Review Article ALS and Oxidative Stress: The Neurovascular ...downloads.hindawi.com/journals/omcl/2013/635831.pdf · ect lower motor neuron (progressive muscular atrophy) or upper

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 635831 14 pageshttpdxdoiorg1011552013635831

Review ArticleALS and Oxidative Stress The Neurovascular Scenario

Akshay Anand Keshav Thakur and Pawan Kumar Gupta

Neuroscience Research Lab Department of Neurology Post Graduate Institute of Medical Education and ResearchSector 12 Chandigarh 160012 India

Correspondence should be addressed to Akshay Anand akshay1anandrediffmailcom

Received 17 August 2013 Revised 7 October 2013 Accepted 17 October 2013

Academic Editor Regina Menezes

Copyright copy 2013 Akshay Anand et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Oxidative stress and angiogenic factors have been placed as the prime focus of scientific investigations after an establishment oflink between vascular endothelial growth factor promoter (VEGF) hypoxia and amyotrophic lateral sclerosis (ALS) pathogenesisDeletion of the hypoxia-response element in the vascular endothelial growth factor promoter and mutant superoxide dismutase1 (SOD1) which are characterised by atrophy and muscle weakness resulted in phenotype resembling human ALS in mice Thisresults in lower motor neurodegeneration thus establishing an important link between motor neuron degeneration vasculatureand angiogenic molecules In this review we have presented human animal and in vitro studies which suggest that molecules likeVEGF have a therapeutic diagnostic and prognostic potential inALS Involvement of vascular growth factors and hypoxia responseelements also highlights the converging role of oxidative stress and neurovascular network for understanding and treatment ofvarious neurodegenerative disorders like ALS

1 Introduction

At the developmental stages the establishment of a neurovas-cular network outside CNS is crucial to the subsequent brainand spinal cord development Molecules deserving specialattention in the course of development and maintenance ofneurovasculature include VEGF (especially VEGF-A)VEGFreceptors Notch ephrin semaphorinsplexin receptorslatent transforming growth factor 120573rsquos [TGF120573rsquos] and TGF120573receptors 120572v1205738 integrin neuropilins and FGF1 [1ndash3] Anydysregulation in the pathways having the above mentionedfactors (responsible for angiogenesis) which contributes tothe development of this communication network has seriousconsequences manifesting in the form of CNS disordersHence angiogenesis is required for vasculature developmentand is governed by the gene expression of vascular molecules[4] Abnormal expression and reduced levels of VEGF havebeen explored to account for devastating disorders of theCNS especially in studies focused on ALS which is desig-nated bymotor neuron degeneration and is fatal in nature [5]Genetic studies in a transgenic mouse and rat model ofALS with mutated superoxide dismutase 1 SOD1G93A haveindicated that inhibition of hypoxia response element (HRE)in the VEGF gene promoter may promote motor neuron

degeneration (since HRE is responsible for inducing angio-genesis throughVEGF as shown in Figure 1) whereas admin-istration of VEGF prolongs survival [6] Hence here wereview the role of neurotrophic and angiogenic factors likeVEGF in the pathogenesis of ALS

2 ALS A Fatal Disease of the Motor Neurons

Motor neuron disease (MND) defines conglomerate ofrelated and progressive degenerative disorders character-ized by selective degeneration of upper motor and lowermotor neuron located in the motor cortex and brain stemand spinal cord respectively [4] The disease may eitheraffect lower motor neuron (progressive muscular atrophy)or upper motor neurons (primary lateral sclerosis) or bothupperlower motor neurons (amyotrophic lateral sclerosis)however careful pathological and clinical studies in MNDhave shown that extra-motor parts of the central nervoussystem are also affected ALS is the most severe MND whereselective degeneration of motor neurons leads to atrophy ofvoluntary muscles followed by paralysis and may prove fatal[5] Mechanisms of selective degeneration of motor neuronsin ALS are obscure Largely ALS symptoms include weaknessof muscles especially those in the hands arms and legs with

2 Oxidative Medicine and Cellular Longevity

ANGANG

ANGNucleus

Endothelial Cell

rRNA

AngiogenesisEvents leading to angiogenesis in cell

Endocytosis

Nuclear ANG

VEGF expression regulated by nuclear ANG

ROS

NO

RNS

Mitochondria

VEGF

ROSHIF-1 alpha

HIF-1 alpha

O2

NF-120581B

Figure 1The role of hypoxia in stimulating theVEGF through an activation ofHIF-1 alpha element HIF-1 alpha gets activated in deficiency ofoxygen in mitochondria leading to creation of oxidative stress This involves the formation of reactive oxygen species which on reaction withfree nitrogen forms NO ultimately leading to reactive nitrogen species (RNS) This RNS further activates NF-120581B pathway which ultimatelyleads to activation of HIF-1 alpha factor The activated form of HIF-1 alpha further leads to VEGF activation thus leading to angiogenesis

or without dysarthria and dysphagia Fasciculation or muscletwitching is also an important clinical finding [7]

3 ALS Contributing Factors

ALS occurs in both sporadic and familial form at an incidencevarying between 04 and 26 for every 100000 individualsand a prevalence rate of 4ndash6 per 100000 population per year[8] The etiology of ALS has been elusive and believed tobe multifactorial Though causes of most cases of ALS areunknown major factors include genetic factors like pointmutations in superoxide dismutase 1 (SOD1) gene accountingfor around 20 of familial ALS (fALS) cases [9] The purelylower motor neuron (LMN) degeneration variant of ALSshows missense mutations in CHMP2B (charged multivesic-ular protein 2B involved in cellular transport) In 10 casesof ALS patients with CHMP2Bmutations are shown to havelower motor neuron degeneration Apart from this othergenes like vesicle-associated membrane protein B (VAPB)(which is involved in providing unfolded protein responseto endoplasmic reticulum) senataxin (SETX) (gene presentin central nervous system involving brain and spinal cord aswell as muscle and play major role in DNA repair to maintainintegrity of cell) and dynactin 1 (involved in cellular transportduring cell division and specially in axonal transport ofnerve cells) with mutations have been shown to play role inaggregate formation andhampering the normal activity of themotor neurons thus contributing to the pathogenesis of ALS

overall in subjectrsquos body [10ndash14] Genes encoding angiogenin(ANG) havingmissense mutations have also been involved inthe pathogenesis of ALS Angiogenin likeVEGF is producedin response to hypoxia and plays a role in neovascularisationas shown in Figure 1 Its importance further stems fromthe fact that it can regulate the expression of VEGF [1516] Hypoxia takes place when oxygen availability is low incell due to which the mitochondria produces ROS specieswhich in turn reacts with nitric oxide (NO) to producereactive nitrogen species RNS and activates HIF-120572 pathwaythrough NF-120581B pathway resulting in stimulation of VEGFThe expression of this VEGF is dependent on the nucleolarANG which directly helps in stimulating the proliferation ofepithelial cells and helps in angiogenesis [17] However thishypothesis raises a question whether angiogenin crosses theblood brain barrier or is retained in cerebrospinal fluid [18]

