Biomarkers and Genomic Medicine (2015) 7, 43e56

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REVIEW ARTICLE

Biomarkers in vascular dementia: A recentupdate

Abhijeet Jagtap, Sonal Gawande, Sushil Sharma*

Saint James School of Medicine, Bonaire, Dutch Caribbean, The Netherlands

Received 30 May 2014; received in revised form 9 October 2014; accepted 14 November 2014Available online 23 December 2014

KEYWORDScerebrospinal fluid;Charnoly body;neuroimaging;serum

* Corresponding author. Saint JamesE-mail address: Sharma@mail.sjsm

http://dx.doi.org/10.1016/j.bgm.2012214-0247/Copyright ª 2015, Taiwan

Abstract Vascular dementia (VaD) affects a broad spectrum of patients with various manifes-tations of cognitive decline, which could be attributed to cerebrovascular or cardiovasculardisease. Diagnosis of VaD depends on the identification of environmental and genetic risk fac-tors including; cerebral autosomal-dominant arteriopathy with subcortical infarcts and leu-koencephalopathy. Mitochondrial oxidative stress, hypoxic ischemia, inflammation,accumulation of advanced glycation products, and proinflammatory cytokines have been impli-cated in the pathogenesis of VaD. Hence it is exceedingly important to determine the risk fac-tors and molecular pathology by identifying specific biomarkers that can be broadly classifiedas: biochemical, molecular, genetic, endocrinological, anatomical, imaging, and neuropatho-logical; for the early differential diagnosis, prognosis, and effective treatment of VaD. The bio-markers of VaD in the serum and cerebrospinal fluid samples include; phosphorylated tau,amyloid-b, matrix metalloproteases, sulfatids, albumin, and proinflammatory C-reactive pro-teins. In addition, Charnoly body (CB) formation and microRNAs can be detected as preapop-totic biomarkers of compromised mitochondrial bioenergetics to further confirm VaD. CBformation occurs in response to nutritional stress and/or neurotoxic insult in the most vulner-able hippocampal neurons due to cerebrovascular insufficiency, and can be attenuated by di-etary interventions, physiological zinc supplementation, and metallothioneins (MTs). MTsprovide ubiquinone-mediated neuroprotection by serving as free radical scavengers, by main-taining the mitochondrial redox balance, by inhibiting CB formation, and by inhibiting progres-sive neurodegenerative a-synucleinopathies. MTs also regulate zinc-mediated transcriptionalactivation of genes involved in cell growth, proliferation, and differentiation, and hencemay be used as novel biomarkers of VaD. In addition to genetic analysis of MTs, Notch3, apoli-poprotein E4, nitric oxide synthase, and cerebral autosomal-dominant arteriopathy withsubcortical infarcts and leukoencephalopathy; omics and microRNA analyses may provide novelbiomarkers of VaD. This review provides recent update on in-vitro biomarkers from the serum

School of Medicine, Plaza Juliana 4, Kralnedijk, Bonaire, Dutch Caribbean, The Netherlands..org (S. Sharma).

4.11.001Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.

44 A. Jagtap et al.

and cerebrospinal fluid samples and in-vivo neuroimaging biomarkers for the differential diag-nosis and effective clinical management of VaD.Copyright ª 2015, Taiwan Genomic Medicine and Biomarker Society. Published by ElsevierTaiwan LLC. All rights reserved.

Introduction

Dementia may be defined as a progressive neurodegenera-tive disease characterized by loss of cognition, significantenough to cause functional disability in everyday life.1,2 Itis a major public health problem affecting over 20 millionpeople around the world and the number is increasingexponentially in industrially-developed countries.3 Theprevalence of two major types of dementia, Alzheimer’sdisease (AD) and vascular dementia (VaD), is around 4.4%and 1e2 % respectively in industrially-developed countries;however, the prevalence is lower in developing countries.4

AD accounts for 70e75% cases of dementia in elderly,whereas VaD comprises a small but significant group ac-counting to around 20% cases, the second most commonform of dementia after AD.4,5 Vascular comorbidity may bepresent in over 30% patients with AD and over 50% patientswith VaD may exhibit pathology associated with AD, sug-gesting a 3.4e73% overlap between AD and VaD.6 Hence,the diagnosis of dementia is not only difficult but alsochallenging as in many elderly patients both the entitiesmay coexist in combination with other neurodegenerativediseases (often termed as mixed dementia). The originalterm multi-infarct dementia is now replaced by a newlyupdated term, vascular cognitive impairment (VCI), whichrefers to any cognitive impairment caused by or associatedwith vascular risk factors and ranges from mild cognitiveimpairment to overt dementia.7 VaD may be caused byhemorrhagic, ischemic, and hypoxic injury to the brain. It isa group of heterogeneous disorders in which the presenceof ischemia/infarction may cause cognitive decline how-ever, the degree of such impairment is directly proportionalto the extent of neuronal damage and location of thelesion.2,8e10 The National Institute of Neurological Disor-ders and Stroke and Association internationale pour laRecherche et l‘Enseignement en Neurosciences (NINDS-AIREN) states that small vessel disease such as microvas-cular angiopathy (lacunar infarction), periventricularischemia, and large vessel athero-embolic disease causingterritorial infarction are sufficient to result in cognitiveimpairment and can be included as a criterion of VaDdiagnosis.1,2,11 Several possible pathogenic factors, such asaccumulation of advanced glycation end-products andactivation of proinflammatory cytokines [interleukin (IL)-1b, tumor necrosis factor (TNF)a, IL-6, and nuclear factor-kB], and experimental studies on animals and culturedneurons have demonstrated that oxidative stress, mito-chondrial dysfunction, inflammatory response and accu-mulation of abnormal amyloid-b have been proposed forthe etiopathogenesis of VaD.4 As per California criteria,diagnosis of VaD requires neuropathological assessment,computed tomography (CT), positron emission tomography

