of AD core biomarkers to VaD and mixed dementia

Approximately 50% of cases of dementia are complicated by multiple causative diseases⁵⁵⁾. The most common cause of mixed dementia is the combination of AD and VaD⁵⁶⁾. Mixed dementia presents more diverse clinical symptoms than single dementia. Moreover, the variation pattern of biomarkers is complicated. No biomarkers useful for mixed dementia have been fully established¹¹⁾.

Whether AD-related pathogenesis and cerebrovascular impairment have independent effects on cognitive dysfunction in mixed dementia remains unknown, as is their synergistic effect^11,57). Reports have demonstrated that AD and cerebrovascular disease independently contribute to cognitive dysfunction⁵⁸⁾. At the same time, reports have upheld the idea that their effects interact with each other⁵⁹⁾. CSF tau, phosphorylated tau (p-tau), and amyloid β42 (Aβ42) can be useful in differentiating AD from VaD⁶⁰⁾, but not AD from mixed dementia^61,62). No significant difference was found in the CSF levels of Aβ42, tau, and p-tau between MCI due to AD and mixed dementia⁶³⁾. Microbleeds are associated with decreased CSF levels of Aβ42 in patients with AD but were not associated with CSF levels of tau and p-tau181⁶⁴⁾. WML was not associated with Aβ42, tau, or p-tau181 levels⁶⁴⁾, and lacunar infarction was associated with low CSF levels of tau rather than Aβ42 and P-tau181⁶⁴⁾. However, these effects may be influenced by whether the patients are APOE ε4 carriers, so further verification in stratified subjects is critically important⁶⁴⁾. The changes in the CSF concentrations of AD core biomarkers over time were not associated with the rate of change in WML volume⁶⁵⁾.

 Summary

In summary, the heterogeneity of VaD pathologies remains a major obstacle to the development of specific therapeutic agents. As biofluid biomarkers can detect pathological changes quantitatively and objectively, they could be useful for the stratification of patients with VaD. Although the CSF levels of the biomarkers of VaD have not been well established, increased CSF/serum albumin quotient, decreased MMP-2 index, and increased NF-L levels are expected to be useful biomarkers that reflect cerebrovascular impairment. Concomitant multiple pathogeneses in dementia are also crucial when considering the significance of biomarkers. The development of biomarkers that specifically and quantitatively reflect the pathophysiology of VaD is essential for the development of new therapeutic agents for VaD.

 Acknowledgements

This work was supported by JSPS KAKENHI Grant Number 21H02828 (grant-in-Aid for Scientific Research (B)) (S.T.) and the research grant from Cell Science Research Foundation (S.T.).

 

Disclosures: Authors have no potential conflicts of interest to declare.

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