Apart from genetic factors the presence of insolubleintracellular protein aggregates in motor neurons and reac-tive astrocytes are considered as the hallmarks for the disease(Figure 3) [19] The other factors include glutamate toxicity[20] lack of trophic growth factors [6 21] autoimmunity[22] toxin [23] and susceptibility ofmotor neurons to neuro-degeneration because of their large size and high energydemands [24]

Currently there is no treatment that could substantiallyalleviate the disease burden because of incomplete under-standing of ALS etiology Food and Drug Administration(FDA) has approved only single drug for the treatment of

Oxidative Medicine and Cellular Longevity 3

ALS a glutamate antagonist that is Riluzole [25 26] Riluzolehas also been studied as a potential inhibitor of VEGFinduced endothelial cell proliferation under both in vitro andin vivo conditions [27] Its neuroprotective effect via sodiumchannel blockage is brought about by the fact that this mech-anism increases resistance to hypoxia through a reduction inenergy demands (a decreased cerebral glucose consumption)[28]

4 VEGF The Neurotrophic andAngiogenic Family

VEGFA gene in humans is positioned at chromosome 6p213with eight exons and is expressed as several isoforms of dif-ferent amino acid chain lengths because of alternative splicing(119881119864119866119865

121119881119864119866119865

145119881119864119866119865

165119881119864119866119865

183119881119864119866119865

189119881119864119866119865

206)

[29] that differ in their ability to bind heparin neuropilin-1(NP-1) and neuropilin-2 (NP-2) Two classes of receptors forVEGF are the tyrosine kinase and the nontyrosine kinasereceptors VEGFR1 (Flt-1 (fms-related tyrosine kinase 1))VEGFR2 (KDRFlk-1 (kinase insert domain receptorfetalliver kinase-1) and VEGFR-3 (Flt-4) are three structurallyrelated receptors present in tyrosine kinase class V whereasneuropilin-1 (NP-1) and neuropilin-2 (NP-2) are part ofnontyrosine kinase receptors VEGF binds to NP 1 and 2 andVEGFR1 and 2 but not to VEGFR-3 as the latter one is nota receptor for VEGF Studies indicate that for transmissionof critical angiogenic signals in response to VEGF VEGFR2plays the role of key mediator [30] However in case ofVEGFR1 the major function is prevention of VEGF bindingtoVEGFR1 thought to be done by a virtue of ldquodecoy receptorrdquoto negatively regulate angiogenesis [31]Neuropilins (NP1 and2) whose primary location is in central nervous system aredescribed as receptor for collapsinsemaphorin family whichare responsible for controlling neuronal cell guidance [32 33]For VEGF165 and a coreceptor ofVEGFR2Neuropilin-1 (NP-1) it is a specific receptor whereas Neuropilin-2 (NP-2) bindsVEGF165 and VEGF145 in isoform specific manner VEGF isthe part of genes which accommodate placental growth factor(PLGF) VEGFB VEGFC VEGFD and VEGFE includingVEGF-A out of which lymphatic vessels development isaffected by VEGF-C [34] Recent evidence from studies alsoindicates that neural cells are directly affected by VEGF-AVEGF-B and VEGF-C [35] In ALS VEGF has been studiedas an important member of gene families impacting thepathology of disease

5 VEGF Molecular Risk Factor in ALS

The lack of trophic (growth) factors has been hypothesized asprobable cause of ALS Since growth factors are neurotrophicand help in growth survival and maintenance of neuronalcells The hypoxia response brings together a cascade ofevents involving angiogenic and inflammatory factors (Fig-ures 1 and 3) Studies have focussed on predictingcorrelatingdisease state with changing levels of such factors in bodyfluids even though these have been conducted utilisingheterogeneous controls

VEGF and its receptors are reported to be localised inneurons and astrocytes [36 37] which in case of ischemiaor spinal cord injuries provides neuroprotection and stim-ulates neuronal growth Decreased VEGF levels may impairperfusion and induce ischemia of motor neurons other thandepriving cells of important survival and neuroprotectivesignals which are VEGF dependent [6]

Cronin et al reported elevated levels of serum angio-genin but no change in serum VEGF levels was observedThe authors also failed to observe any correlation betweenserum angiogenin andVEGF levels [16] In another study thepatients with limb onset and long duration of ALS showedhigher concentration of CSFVEGF as compared to thosewithbulbar onset of ALS and patients with short duration illnessrespectively [38] It may be possible that significant increasein cerebrospinal fluid (CSF)VEGF levels may have protectiverole against over-excitation ofmotor neurons (excitotoxicity)This overexitation may be mediated by excessive accumula-tion of glutamate at synaptic cleft in patients with limb onsetof ALS and those with long duration of the disease since itwas suggested that the increased levels of VEGF account fora compensatory mechanism and may be required to stabilizeneuronal excitation [39]The rationale was further supportedby Bogaert et al who reported thatVEGF protectsmotor neu-ron against excitotoxicity by upregulatingGlutamate receptor2 [40] Significantly lower baseline CSF VEGF levels in caseof patients with ALS in comparison to normal controls andneurologic controls during early phase of disease have beenobserved suggesting the possible link of ALS pathogenesiswith VEGF gene regulation [41]