(PET), magnetic resonance imaging (MRI), and magneticresonance spectroscopy (MRS).2,12e14 Fig. 1 is a systematicdiagram illustrating various factors involved in the patho-genesis and risk factors of VaD. In principle, the risk factorsfor stroke are also the risk factors for dementia. Clinically-evident hypertension has been shown to have significantassociation with dementia while hyperlipidemia andmetabolic syndrome could be predictive of dementiarisk.2,15e17 Transient ischemic attacks also predispose toincrease the risk of stroke and 30% of the patients whosuffer stroke develop dementia after a period of 6e12months. Although the exact etiopathogenesis of VaD re-mains unknown, diabetes mellitus, hormone replacementtherapy for postmenopausal women, obesity, improper di-etary habits including: food rich in trans-saturated fats andlacking omega-3 fatty acids (docohexanoic acid and eico-sapentanoic acid) and polyunsaturated fatty acids (linoeicacid, linolinic acid, and arachidonic acid), various envi-ronmental neurotoxins; drug addiction; aging; and un-healthy life style including sedentary life style, lack ofexercise, physical and mental stress, overmedication, andlate night sleep have been proposed in the etiopatho-genesis of VaD and can enhance the disease process.Several of these risk factors, apart from genetic, may beprevented by diet manipulation, moderate exercise, andlifestyle modifications. Age of onset of stroke and lack ofeducation have also been associated with higher risk ofdementia.2 Cerebral autosomal-dominant arteriopathy withsubcortical infarcts and leukoencephalopathy (CADASIL), aninherited disorder, manifested as syndrome of migraine,mood disorder, recurrent transient ischemic attacks,stroke, and early development of dementia is an indepen-dent age-related pathogenesis of AD and VaD; the rootcause is primarily microvascular disease. Due to thenumerous attributable risk factors detailed studies areneeded to understand the exact etiopathogenesis of VaD.

In the present report, we describe systematically recentupdates on various in-vitro biomarkers from serum andcerebrospinal fluid (CSF) samples and in-vivo multimodalityneuroimaging biomarkers for the effective clinical man-agement of VaD. It is expected that the information in thisreview will be of significant interest to medical students,clinicians, and researchers interested in understandingfurther about this devastating progressive neurodegenera-tive disorder of unknown etiopathogenesis.

Classification of biomarkers in VaD

Accurate classification of dementia would significantlyimpact its treatment. Currently, there is no approved drugfor the treatment of VaD, so it is exceedingly important toreduce the risk factors and provide adequate treatment

Figure 1 Risk factors and pathogenesis of vascular dementia (VaD). A pictorial diagram illuatrating various risk factors includinghypertension, hyperlipidemia, obesity, drugs of abuse, overmedication, hormone replacement therapy, and environmental factorscould lead to the pathogenesis of VaD. Activation of proinflammatory cytokines, abnormal accumulation of amyloid-b, accumu-lation of advanced glycation products, inflammation, and mitochondrial oxidative stress are caused by hypoxia, hemorrhage, andischemia, resulting in the pathogenesis of VaD.

Biomarkers in vascular dementia 45

with proper vascular agents for preventing and/or pro-longing the onset of dementia. The major risk factors, hy-pertension, and hyperlipidemia, can be controlled onlywith conventional treatment to reduce the risk of VaD.Therefore there is a dire need to classify and determine thespecific etiology of dementia based on the biomarkers an-alyses. These biomarkers must be easily measurable tofacilitate early and accurate diagnosis. In this regard, thesurrogate biomarkers of VaD, based on functional neuro-imaging, and CSF- and blood-based analysis have gained

importance as these are noninvasive or minimally invasive,easy to perform as compared to pathological analysis onpostmortem brain samples. Moreover, premortem analysisof these biomarkers would enhance the diagnostic capa-bility of VaD.18 The most significant VaD biomarkers thatcould be used for the early clinical diagnosis, prognosis, andtreatment of VaD are presented in Fig. 2. These can bebroadly classified as: clinical biomarkers (neurobehavioralassessment); pathological biomarkers (identifying cellular/histological changes); biochemical biomarkers (serum,

46 A. Jagtap et al.

plasma, CSF biomarkers); neuroimaging biomarkers, whichinclude functional multimodality fusion imaging with CT,MRI/MRS, PET, and single photon emission CT (SPECT) toderive structural as well as functional information simul-taneously regarding the diseases process, genetic bio-markers (identifying genes involved in cerbrovasculardisease); and omics and microRNA biomarkers (identifyingsubcellular components of VaD). Simonsen et al recentlydescribed laboratory methods for collection, detection ofproteins in CSF and plasma along with purification ofcandidate biomarkers (chromatographic and electropho-resis technique) and protein profiling using multiplexenzyme-linked immunosorbent assay, surface enhancedlaser desorption/ionization time of flight mass spectrom-etry, and peptide mass fingerprinting.5 Various statisticalmethods to calculate probability, sensitivity, and specificityhave been described to determine the utility of these bio-markers. Although several biomarkers have been correlatedwith underlying pathological processes at the cellular andmolecular level; the biochemical biomarkers have beendirectly correlated with clinical and imaging findings indementia. However, their pathological correlation is yet tobe established.19,20 Humpel reported that the detection ofbiomarkers in CSF can only support the clinical diagnosis ofVaD.21 Recent studies on biomarkers have emphasized oninflammation, hemostasis, oxidative stress, hypo-xiaeischemia, accumulation of biochemical substances,complex proteins, and other metabolites in the hyper-tensiveeatheromatous disease and hyperlipidemia in tissueand CSF.4,22

Figure 2 Classification of vascular dementia (VaD) bio-markers. A diagram demonstrating various biomarkers in VaDsuch as: omics/microRNA, genetic biomarkers, neuroimagingbiomarkers, cerebrospinal fluid (CSF) and serum biochemicalbiomarkers, pathological biomarkers, and clinical biomarkers,which can be used for the differential diagnosis, prognosis, andeffective treatment of VaD.

CSF biomarkers of VaD

Although biomarkers can be measured in various body fluidssuch as saliva, blood, and urine and tissue, CSF has beenstudied extensively because it drains the ventricular systemof the brain and concentration of various metabolites maydirectly reflect various pathological processes in the brainproviding a lead to develop sensitive and specific biomarkerin CSF to differentially diagnose various etiological types ofdementia.21 CSF biomarkers in dementia have been re-ported, especially in patients with AD on a larger scale andhave shown promise as sensitive diagnostic tool; however,very few studies are yet available and the candidate CSFbiomarkers studied so far show conflicting results and lackspecificity due to the heterogenous nature of VaD. As Table1 summarizes, the protein biomarkers that can be usedqualitatively and quantitatively; although they are notspecific to VaD when used in combination, they can in-crease the diagnostic certainty of VaD. CSF serum albuminratio, CSF index, and CSF total protein are biomarkershaving high diagnostic value as these can identify structuraland functional integrity of the bloodebrain barrier andmicrovascular damage. An increased albumin level andincreased index in VaD patients are well established, butthey are nonspecific and may not distinguish VaD fromAD.23,24 Sulfatide a maker for demyelination, is used toidentify the extent of demyelination in the white matterand it is found to be elevated in VaD as reported by Tullberget al25 and Fredman et al.26 The cytoskeletal organelle,neurofilament is estimated to identify axonal degenerationand the extent of white matter damage, and is found to beincreased in CSF of patients with VaD but not with AD pa-thology, reflecting the axonal damage that is characteristicof VaD.24,27 Furthermore, the matrix metalloproteases(MMPs) in the CSF, can be estimated to identify changes inthe extracellular matrix associated with vascular diseaseswith inflammation.28 MMPs attack the myelin and areregarded as biomarkers of demyelination. Various studiesincluding autopsy studies have shown that MMPs areincreased in patients with VaD.29,30 Certain CSF biomarkerswere used earlier for the evaluation of diagnostic utility inAD. Conflicting results in multiple studies have been re-ported and their potential utility lies in differentiating VaDfrom AD and the other neurodegenerative diseases. How-ever, many studies show significant overlap between levelsin VaD and AD.5,6 Serum to CSF folate ratio can be used todifferentiate VaD from AD. This ratio is significantlyreduced in VaD. The reduced folate ratio has been found tobe a characteristic of VaD.5,31 In addition AD is character-ized by amyloid (A)b plaque deposition irrespective of itsetiopathogenesis. Amyloid-b peptide (1e42) (Ab-42) isformed after amyloid-b is cleaved from amyloid precursorprotein by secretases. A significant reduction of Ab-42 inpatients with AD as well as VaD suggests a significantoverlap making it difficult to distinguish AD from VaD.5,6 Ab-42, total tau and phosphorylated tau (p-tau) have beenextensively studied in AD and there are several reports ontheir utility in diagnosis and prognosis of AD. Increasedlevels of tau and decreased levels of Ab-42 have beendetected in AD as well as VaD but more specifically in AD.Hence a combined analysis of these CSF biomarkers has

Table 1 Cerebrospinal fluid (CSF) biomarkers with high diagnostic utility: (Biomarker levels in CSF are raised in vasculardementia, VaD).

Biomarkers Diagnostic utility

CSF:serum albumin ratio,CSF total protein

To identify bloodebrain barrier damage to the small intravascular vessels

Sulfatide To identify demyelination of white matterNeurofilament To identify axonal degeneration (marker of white matter damage)Matrix metalloproteases To identify changes in the extracellular matrix associated with cardiovascular disease (i.e.

vascular disease with inflammation)Serum to CSF Folate ratio Low ratio in VaDIncreased total tau, p-tau,

decreased amyloid b42May differentiate VaD from Alzheimer’s disease and other NDD(Neurodegenerative Diseases)

Biomarkers in vascular dementia 47

been recommended for the differential diagnosis ofVaD.20,32e35 The protein biomarkers as mentioned in Fig. 3represent various physiological processes such as proteindegradation (ubiquitin), protease inhibition (cystatin C anda1 anti-chymotrypsin), inflammation (C3a, C4a) are knownto be associated with neurodegenerative diseases includingall forms of dementia. However, their diagnostic utility isenhanced when used in combination with folate ratio, Ab-42, total tau, or p-tau levels. Simonsen et al conducted thefirst study to establish the status of these candidate bio-markers in VaD patients.5 These markers lack specificityand need to be validated and investigated in large pro-spective multicentric trials. A biomarker of neuronal death,heart fatty acid binding protein is elevated in CSF frompatients with various neurodegenerative diseases. Althoughheart fatty acid binding protein can be detected in earlyVaD and AD, it lacks specificity.36

Serum and plasma biomarkers of VaD

Apart from CSF, certain biomarkers have been identified inthe serum and plasma from the blood samples of patientswith VaD, AD, and other neurodegenerative diseases. C-reactive protein is an inflammatory biomarker and its levelsare elevated in VaD. Hyperhomocysteinemia is a well-established vascular risk factor and increased level ofserum homocysteine proves a causal relationship withvascular lesions and thereby VaD.37,38 Elevated levels ofserum homocysteine were also seen in AD patients and areconsidered to contribute to vascular pathogenesis of AD.Recently, many studies have shown that elevated serumhomocysteine is associated with hippocampal and corticalatrophy in patients with VaD.39 Although deficiency ofvitamin B12 and folate causes hyperhomocysteinemia, thesupplementation of these vitamins failed to produce anyimprovement in patients with dementia; hence role of ho-mocysteine remains controversial.40 Elevated lipoprotein-ais considered an independent genetic risk factor for VaD butnot in AD, which helps in understanding the pathogenesis ofatherogenic processes in VaD.41,42 Dehydroepiandrosterone(DHEA), a neurosteroid, and its metabolite, DHEA sulfate(DHEA-S) have neuroprotective effects and their levels inthe central nervous system are raised in neurodegenerativediseases however, the reason for their altered levels inblood as a cause or as an effect remains uncertain.43e45