Moreau et al demonstrated that hypoxaemic ALSpatients had lower VEGF levels in CSF from normoxaemicALS patientsThis happened due to an early defect in hypoxiainduced factor-1 (HIF-1) mediated regulation of VEGF Incontrast higher levels of VEGF in CSF were demonstratedin hypoxaemic neurological controls than normoxaemic neu-rological controls Hypoxaemia severity in ALS is explainedby dysregulation of VEGF in ALS This association of VEGFexpression and hypoxia (Figure 1) in ALS introduced aconcept of incongruous response [42] Nagata et al failed toreproduce the above results as no significant difference wasobserved in CSF VEGF levels between ALS patients normalcontrols and controls with other neurological disorders [43]It was argued by Cronin and coworkers that the conflictingreports of elevated normal and decreased VEGF mighthave resulted from different study designs and ELISA kitemployed with varying diagnostic criteria of ALS patientsdiverse clinical details of ALS patients including definite andprobable forms of disease [16] In a unique histochemicalstudy a markedly elevated level of VEGF was detected in theskin of ALS patients when compared with normal subjectssuggesting a positive correlation of VEGF levels in skin andseverity of ALS patients [44] The finding suggests systemicdysregulation of VEGF expression in ALS Recently it hasbeen observed that elevated levels of VEGFA in CSF serumand peripheral blood mononuclear cells may account forsubstantially prolonged life span of Indian ALS patients ascompared to their Western counterparts [45ndash47] Surpris-ingly longer survival is shown in Indian ALS patients after


Hypoperfusion

Decreased blood flow to cells

Glucose

Restricted glucose availability to cells

No glycolysis

No ATP formation leading to energy failure

Ferritin

Ferritin

Reduced ferritin protein

Increased amount of unbound iron molecules resulting in formation of ROS leading to increased

oxidative stress

ROSROS

ROSROS

Oxidative stress

Fe2+

Fe2+ Fe2+ Fe2+

Fe2+

Fe2+Fe2+

Fe3+

Fe3+


Acetyl CoA

Citricacidcycle NADH

ADP

ATP

NAD+

Oxidativephosphorylation

Figure 2 Role of hypoperfusion in elevation of oxidative stress and energy failure As hypoperfusion reduces bloodflow towards cells resultingin reduced ferritin Fe3+ protein it releases unbound iron Fe2+ molecules resulting in formation of ROS thus increasing the oxidative stressHypoperfusion also leads to unavailability of glucose to brain cells thus leading to energy failure

onset (sim9 year) of ALS [45 46 48 49] Further reduced levelsof soluble VEGFR1 (sVEGFR1) an inhibitory receptor ofVEGF have been observed in these patients supporting theneurotropic nature ofVEGF [50] However these results needconfirmation in comparable Caucasian ALS population

6 ALS VEGF and Oxidative Stress

Lowering of VEGF levels places neural tissue at the risk oflimited perfusion thus making way for motor neuron degen-eration [51] This degeneration is a direct consequence ofthe fact that the deficient oxygen and glucose levels createdas a result of decreased vascular perfusion can hardly meetthe energy demands of motor neurons [52] Oxidative stressdue to hypoperfusion has been reported in cases of otherneurodegenerative disorders such asAlzheimerrsquos disease [53]Oxidative stress is one of the outcomes of hypoperfusionapart from energy failure as blood is known to carry severalvital components essential for cell survival including glucoseand ferritin As glucose is able to readily cross blood brainbarrier (BBB) the deficiency of blood flow leads to reducedsupply of glucose to brain resulting in limited energy pro-duction for cells Similarly the deficiency of ferritin whichis responsible for binding of free iron results in formation

of reactive oxygen species as shown in Figure 2 [54] At leastone study has reported that the variable levels of VEGF leadto altered ferritin levels [55] Therefore it is safe to say thatoxidative stress deserves special significance in the patho-genesis of neurodegenerative diseases like ALS since motorneurons are particularly susceptible to oxidative damage

This significance is born out of the fact that the firstevidence of association between ALS pathology and VEGFcame when Oosthuyse et al created homozygous VEGF(119881119864119866119865120575120575) knock-inmice by introducing homozygousmuta-tion of hypoxia response element (HRE) in the VEGFgene promoter to study angiogenic property of VEGF Theyobserved that almost 60 of mice did not survive before oraround birth due to vasculature aberrations in lungs The40 who survived began to develop symptoms like classicalALS around five months of age [6] This unusual findingcompelled researchers to explore significance of growthfactors in pathology of ALS utilising a variety of tools suchas those discussed below

61 Autopsy Based Studies Spinal cord tissue analysis of ALSpatients has revealed elevated dendritic cell marker tran-scripts (like CD83) and monocyticmacrophagemicroglialtranscripts [56] expression of cyclooxygenase-2 (COX-2)


Na+K+

Na+

K+

Astrocyte

Astrocyte Breaking of

homeostasis

Hypoxia

DNA damage

Motor neuron

Motor neuron

Blocking of synapse

formation

AstrocyteImbalance of

load outside cytoplasm detected by astrocyte

Stimulation to produce more

astrocytes in the vicinity of dying

cell

Na+

K+K+

K+

K+

K+

K+

K+

K+

K+

K+ K+

K+

K+

K+

Na+K+

Figure 3 Hypoxia induced mobilisation of astrocytes Astrogliosis is the result of aggressive increase of astrocytes number in the vicinity ofdamaged neuron cell Synapse formation is hampered when there is neuronal damage thus leading to breakdown of Na+K+ homeostasisThisK+ concentration is detected by the astrocytes

[57] connective tissue growth factor (CTGF) [58] monocytechemoattractant protein-1 (MCP1) [56] and VEGF receptor(VEGFR)-1 [59] and activity of glutamate dehydrogenase(GDH) accompanied by reduced levels of glutamate andaspartate [60]

The increase in CTGF expression is explained by the factthat CTGF plays an important role in astrogliosis which isoften seen as a consequence of hypoxic conditions and istherefore a pathological hallmark of ALS [58] As depictedin Figure 3 astrogliosis is the result of aggressive increaseof astrocytes number in the vicinity of damaged neuroncell Hypoxia generally induces damage in the DNA of theneuronal cells Since the neuronal damage has taken place itsnormal activity of synapse formation is hampered affectingthe Na+K+ activity in those cells leading to breakdown ofNa+K+ homeostasis This change in balance of K+ concen-tration is detected by the astrocytes This alteration results inthe activation of astrocytes by initiation of clustering aroundthe damaged cells in order to restore the functioning of thosedamaged cells [61ndash63]