Serum level of DHEA-S is unaltered as reported by a few

studies in patients with VaD. A detailed study is needed todetermine the exact clinical significance of these and otherbiomarkers in the differential diagnosis of VaD, as severalfactors may influence their levels in blood. Furthermore,these biomarker studies should be correlated andconfirmed with imaging and histopathological evidence toauthenticate VaD diagnosis. Similarly oxidative stressorssuch as malondialdehyde (MDA), thyroid stimulating hor-mone (TSH), calcium, and magnesium have been found tobe nonspecifically elevated in patients with dementia,suggesting vascular etiopathogenesis in dementing ill-nesses.46 The receptor for advanced glycation end products(RAGE) is a cell-bound receptor of the immunoglobulin su-perfamily that may be activated by proinflammatory li-gands including advanced glycol-oxidation end productsand amyloid-b peptide. Clinical studies have shown thathigher plasma levels of RAGE are associated with reducedrisk of coronary artery disease, hypertension, metabolicsyndrome, arthritis, and AD.47 Similarly, atheroscleroticcerebrovascular disease is a significant cause of VaD. So,the protective nature of this biomarker requires furthervalidation. Geroldi et al recently demonstrated that onlyRAGE and b-site amyloid precursor protein (APP) cleavingenzyme 1 (BACE-1) proved to be predictor of cognitiveimpairment after stroke but there was no association withneprilysin or apolipoprotein E (ApoE).48 It needs to bedetermined whether these biomarkers help in distinguish-ing VaD from vascular cognitive impairment after acuteischemic stroke. Increased levels of thrombin, D-dimer, andthrombin fragment 1þ2, and biomarkers of endothelialdysfunction (von Willebrand factor and plasminogen acti-vator inhibitor) are associated with cerebrovascularthrombosis and thereby VaD as illustrated in Fig. 4. Suchassociation may be secondary to chronic inflammation.These mechanisms may underlie prothrombotic state, ce-rebral microinfarction, and eventually subcortical smallvessel infarction. Most of the cases of dementia have mixedetiopathogenesis, contributing a variable amount ofvascular pathology (Neuropathology group of the MedicalResearch Council Cognitive Function and Ageing Study).49,50

Genetic biomarkers of VaD

Identifying new risk factors for ischemic stroke could helpimprove prevention strategies and identify new therapeutictargets. Genetic risk factors are particularly interesting,

48 A. Jagtap et al.

because they can offer a direct clue to the biologicalpathways involved. Ischemic stroke is a heterogeneousdisorder, and must be considered for genetic susceptibilityfactors. In Western countries, most ischemic strokes can beattributed to large-artery atherosclerosis (athero-thrombotic stroke) and small-artery occlusion (lacunarstroke).51 The genes underlying VCI must be of two exclu-sive classes: (1) genes that predispose individuals to cere-brovascular disease, and (2) genes that determine tissueresponses to cerebrovascular disease (e.g. genes conveyingischemic tolerance or susceptibility, or the ability torecover from ischemic insult).52 In the first category, genesthat confer susceptibility to hypertension and atheroscle-rosis have been identified with some monogenic forms ofdisease such as CADASIL caused by mutations in NOTCH 3gene. From the second category; genes that modify tissueresponses to injury have also been identified and at leastthree sets of genes in the AD pathway, the presenilins, APP,and APOE are known to interact with the VCI diseasepathway. The presenilin mutations causing AD have beenshown to interact directly with Notch proteins, includingNotch 3 (mutations of which cause CADASIL).53e55 There isdirect evidence from both human and animal studies forspecific non-AD genes that play a significant role in tissueresponses in ischemia. Earlier studies in humans suggestthat variants in the genes for platelet glycoprotein and a-fibrinogen affect post stroke outcomes without affectingstroke risk per se. Animal studies have suggested glutamate

Figure 3 Cerebrospinal fluid (CSF) protein biomarkers mainly usethat are altered in vascular dementia (VaD) and Alzheimer’s diseaApolipoprotein-A1, dimers of apolipoprotein-A2, albumin, and imcompared to AD patients, whereas integral membrane 2BeC, terminC, ubiquitin-3a from computed tomography, neuroendocrine protelower in VaD as compared to AD patients.

and g-aminobutyric acid receptors, acid-sensing ion chan-nels, proteases, growth factors and their receptors, andtranscription factors as the major molecules involved ininfluencing brain responses to cerebrovascular injury.56,57

In addition, chromosome 9p21.3 genotype has been asso-ciated with VaD and AD.58 Linkage and association analyses(including single nucleotide polymorphism EDN1, MHTFR,NOS3, and ApoE 4) and AGTR1, AGTR2 of renal angiotensinsystem) have shown the association of these genes withpathogenesis of small vessel disease, cardiovascular disease(CVD), and VaD. The genes/molecules described in Fig. 5have been studied extensively in relation to CVD and at-tempts are being made to determine predisposition to CVDand VaD.51,59 Genetic diseases such as sickle cell disease,Fabry disease, and homocysteinuria, and genes involved ininflammation (LTC4S, IL-6), thrombosis (TGB3, factor VIII),lipid metabolism (ApoE, PON 1 PON2, PON3, ApoA5, LPL,LDL), endothelial function and oxidative stress (NOS3,MTHFR) and genes identified through linkage analysis in anIcelandic population (ALOX5, PDE4d) are all candidatebiomarkers to establish association with cerebrovasculardisease, ischemia-stroke, and VaD.51 There are limitedstudies available regarding the genetic biomarkers in VaD(Fig. 6). Hence potential genetic and molecular biomarkersof VaD such as genes responsible for cerebrovascular dis-ease, genes influencing the native tissue response andmolecules such as soluble receptors for various metabolitesand enzymes identified in VaD, have been proposed as

d in combination. A pictorial representation of CSF biomarkersse (AD). These biomarkers possess moderate diagnostic utility.munoglobulin-G have higher levels in the CSF samples of VaDal fragment, C3a peptide lacking c-terminal arginine, cystatin-in 7B2 (secretogranin V), and C4a peptide des-Arg levels are

Figure 4 Candidate vascular dementia (VaD) biomarkers in plasma/serum. C-reactive protein, homocysteine, lipoprotein-A,malondialdehyde, total eSH, calcium, magnesium, thyroid stimulating hormone (TSH) b-secratase, neprilysin levels areincreased in VaD as compared to normal control and AD patients. DHEA-S levels remain unaltered in VaD but are reduced in ADpatients. Folate and vitamin B12 and s-RAGE are lowered in VaD as compared to AD patients.

Biomarkers in vascular dementia 49

potential biomarkers to correlate with the pathogenesis ofVaD. However, further studies are needed to establish theirclinical significance.47,60 The proposed biomarker genespredisposing to cerebrovascular disease are ACE, AGT,eNOS, PON, MTHFR, MEF2A, ALOX5, LTA, APOM, and PDE4D.Certain genes can influence the brain tissue response toVaD such as neurotrophic factors: brain-derived neuro-trophic factor (BDNF), nerve growth factor (NGF), vasculargrowth factors, ApoE, MMPs, glutamate, and GABA re-ceptors, adhesion molecules, transcription factors, ionchannels, and NOS pathway genes. Genes/loci that areknown to alter risk of VaD in a community are PDE4D,ALOX5AP, LTA4H, 9p21, 4q25, ApoE 4 in CADASIL, specificpathway genes, APP, PPAR-g, LPL, and LIPC.51,59

MicroRNA biomarkers in VaD

Various types of microRNAs (miRs) are impaired due toabnormal adipogenesis in obesity to influence the geneticpredisposition of VaD. A further study is required todetermine their exact significance in the clinical manage-ment of VaD. Further studies employing omics

biotechnology and miR analysis would provide preciseknowledge regarding the exact etiopathogenesis and clin-ical management of VaD in future. Early mild cognitiveimpairment syndrome in vitro can be estimated by quan-titative analysis of brain-enriched cell-free miR in the bloodusing quantitative real-time polymerase chain reaction. AsmiRs are important epigenetic regulators of numerouscellular processes including neurodegenerative diseases,specific miRs such as the miR-132 and miR-134 familiespaired with miR-491-5p and miR-370, respectively, haveproven to be the best, detecting mild cognitive impairmentof varied etiology and AD. The use of brain-enriched neu-rites/synapses miR enables detection of early pathologicevents occurring in degenerating neurons.61 Numerous miRsincluding guardian of endothelial cells, miR 126 (loweredlevel) and others are found in vascular inflammatory pro-cesses, and could serve as biomarkers of early detection ofvascular cognitive impairment. Also, therapeutic potentialof miRs is a future challenge. The invention of novel mod-ifications of RNA bases and the synthesis of artificial anti-sense miR or antagomir, may be used as novel therapeutictools to manipulate miR and control vascular inflammatorydiseases.62 Although free radicals can induce inflammation

Figure 5 Molecular biomarkers in vascular dementia (VaD). A pictorial diagram illustrating genes such as ACE, AGT, eNOS, PON,MTHFR, MEF2A, ALOX5, LTA, APOM, and PDE4D are the genes predisposing to cerebrovascular disease. Genes which influence thebrain tissue responses include neurotropic factors brain-derived growth factor (BDNF), nerve growth factor (NGF), and vascularendothelial-derived growth factor (VEGF), apolipoprotein-E (APOE), and matrix metalloproteinases, glutamate and GABA re-ceptors, adhesion molecules, transcription factors, ion channels, NOS pathways genes. The genes known to alter risk of cere-brovascular diseases in community are: PDE4D, ALOX-5AP, LTA4H, chromosome-9 p21, 4q25, APO-4 in CADASIL, and specificpathway genes such as APP, PPAR-g, LPL, and LIPC.

50 A. Jagtap et al.

by activating redox-sensitive proinflammatory transcriptionfactors, the endothelial dysfunction induced by oxidativestress can release vascular endothelial-derived growthfactors (VEGFs) and prostanoids promoting vascularleakage, protein extravasation, and cytokine production.Inflammation enhances oxidative stress by upregulating theexpression of reactive oxygen species-producing enzymesand downregulating antioxidant defenses. miRs of thesetranscription factors can act as potential biomarkers incirculation for VaD. A study published by Ungvari et al,suggests Dicer1 (ribonuclease III) is a key enzyme of the miRmachinery, which is responsible for synthesis of maturefunctional miRs.63 There is evidence that Dicer1 in endo-thelial cells may regulate angiogenic processes, abiomarker to be explored as therapeutic target. Role ofdysregulation of Dicer1 in age-related impairment ofangiogenesis identified a number of miRs that are down-regulated in cerebromicrovascular endothelial cells in

dementia. Aging results in cerebromicrovascular rarefac-tion and cerebral angiogenesis is impaired in response tohypoxia or VEGF administration. This plays a prominent rolein impairment of regional cerebral blood flow and theoccurrence of VCI with age. Because the role of miRNAregulation and function in the aging vascular system is anemerging area, further research is needed to study thecontribution of individual miRs or miR families in geneexpression that underlie microvascular aging and thereby,VaD.