Gliosis is also related to the enhanced GDH activity asreported byMalessa et al [60]The function of the GDH is toenhance the availability of the glutamate This glutamatefurther acts as neurotransmitter or gliotransmitters since itincreases the availability of Ca+ required by glial cells toperform their normal function of providing protection nutri-tion and avoiding accumulation of any chemicals involved

in synapse formation which may later lead to toxication ofneuron cell Recruitment of glial cells to the site of damagemay be considered as the bodyrsquos primary response to save thedying neurons [64 65] and thus the fact may be related tothe point of association of enhanced GDH activity to gliosisThe authors also suggested a disturbance in cholinergic trans-mission in ALS spinal cord thus contributing to the reducedamino acid levels [60] Glutamate and aspartate amino acidsare linked with the neurotransmitters in the body They aremainly the excitatory neurotransmitters which utilise theNa+K+ pump to maintain their flow to the postsynaptic cleftduring the nerve transmission Li and Zhuo demonstratedthat cholinergic transmitters play a role in inhibiting theglutamate based transmission Release of acetylcholine leadsto the activation of the muscarinic receptors resulting inan inhibition of AMPA receptors (also called as glutamatereceptors) and it increases the nonavailability of glutamateThis evidence also supports the fact mentioned in the abovestudy that disturbance in cholinergic transmission may leadto reduced amino acid levels [66]

VEGF was first measured in spinal cord and serum ofALS patients by Nygren and colleagues Authors did notobserve any significant alteration in spinal cord VEGF levelsbut they were able to observe higher serum VEGF levels inALS patients in comparison to controls similar to those laterreported by Gupta et al in case of Indian ALS patients [46]Considering the higher levels ofVEGF in serum suggests that


the cells other than central nervous system or which are notpart of CNS are involved In case of ALS skeletal muscles arethe most affected region of body Regional ischemia a con-dition in which the blood supply is halted in specific regionof brain has been reported in case of ALS [67] Rissanen etal observed higher levels of VEGF in skeletal muscles withacute phase of ischemia [68] Thus it was hypothesized thatVEGF is expressed in skeletal muscles in response to hypoxiaand the increase was also reflected in serum [69]

The autopsy samples depict the terminal stage of thedisease and provide a reliable proof of the disease and itssignatures [70]

62 Muscle Biopsy Based Studies In contrast to the increasedcyclooxygenase (COX) activity in spinal cord of ALS patientsas discussed above Crugnola et al reportedCOX deficienciesin 46 patients based on their histochemical analysis ofmuscle specimens Moreover molecular studies and bio-chemical analysis on the selected specimens displaying severeCOX deficiencies even correlated with mutations in SOD1and TARDBP genes and mitochondrial DNA defects thuspointing towards the secondary nature of COX deficienciesin the pathogenesis of ALS in light of the genetic natureof defects [71] This is also confirmed by the findings ofVielhaber et al who observed mitochondrial DNA damagein skeletal muscle along with lowered levels ofmitochondrialMn-SOD [72]The specific nature of mitochondrial dysfunc-tion is further revealed by studying mitochondrial markerslike citrate synthase and succinate dehydrogenase in musclehistochemically However such a study by Krasnianski et alrevealed that one cannot narrow down the observed mito-chondrial changes to only depict ALS but in fact view themas an indication of other neurogenic atrophies too [73] Inview of neurotrophic support provided by muscle tissuethe findings by Kust et al depicted enhanced expressionof nerve growth factor (NGF) and neurotrophins such asbrain-derived neurotrophic factor (BDNF) in postmortembicep tissue of ALS patients Even so externally administeredneurotrophins have not shown promising results in humantrials or animal models of ALS [74]

63 Polymorphism Based Studies Increased oxidative stressimplies consequent increased oxidative damage for motorneuronal DNA Such oxidative damage of DNA is driven bythe base excision repair (BER) system One such product ofoxidative damage of DNA is 8-hydroxy-21015840-deoxyguanosine(8-OHdG) which is regulated by two enzymes namelyhuman 8-oxoguanine DNA glycosylase 1 (hOGG1) andapurinicapyrimidinic endonuclease APE1 Consequentlymutations and polymorphisms in coding area of genes codingfor both of these enzymes are of interest to researchersConcurrent oxidative stress conditions and a faulty DNArepair system are a risk factor for motor neurons

In most studies concerning hOGG1 Ser326Cys polymor-phism levels of 8-OHdG are taken into account as 8-OHdGis the product of DNA oxidation [75] A study conductedby Chen et al showed the reduced activity of hOGG1 inpatients with 326 CC polymorphisms (119875 = 002) as comparedto those with 326 SC polymorphisms (119875 = 005) [76]

Similar observations were made by authors in current studyIn a Caucasian study Coppede et al studied the distributionof allele frequencies and genotypes in sALS patients andcontrols for the hOGG1 Ser326Cys polymorphism in sALSpatients and controls The authors reported a significantlyincreased sALS risk associated with a combined Ser326Cys+ Cys326Cys genotype However the Ser326Cys genotypeshowed nonsignificant results predicting that the hOGG1Ser326Cys polymorphism in patient also pose a risk factorfor ALS Ser326Cys polymorphism takes place when at exon7 position 1245 C to G substitution occurs and as a result S issubstituted to C in codon 326