Biomarkers of cell-based therapy for VaD

There are few studies as yet available on the therapeuticpotential of cell-based therapy in VaD. Laboratory studieshave shown that transplanted bone marrow stem cellsimprove neurological diseases of the CNS by generating

Biomarkers in vascular dementia 51

neural cells or myelin-producing oligodendroglial cells andenhancing neural plasticity.64e68 However there are fewobjective data providing evidence for clinical improve-ment. Sharma et al administered autologous bone marrowderived mononuclear cells, intrathecally to a 61-year-oldwoman who was diagnosed with VaD.69 After follow-up of 2years she showed clinical improvements as assessed bymini-mental state examination and functional indepen-dence measure along with PET/CT neuroimaging exhibitingimproved metabolic activity providing evidence of benefitsof cell-based therapy and suggestion to investigate variousstem cell biomarkers employing omics biotechnology infuture studies on VaD.

Recently, significant efforts have been made to explorethe basic molecular mechanisms of atherosclerosis (theunderlying cause of cerebrovascular and cardiovasculardisease), which remains a major cause of morbidity andmortality worldwide. Because of the complex pathophysi-ology of cardiovascular disease, different research methodshave been combined to unravel genetic aspects, molecularpathways, and cellular functions involved in atherogenesis,vascular inflammation, and dyslipidemia to gain a multi-faceted picture addressing this complexity. Recent evolu-tion of high-throughput technologies is able to generatedata at the DNA, RNA, and protein levels with sophisticatedcomputational technology. These data sets are integratedto enhance information and are being used as regulatednetworks. Doring et al described genomics, tran-scriptomics, proteomics, and epigenomicsdand systemsbiology to explore pathomechanisms of vascular inflam-mation and atherosclerosis.70 Cerebrolysin, a naturally

Figure 6 Regulation of vascular dementia (VaD) biomarkers bydemonstrating various risk factors including altered circadian rhytlack of exercise can induce CB formation, and obesity due to abnorleptin and orexin gene dysregulation. Omega-3 fatty and PUFA preand stabilization.

occurring substance represents a therapeutic strategy forneurological disorders like dementia, stroke, and traumaticbrain injury.71 It is a neuropeptide mimicking the action ofneurotrophic factors that enhances neurogenesis, sustain-ing the brain’s self-repair, promotes neural progenitor cellmigration, synaptic density rebuilding neuronal cytoarchi-tecture, restorative processes, decreases the infarct vol-ume and edema formation and promotes functionalrecovery. Since mitochondrial redox balance is impaired asa consequence of brain regional cerebrovascular insuffi-ciency, antioxidants such as quercetin and isoquercitrin asnatural flavonoids help to provide mitochondrial neuro-protection in VaD. Similarly, melatonin reduces free radicalgeneration by enhancing glutathione levels. Neurotrophicfactors such as NGF, glial cell-derived neurotrophic factor,and BDNF have already been implicated as targets fortreatment of degenerative diseases. These neurotropicfactors are generally present in significantly high amountsin the bone marrow-derived mononuclear cells. A recom-binant DNA vaccine composed of domains of neuriteoutgrowth inhibitors. The immunological mechanisminducing effective antibodies against the specific domainsand the modulation of mRNA expression regarding neuriteoutgrowth inhibitors, which help in repair/regeneration ofneural and oligodendrocytic damage. Stem cells might bean alternative to brain regeneration. In experimentalmodels of acute ischemic stroke using Q-dot labeledmononuclear cells, we have established that these cellsexhibit preferential chemotaxis in the peri-infarcted regionand are exponentially eliminated as a function of time.Although the exact molecular mechanism of

omega-3 and polyunsaturated fatty acid (PUFA). A diagramhms, drug abuse, environmental and psychological stress, andmal microRNA and abnormal adipogenesis as a consequence ofvent vascular dementia by providing new membrane synthesis

52 A. Jagtap et al.

neuroprotection offered by mononuclear cells remainsenigmatic, it is assumed that the neuroprotection is pro-vided by autocrine and paracrine mechanism by localrelease of neuroprotective biomarkers such as insulin-likegrowth factor, endothelial derived growth factor, vonWilebrand factor, and granulocyte colony stimulating fac-tor, IL-4, and IL-10 as anti-inflammatory cytokines. Naivehuman chorionic villi and amniotic fluid derived cellsrelease significant amounts of BDNF, as well as VEGF.Nimodipine, as an L-type voltage-dependent Ca2þ channelantagonist and an antihypertensive agent, can also reduceischemic nerve cell death in VaD. Further studies in thisdirection promise to discover sensitive and specific bio-markers of VaD.

Neuroimaging biomarkersNeuroimaging has been extensively studied in various typesof dementia including VaD. In particular, CT and MRI spe-cific changes have been identified as potential biomarkersdemonstrating mechanisms of vascular injury and their ef-fects in the parenchyma, which can be detected in all thestages of VaD.1 Phase contrast MRI and the analyses ofhemodynamics in the brain have also been regarded aspotential biomarkers, although their sensitivity and speci-ficity need considerable evaluation. Neuroimaging findingscorrelate very well with the underlying pathological pro-cesses and hence have gained importance in research andclinical investigation on VaD.1,28 Significant findings onstandard and routine neuroimaging techniques that can beutilized as biomarkers in the diagnosis of VaD are summa-rized in Table 2. It has been proved that T2 weighted MRIsequences alone or in combination with CT can identifyleukoaraiosis (white matter lesions), microvascular angi-opathy, lacunar infarction, dilation of VirchoweRobinspaces, pulse wave encephalopathy, parameters of cere-bral embolic disease, which have been correlated with

Table 2 Imaging biomarkers in vascular dementia (VaD).

Imaging method Biomarkersdsalient features in VaD

Magnetic resonanceimaging (T1,T2weighted and FLAIRimages)

Deep white matter hyperintensity

Periventricular hyperintensityInfarction (lacunar, site specific suchgangliaecystic lesions, number, andHigh signals in basal gangliaDilated VirchoweRobin spacePulse wave encephalopathy seen asinfarction and white matter hyperinHemorrhage (number, size and locatBrain atrophy

Computed tomographyscan

Ventricular size, medial temporal atacute or chronic hemorrhage, hypoddefining infarction as per size, locat

Transcaranial Doppler Spontaneous cerebral emboli

CVD Z cardiovascular disease.

postmortem findings of vascular pathology of dementia.1

However, there is still a need for the development of im-aging parameters having diagnostic utility but also havingcapacity of determining etiopathogenesis, differentiatingVaD from AD and other neurodegenerative diseases. Ligand-specific PET and SPECT will serve as future diagnosticmethods when these ligands are developed for differentproteins found in VaD e.g. Tau, Ab 40 and many others.Arterial spin labeling, which measures absolute blood flowthough cerebral vessels may offer better results than SPECTin detecting hypoperfused areas. Moreover it is costeffective and avoids use of radioactive substances. Func-tional MRI can assess neuronal function through blood ox-ygen level-dependent changes. Although the neurovascularmechanism underlying blood oxygen level-dependentchanges is still poorly understood, functional MRI is beingused in neurological research. T2-T2

0-T2 relaxometry andsusceptibility-weighted MRI takes into account not only themagnitude but also the phase and signals for gradient echoMRI sequence. Susceptibility-weighted MRI, which hasability to differentiate calcium, iron and hemorrhagicproducts, can be a promising biomarker in differentiatingthe aging brain from VaD.72

Pathological biomarkers of VaD

The definitive diagnosis of VaD depends on the histo-pathological analysis of postmortem brain samples or an-imal models, which not only confirm the specificity ofbiomarkers but also facilitate classifying the disease pro-cess at the cellular and molecular level. The character-istic pathology such as microvascular angiopathy,CADASIL, hypertensive vasculopathy, cerebral amyloidangiopathy (CAA), and atheroembolic or thrombotic dis-eases have been identified and well-documented.1,7,73

Diagnostic utility

Strongly correlated with small vessel CVD,embolic disease and VaD (Mills)

Associated with CVD, ischemic diseaseas basalsize)

Strongly associated with CVD and VaD

Represents atherosclerotic arteries and VaDStrongly related to VaD on autopsy studies.

lacunartensity

Related to abnormal pulse pressure andWindkessel effect leading to CVD

ion) Associated with CVDEstimates age related, site related changes andvascular pathology

rophy,ensitiesion

Data not validated. Association with VaD is notyet proven.

Related to embolic infarct and VaD

Biomarkers in vascular dementia 53

Various pathological VaD biomarkers are illustrated inTable 3. Theses biomarkers can be divided into six majorcategories: (1) biomarkers of CADASIL; (2) biomarkers ofmicrovessel angiopathy; (3) biomarkers of hypertensivevasculopathy; (4) biomarkers of cerebral amyloid angiop-athy; (5) biomarkers of atherosclerosis or thromboticdisease; and (6) CB formation due to mitochondrialdegeneration and eventually apoptosis of the mostvulnerable cells in the hippocampal dentate gyrus and CA-3 regions due to cerebrovascular insufficiency inVaD.14,74e80

Clinical biomarkers in VaD

Clinical assessment of VaD is based on neurobehavioralbiomarkers that are assessed by performing mental statusexamination (MSE). MSE evaluates the extent of intellec-tual deterioration and personality change. This is followedby language performance test to acquire high yield resultsfor the clinical assessment of VaD.81 Among the differenttypes of MSEs, Mini MSE of Folstein, Hachinski ischemicscore scale, and Wechsler adult intelligence scale havebeen found to be most useful. These tests use evaluation ofattention span, temporal, and spatial orientation andretentive (declarative) memory. A score < 23 on Mini MSEof Folstein is usually diagnostic of dementia. Further typingis based on identification of risk factors as in VaD.82,83

Table 3 Neuropathological biomarkers.

Category Biomarkers

Biomarkers of CADASIL(Cerebral autosomaldominant arteriopathy withsubcortical infarcts andleukoencephalopathy)

CADASIL, CRV, HERNS

Biomarkers of microvascularangiopathy

Thickening of small vessel wall,narrowing, degeneration of tunfibrinoid necrosis, inflammationVasculitides

Biomarkers of hypertensivevasculopathy

Circle of Willis assessment, Presaneurysm, stenosis of vessels

Biomarkers of CAA (Cerebralamyloid angiopathy)

Amyloid deposition in vessels onH&E staining and Congo red þ b

antibody stainingBiomarkers of atherosclerosis

or thrombotic disease1. Presence of ischemia or hem2. Presence of infarcts: numberlocation, acute or chronic, cystwatershed, lacunar (white mattmatter, brain stem), laminar nehippocampal injury, cribriform3. Incomplete ischemic injury4. Loss of myelin on H&E and spsuch as LFB

Charnoly body (CB): A universalpre-apoptotic biomarker ofcompromised mitochondrialbioenergetics and cell injury