Another interesting observation (though not significantas the test group of subjects used for the study was smallmore significant results can be obtained if the studywith largenumber of patients is conducted) in the above study was thefact that sALS patients as opposed to those bearing one ortwo copies of the 326Cys mutant allele bearing the Ser326Sergenotype displayed lower levels of AOPP (advanced oxi-dation protein products believed to be stable markers ofoxidative damage to proteins) [77] Since abnormal levelsof VEGF are implicated as risk factor in ALS it is evidentthat mice with hypoxia response element deletion in vascularendothelial growth factor gene develop features reminiscentof ALS [5] although no spontaneous mutations have beenobserved in HRE in ALS patients [78 79] Large family-based and case-control cohort of North American whitesubjects (119899 = 1603) were studied for the association of sALSwith promoter polymorphisms of three VEGF genes VEGFpromoter polymorphisms do not find their casual role inALS in light of absence of their association with sALS [80]Risk of developing ALS has been associated to VEGF dueto alterations in sequence in the promoter region of geneIn The Netherlands 373 patients with sporadic ALS alongwith 615 matched healthy controls were found to have VEGFpromoter haplotypes No significant association between thepreviously reported at-risk haplotypes and ALS was found[81] However in some studies ALS has been found to beassociated with VEGF C2578A polymorphism In a studyof Chinese population by Zhang et al 115 sALS patientswith 200 healthy individuals were analyzed for C2578Apolymorphism (by amplifying 2705 to 2494 bps of VEGFgene promoter) Reports were in disagreement to previousstudies from Caucasian populations as Chinese populationdid not fall susceptible for ALS due to C2578A polymorphism(attributing the effect to different genetic background inChinese population) [82] No significant association of ALSwith three common VEGF variations [-2578CA -1154GAand -634GC] in original form or in haplotype combinationin a recent meta-analysis study comprising of over 7000individuals involving three North American population andeight European populations was reported However in males-2578AA genotype increased the risk of ALS in subgroupanalyses by gender [83] in contrast to a German study whichsuggested that risk of ALS in case of female patients mightbe higher as the VEGF role might be gender dependent[84] Oates and Pamphlett did not observe any alteration offunctioning of motor neurons by epigenetic transcriptionalsilencing of VEGF gene by methylation [85] Additionally


screening of regulatory sequences of VEGFR2 found noassociation of polymorphism of VEGFR2 gene with risk ofALS [86] Although association of VEGF with ALS has beenwell established by culture and animal studies evidence fromgenetic studies in human cohorts suggests only a minorassociation between VEGF and the risk of developing ALS

The role of VEGF involvement in ALS is questioned dueto lack of association of VEGF genotypes and haplotypes inlarge meta-analysis study Possibilities for VEGF role inpredisposed patients to ALS cannot be ruled out Morestudies are needed to discern the actual role of VEGF inpathogenesis of ALS

64 Animal Model Based Studies

641 Primates The concept of utilizing the cytotoxic prop-erties of the extract obtained from the spinal cord of ALSpatients was applied by Zilrsquober et al as early as in 1963 soas to reproduce the disease in rhesus monkeys The authorscould only conclude to a viral nature of this disease but atthe same time they recognised that the high incidence pre-viously reported in the Chamorro tribe of Guam suggesteda unique basis [87] Another study conducted on rhesusmonkeys attempted to validate the efficacy of bovine SOD as atherapeutic agent to compensate for the functions of themutated form of the enzyme SOD being a locally actingenzymewas administered intrathecally and intraventricularlyso as to bypass the blood brain barrier The injected bSODshowed commendable tolerance though its clearance wasslower when compared with results obtained from rats Butthe therapy when administered into a late stage FALS patientdid not show promising results [88]

642 Rodents The neuroprotective effect of VEGF suggeststhat exogenous VEGF administration may prevent degen-eration of motor neuron In a SOD1Gly93Ala rat model ofALS it was shown that onset of paralysis was delayed by 17days improved motor performance and extended lifespanby 22 days due to intracerebroventricular (icv) delivery ofrecombinant (VEGF) The study demonstrated the high scaleeffect in animal models of ALS achieved by protein delivery[89] Intrathecal transplantation of human neural stem cellsoverexpressing VEGF increased the duration of survival of atransgenicALSmousemodel [90] Similarlymice after spinalcord ischemia showed susceptibility to paralysis in nervoustissue with reduced VEGF-A expression levels whereas aftertreatment with VEGF-A showed protective effect againstischemicmotor neuron death [91]These results unveil a ther-apeutic potential of VEGF for degenerating motor neuronsin case of human ALS In similar study authors investigatingthe protective role of VEGF during ischemia has shown toreduce infarct size improve neurological performance andenhance the survival of newborn neurons in the dentate gyrusand subventricular zone in adult rat brain with focal cerebralischemia Thus VEGF shows acute neuroprotective effectand prolongs survival of new neurons in the ischemic brain[92]

Zheng et al demonstrated for the first time in CuZnSOD1 transgenic mouse model of ALS that VEGF delayed

diseased symptoms progression and prolonged survivalsuggesting the importance of VEGF or related compoundsin the treatment of ALS patients [93] Rats having VEGFtreatment showed significantly improved performance up to6 weeks after spinal cord contusion injury compared withcontrol animals Furthermore the group showed that VEGFtreated animals had increased amount of spared tissue inthe lesion centre with higher blood vessel density in parts ofthe wound area compared to controls proving neurogenicand angiogenic capacity of VEGF [94] Enhanced expressionof VEGF by intramuscular administration of zinc fingertranscription factor in SOD1 rats has been shown to improvefunctional disability [95] Nitric oxide is known to decreasepressure in blood vessels [96] and it is possible that lowVEGF adversely affects vasculature via changing the amountof nitric oxide released from endothelial cells which furtherimpairs perfusion and causes ischemic damage of motorneurons [91] Moreover decreased flow of blood has beenobserved in patients with ALS [97] Both mechanismsmay contribute to adult-onset progressive degeneration ofmotor neurons muscle weakness paralysis and death atypical feature of amyotrophic lateral sclerosis It was earlierdemonstrated that exposure to low levels of lead prolongssurvival of ALS transgenic mouse possibly mediated byupregulation of VEGF which in turn reduces astrocytosis[98] In another case retrograde delivery of lentivirus intomouse model of ALS prolonged survival in animals Authorsreported that lentivirus helped in stimulation of VEGF levelsduring diseased condition in animals [99] Although in ALSanimal models VEGF delivery has been successful dose ofdelivery of VEGF should be adequately optimized to preventadverse effects on the vascular system It is possible thatlevels of VEGF higher than a certain threshold value mayincrease leakiness of blood vessels andmodulate permeabilityof blood brain barrier [100] and therefore result in intrathecalaccumulation of fluid The presence of the blood breakdownproduct hemosiderin in and around spinal cord motorneurons supports increased leakiness and malformed bloodvessels in ALS mouse models [101]