Hippocampal lesions

Miscellaneous Mixed-multiple pathology

CVD Z cardiovascular disease; H&E Z hematoxylin and eosin; VaD Z

Furthermore, the Hachinski ischemic score scale is a sim-ple bedside clinical biomarker and currently used fordifferentiating types of dementia (primary degenerative,vascular, multi infarct, mixed type). A cut-off score � 4 fordementia of other types and � 7 for VaD has a sensitivity of89% and a specificity of 89%.84,85 The Diagnostic and Sta-tistical Manual of Mental Disorders, 4th Edition (DSM-IV)Criteria for VaD take into account memory impairment, oneof the cognitive disturbances such as aphasia, apraxia,agnosia and laboratory imaging findings in support ofvascular etiology.13 International Classification of Diseases-10 Research Criteria (DCR-10) for VaD is similar to DSM IVcriteria with additional evaluation of consciousness, anddecline in emotional and social behavior. AD Diagnostic andTreatment Centers Criteria for the Diagnosis of ProbableIschemic VaD takes into consideration memory decline,history of vascular risk factors, neurological signs, andneuroimaging findings, relatively early appearance of gaitdisturbance and urinary incontinence to favor diagnosis ofdementia with probable ischemic etiology. Ischemic scoresclassified as VD by different diagnostic guidelines set byHachinski as follows: score indicating VaD � 7; AD Diag-nostic and Treatment Centers criteria: 10.3 � 3.4; DSM-IVcriteria: 6.5 � 4.4; DCR-10 criteria: 7.9 � 4.0; NINDS-AIREN criteria: 12.5 � 2.6. Orthostatic circulatory distur-bances such as alteration in mean arterial pressure,postural hypotension have been shown to be associated

Diagnostic utility

Familial small vessel diseases

luminalica media,

Assesses microvascular angiopathyNoncerebral amyloid angiopathy associatedangiopathy

ence of Marker for atherosclerosis

routineamyloid

Indicator of CAA

orrhage, size,ic,er, greycrosis,change.

ecial stain

Assesses cerebrovascular injuryStrong parameters of CVD and VaD, correlatedclinically and with imaging findingsCVDLeukoencephalopathy.

Need to be validated and distinguished formlesions in Alzheimer’s disease (CB as abiomarker of compromised mitochondrialbioenergetics)CVD

vascular dementia.

54 A. Jagtap et al.

with development of VaD and in some other neurodegen-erative diseases.86e88

Limitations in biomarker studies

Although biomarkers have been studied extensively, thereis no consensus on several issues such as definitions ofstandard procedures, uniqueness of processing and stor-age, analysis and interpretation of results and theirdiagnostic utility. It is important to identify a biomarkerthat is not only specific but also stable. In routine clinicalpractice it is desirable to use the biomarkers with stabilitydue to constraints of time and handling of specimens e.g.RNA stabilizers or exclusion of RNA chips, use of antico-agulants may give rise to variable results. Large multi-centric trials are necessary to compare diagnosticaccuracy of different laboratories all over the world.21

There are limitations in use of different analyticalmethods. For instance, enzyme-linked immunosorbentassay, used routinely may differ from Luminex’s xMAPtechnology when CSF and plasma samples are analyzed,which also affects the cut off values hence, standardiza-tion of international standard values is required. Valida-tion of biomarkers in body fluids need standardizationwith universal and valid criteria for clinical diagnosis,defining healthy controls, reproducibility in multiplecenters and correlated with standard postmortem diag-nosis. CSF diagnosis of dementia supports only clinical andnot postmortem diagnosis. Novel potential biomarkers ofVaD such as asymmetrical dimethylarginine, which is abiomarker of endothelial dysfunction, adhesion moleculeP-selectin may contribute to vascular processes andthereby dementia need larger studies and validation.7

Currently CSF isoprostane, a biomarker of oxidativestress, Ab oligomer, a synuclein, TDP-43, CSF DJ-1, TDP-43 are being investigated in AD. As dementia is defined asmixed etiology, there is a dire need to consider all thesebiomarkers along with newer biomarkers of vascular injuryin determining causal relationship to VaD. Innovativeneuroimaging techniques such as diffusion tensor imaging,MRS, functional MRI, amyloid-b PET imaging may providenewer insights in the etiopathogenesis of VaD.89 Gadolin-ium diethylene triamine penta-acetic acid also has shownpromise in detecting vascular pathology in dementia;however, all these imaging biomarkers need furtherevaluation through multicentric trials. Dysfunction ofautonomic regulation of cerebral blood flow is also asso-ciated with VaD where imaging biomarkers may providediagnostic utility.7 There has not been any consensus oncriteria, definitions, or analysis of VaD in neuropatholog-ical assessment. Although numerous gross and microscopicchanges have been identified as diagnostic of VaD, there isa need of multidisciplinary team performing large multi-center clinicopathological studies and harmonize thediagnostic approach and validate the biomarkers underinvestigation; e.g. abandonment of the term lacunae,which is a source of confusion; reducing interobservervariability.24 Genetics and molecular biology may show adefinitive avenue towards diagnosis and behavior of VaD;genome-wide association studies have become technicallyfeasible but are still expensive.

Conclusion

In this communication, we have reviewed the recent liter-ature on the development of VaD biomarkers. A variety ofcandidate biomarkers identified in CSF and blood by neu-roimaging methods, neuropathological examination, andgenetic analysis have shown promise in their utility asbiomarker for etiological diagnosis and behavior of vascularcognitive impairment and VaD however, lack of specificity,lack of criteria to identify and define the components ofVaD prompts further large scale studies and evaluation ofthese biomarkers and need to develop novel biomarkers.The recent discovery of CB formation as a preapoptoticbiomarker of oxidative stress and compromised mitochon-drial bioenergetics may serve as a novel biomarker of VaD.In addition, biomarkers of oxidative and nitrative stress inserum and CSF samples can be detected for the earlydiagnosis, treatment and prognosis of VaD. A further studyin this direction will go a long way in the clinical manage-ment of VaD.

Conflicts of interest

All contributing authors declare no conflicts of interest.

Acknowledgments

The authors express their sincere thanks to Kallol Guha,President, Saint James School of Medicine, Bonaire for hismoral support and encouragement.

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