It must be noted that a drawback with using SOD1 basedtransgenicmodels is that SOD1 genemutations represent only20 of cases of familial ALS which themselves represent just10 of the total ALS cases Therefore remaining 90 of ALScases sporadic in nature are difficult to mimic using suchanimal models [70]

65 Cell Culture Based Studies Owing to a translational gapfrom animalmodels of ALS to humans in vitro investigationsutilising human motor neurons and astrocytes purified fromthe human embryonic spinal cord anterior horns allow forgreater manipulations and are therefore a critical tool indiscerning mechanisms pertaining to motor neuron degen-eration in ALS [102]

The mRNA level of VEGF has been an important markerto analyse the role of VEGF in ALS Destabilization anddownregulation of VEGF mRNA with concomitant loss ofprotein expression in glial cells expressing mutant SOD1in vitro are in consensus with many reports on the roleof reduced VEGF expression in ALS pathogenesis [103] In


contrast it was reported that hypoxia induced proteins bindand stabilize VEGF mRNA transcript resulting in increasedexpression of VEGF as a compensatory protective mecha-nism in later stages of disease [104]

The potential role of VEGF in preventing cell death bySOD-1 mutation has been studied in NSC-34 motor neuroncell line from mouse Infection by adenovirus containingmutant Gly93Ala-SOD1 was shown to increase cell death andcellular oxidative stress However VEGF showed a dosedependent resistance to oxidative damage from hydrogenperoxide TNF-alpha and mutant Gly93Ala-SOD1 in NSC-34 cells treated with VEGF Both phosphoinositide-3-kinase(PI3-K) and mitogen activated protein kinase (MAPK) activ-ities inmouse NSC-34motor neuron-like cells were activatedby VEGF [105] Recently a culture study using primaryculture of SOD1 mutated rat motor neurons has shown thatdecrease in VEGF before or during motor neuron degener-ation amplifies the risk of mutated SOD1 induced toxicity inmotor neurons [106] Thus the in vitro study shows VEGFas an antiapoptotic molecule Overexpression of VEGF inthe hippocampus using recombinant adeno associated virusvector in adult rats has been reported to result in improvedcognition in associationwith approximately 2-fold increase inneurogenesis Moreover environmental induction of neuro-genesis is completely blocked RNA interference based inhibi-tion ofVEGF expressionThis data supports amodel wherebyVEGF acting via kinase insert domain receptor (KDR) is amediator of the effect of the environment on neurogenesisand cognition [107] Meng et al investigated in vitro the pro-liferation and differentiation of subventricular zone neuralprogenitors of adult mouse by virtue of direct effect ofVEGFDownregulation of endogenous VEGF receptors 1 and 2 inassociation with reduced neural progenitor cell proliferationand enhanced neuronal differentiation was reported as aresult of high dose (500 ngmL) of VEGF whereas endoge-nous VEGF receptors 1 and 2 were significantly upregulatedwithout increased proliferation and differentiation at lowdose (50 ngmL) of VEGF Above given experiments suggestthatVEGF regulates neurogenesis and its high dose enhancesadult neural progenitor cell differentiation into neuronsshowing exogenous VEGF to exert a biphasic effect on theexpression of endogenous VEGF receptors [108] It has beenshown that VEGF induces differentiation of stem cells inendothelial cells which in turn secrete various neurotrophicfactors and infers a novel mechanism of neuroprotection byVEGF [109] Apart from VEGF recently VEGFB was shownto protect cultured primary motor neurons Further it wasobserved that mutated SOD1 ALS mouse without VEGFBgene developed more severe form of ALS than ALS mousewith VEGFB [110]

7 VEGF in Blood Brain Barrier (BBB) andBlood Spinal Cord Barrier (BSCB)

Blood brain barrier (BBB) is the only checkpoint thatstops inflammatory agents to reach central nervous system(CNS) as it contains a balanced interaction of microvascularendothelial cells and other components such as astrocytes

pericytes neurons and basement membrane These com-ponents are collectively called as neovascular unit NVUTight junctions among NVU make the entry of undesirablecomponents restricted to CNS [111] BBB breakdown maylead to disruption of various biochemical reactions or maylead to accumulation of various inflammatory proteins thatmay aggravate the disease conditions of CNS [112ndash114]Similarly blood brain and spinal cord barrier which can bea morphological isotype for BBB performs same function inseparating the spinal cord from all harmful components thatmay lead to diseased conditions of nervous system [115] Ithas been observed that in case of human and animal modelstudies both the infiltration of brain and spinal cord with Tcell dendritic cells or IgG have resulted in degeneration ofmotor neurons [116] Claudins play a major role in formingtight junctions in the body among the cells to function asa barrier or act as a filter for these inflammatory factors toenter CNS [117] Earlier studies have shown that astrocytesproduce certain chemokines which play a role in attractingthe dendritic cells to the CNS [118] Recently a link betweenthe reactive astrocytes and disruption of these barriers hasbeen reported Argaw et al tried to examine a link betweenastrocyte derived VEGFA and BBB permeability Astrocyticexpression of HIF-alpha and VEGFA leads to downregula-tion of claudins CLN-5 and their regulatory protein OCLN[119] VEGFA by the virtue of tyrosine phosphorylationdownregulates the expression of CLN ultimately resultingin disruption of permeability barrier VEGF induces themigration among the endothelial cells and increasing thepermeability to CNS [120] (Figure 4) However theis link ofVEGF is conflicting with the earlier reports in this paperregarding the protective role of VEGF in ALS pathogenesis

8 Natural Products and Regulation ofVEGF Expression

Naturally occurring compounds are also in current focus toexamine their role in VEGF expression The mechanism hasbeen postulated to be common in all cases for those whichare known to be responsible for increase in the expressionof VEGF All of them have been shown to affect the HIFpathway inducing the expression of VEGF It is not clearhow these natural compounds can be successfully translatedfor clinical use in near future which will need more studiesCertain extracts like turmeric gigko biloba and ginseng havebeen shown in mice studies to delay the disease onset orprolong survival in mice studies However recently a groupfrom China reported a component Baicalin in the roots ofplant Scutellaria baicalensis which enhances the expressionof VEGF [121] Although the HIF expression was less ascompared toVEGF authors reported that other transcriptionfactors such as oestrogen-related receptors (EERs) exerttheir effects via VEGF promoters Peroxisome proliferator-activated receptor-120574 coactivator-1120572 (PGC-1120572) an importantmolecule independent of activator is shown to interact withERR120572 [122 123]These PGC-1120572 are shown to enhance expres-sion ofVEGF in culturedmuscle cells in vivo in HIF indepen-dent pathway [121 124] In contrast grape seed extract (GSE)


Hypoxia

Astrocytes

PP

P

P

PP

P

P

Phosphorylation

Mobilisation of endothelial cells

BBBBSCB

Breakdown of barrier

HIF

-alp

ha

VEGF-A

Figure 4 VEGF and permeability of blood brain barrier Astrocytic expression of HIF-alpha and VEGFA leads to downregulation ofclaudins CLN-5 and their regulatory protein OCLN VEGFA by the virtue of tyrosine phosphorylation downregulates the expression ofCLN ultimately resulting in disruption of permeability barrier VEGF induces the migration among the endothelial cells and increases thepermeability to CNS

is known for its antitumor properties and is shown usefulin case of breast lung skin or gastrointestinal cancer [125ndash127] Lu and group recently showed that GSE reduced theVEGF expression by inhibiting the HIF expression in humanbreast tissue cancer cells Authors argued that it involved theblockade of HIF expression by inhibiting AKT-3 pathwaynormally known for supporting the cell survival [128] Apartfrom these other natural components have been shown toprovide nonsatisfactory results in certain trials conducted indifferent human population Vitamin E the most commonlystudied antioxidant has been implicated with role of slowingdown the disease progression in its severe form DesnuelleC and colleagues conducted a study in French population ofALS patients 289 patients were recruited for the study Allof them were randomly assigned the dose of Vitamin E andwere assessed after every 3 months The results did not showthe effect in survival of muscle cells except for the fact thatpatients who were administered the Vitamin E stayed in themilder form of disease for longer time [129] Another studydone in German population with high dose of Vitamin-E

(5000mgday) showed ineffective results in comparison tothe placebo effect [130] However in one of themeta-analysesof 23 studies published in year 2008 it was stated thatantioxidants whether in combination or during individualadministration do not show effective results [131] Creatineone of the sports supplement has been known to increasethe muscle strength One study that came up in 1999 wasconducted in animal model of ALS Transgenic mice of ALSwas administered with creatine dose Authors reported thatcreatine was helpful in saving the mice neurons from dyingin the age of 120 days The group reported that creatine wasalso helpful in saving the mice from oxidative stress as well[132] Later in 2004 a translational study performed with thesame idea in human subjects demonstrated totally oppositeresults 175 probable laboratory supported ALS patients wereadministered the 10 gm dose of creatine daily The studyshowed no effect on survival rate neither it helped in revivingthe rate of functional activities in patients [133] Cannabinoidanother naturally produced chemical present in humans aswell as animals was studied by a group in 2004 in ALS mice


modelThey reported that Cannabinoid helped in prolongingsurvival of animals Authors also reported reduced oxidativedamage in spinal cord cell cultures of ALS mice and showedthat it acts as an antiexcitotoxic agent in vitro [134] Similarresults have been shown in case of synthetically producedchemical called as cannabinol with dosage of 5mgkgday forover a period of 12 weeks although no effect was there onsurvival [135] Details for the functioning of these agents havenot been mentioned but all of them report to choose or affectthe oxidative stress pathway although the oxidative stressbased stimulation pathway by these compounds for VEGFcannot be ignored Several studies report a common path forVEGF enhanced expression but validity of usefulness of thesenatural components in case of ALS still needed to be studied

9 Concluding Remarks

The human animal and culture studies have shown thatVEGF could be a promising therapeutic target inALS Upreg-ulation of VEGF by different means such as genetic engi-neering transplantation of stem cells overexpressing VEGFandor direct infusion of VEGF may rescue the damage ofmotor neurons and enhance the survival of patients with ALSeither by increasing blood perfusion or direct neuroprotec-tive effect on motor neurons However additional blindedpreclinical studies ofVEGF particularly among primates arestill needed in ALS and other neurodegenerative disordersincluding Alzheimerrsquos and Parkinsonrsquos disease before startingclinical trials Regardless of the conflicting reports describingthe role of oxidative stress and role of VEGF in various ALSinvestigations both human and in vivo studies suffer fromlongitudinal analysis including the prospective nutritionalinterventional studies Besides the patient oriented geneticprofiling studies have failed to include large cohort of homo-geneous populations thus impacting the understanding ofthe demographic-SNP link in motor neuron degenerationNonpharmacological therapeutic approaches inALS have notbeen adequately addressed and need new research focus fordevelopment of therapeutics

Acknowledgments

Akshay Anand and Keshav Thakur are the first authors Theauthors thank Dr Peter Carmeliet and Dr Strokebaum forreviewing the paper and providing their expert comments

References

[1] J M James C Gewolb and V L Bautch ldquoNeurovasculardevelopment uses VEGF-A signaling to regulate blood vesselingression into the neural tuberdquo Development vol 136 no 5pp 833ndash841 2009

[2] V L Bautch and J M James ldquoNeurovascular developmentthe beginning of a beautiful friendshiprdquo Cell Adhesion andMigration vol 3 no 2 pp 199ndash204 2009

[3] J H McCarty ldquoIntegrin-mediated regulation of neurovasculardevelopment physiology anddiseaserdquoCell Adhesion andMigra-tion vol 3 no 2 pp 211ndash215 2009

[4] D Lambrechts and P Carmeliet ldquoVEGF at the neurovascularinterface therapeutic implications for motor neuron diseaserdquoBiochimica et Biophysica Acta vol 1762 no 11-12 pp 1109ndash11212006

[5] D C Dugdale D B Hoch and D Zieve Amyotrophic LateralSclerosis ADAM Medical Encyclopedia 2010

[6] B Oosthuyse L Moons E Storkebaum et al ldquoDeletion of thehypoxia-response element in the vascular endothelial growthfactor promoter causes motor neuron degenerationrdquo NatureGenetics vol 28 no 2 pp 131ndash138 2001

[7] K R Mills ldquoCharacteristics of fasciculations in amyotrophiclateral sclerosis and the benign fasciculation syndromerdquo Brainvol 133 no 11 pp 3458ndash3469 2010

[8] G C Roman ldquoNeuroepidemiology of amyotrophic lateral scle-rosis clues to aetiology and pathogenesisrdquo Journal of NeurologyNeurosurgery and Psychiatry vol 61 no 2 pp 131ndash137 1996

[9] D R Rosen T Siddique D Patterson et al ldquoMutations inCuZn superoxide dismutase gene are associated with familialamyotrophic lateral sclerosisrdquoNature vol 362 no 6415 pp 59ndash62 1993

[10] L E Cox L Ferraiuolo E F Goodall et al ldquoMutations inCHMP2B in lower motor neuron predominant amyotrophiclateral sclerosis (ALS)rdquo PLoS ONE vol 5 no 3 article e98722010

[11] P F Chance B A Rabin S G Ryan et al ldquoLinkage of the genefor an autosomal dominant formof juvenile amyotrophic lateralsclerosis to chromosome 9q34rdquoTheAmerican Journal ofHumanGenetics vol 62 no 3 pp 633ndash640 1998

[12] Y Z Chen C L Bennett H M Huynh et al ldquoDNARNAhelicase gene mutations in a form of juvenile amyotrophiclateral sclerosis (ALS4)rdquo The American Journal of HumanGenetics vol 74 no 6 pp 1128ndash1135 2004

[13] A L Nishimura M Mitne-Neto H C A Silva et al ldquoA muta-tion in the vesicle-trafficking protein VAPB causes late-onsetspinal muscular atrophy and amyotrophic lateral sclerosisrdquoTheAmerican Journal of Human Genetics vol 75 no 5 pp 822ndash8312004

[14] I Puls C Jonnakuty B H LaMonte et al ldquoMutant dynactin inmotor neuron diseaserdquo Nature Genetics vol 33 no 4 pp 455ndash456 2003

[15] R Fernandez-Santiago S Hoenig P Lichtner et al ldquoIdentifi-cation of novel Angiogenin (ANG) gene missense variants inGerman patients with amyotrophic lateral sclerosisrdquo Journal ofNeurology vol 256 no 8 pp 1337ndash1342 2009

[16] S Cronin M J Greenway S Ennis et al ldquoElevated serumangiogenin levels in ALSrdquo Neurology vol 67 no 10 pp 1833ndash1836 2006

[17] KKishimoto S Yoshida S Ibaragi et al ldquoHypoxia-induced up-regulation of angiogenin besides VEGF is related to progres-sion of oral cancerrdquoOral Oncology vol 48 no 11 pp 1120ndash11272012

[18] D Lambrechts P Lafuste P Carmeliet and E M ConwayldquoAnother angiogenic gene linked to amyotrophic lateral sclero-sisrdquo Trends in Molecular Medicine vol 12 no 8 pp 345ndash3472006

[19] L H Barbeito M Pehar P Cassina et al ldquoA role for astrocytesin motor neuron loss in amyotrophic lateral sclerosisrdquo BrainResearch Reviews vol 47 no 1ndash3 pp 263ndash274 2004

[20] A Plaitakis and J T Caroscio ldquoAbnormal glutamatemetabolism in amyotrophic lateral sclerosisrdquo Annals ofNeurology vol 22 no 5 pp 575ndash579 1987


[21] B K Kaspar J Llado N Sherkat J D Rothstein and FH GageldquoRetrograde viral delivery of IGF-1 prolongs survival in amouseALS modelrdquo Science vol 301 article 5634 pp 839ndash842 2003

[22] I Niebroj-Dobosz Z Jamrozik P Janik I Hausmanowa-Petrusewicz and H Kwiecinski ldquoAnti-neural antibodies inserum and cerebrospinal fluid of amyotrophic lateral sclerosis(ALS) patientsrdquo Acta Neurologica Scandinavica vol 100 no 4pp 238ndash243 1999

[23] S J Murch P A Cox S A Banack J C Steele and O WSacks ldquoOccurrence of 120573-methylamino-L-alanine (BMAA) inALSPDCpatients fromGuamrdquoActaNeurologica Scandinavicavol 110 no 4 pp 267ndash269 2004

[24] P J Shaw and C J Eggett ldquoMolecular factors underlyingselective vulnerability of motor neurons to neurodegenerationin amyotrophic lateral sclerosisrdquo Journal of Neurology vol 247supplement 1 pp I17ndashI27 2000

[25] G Bensimon L Lacomblez and V Meininger ldquoA controlledtrial of riluzole in amyotrophic lateral sclerosis ALSRiluzoleStudy Grouprdquo The New England Journal of Medicine vol 330no 9 pp 585ndash591 1994

[26] L Lacomblez G Bensimon P N Leigh P Guillet and VMeininger ldquoDose-ranging study of riluzole in amyotrophiclateral sclerosis Amyotrophic Lateral SclerosisRiluzole StudyGroup IIrdquoThe Lancet vol 347 no 9013 pp 1425ndash1431 1996

[27] M H Yoo H J Hyun J Y Koh and Y H Yoon ldquoRiluzoleinhibits VEGF-induced endothelial cell proliferation in vitroand hyperoxia-induced abnormal vessel formation in vivordquoInvestigative Ophthalmology and Visual Science vol 46 no 12pp 4780ndash4787 2005

[28] T P Obrenovitch ldquoAmyotrophic lateral sclerosis excitotoxicityand riluzolerdquo Trends in Pharmacological Sciences vol 19 no 1pp 9ndash11 1998

[29] H Takahashi andM Shibuya ldquoThe vascular endothelial growthfactor (VEGF)VEGF receptor system and its role under physi-ological and pathological conditionsrdquo Clinical Science vol 109no 3 pp 227ndash241 2005

[30] F Shalaby J Rossant T P Yamaguchi et al ldquoFailure of blood-island formation and vasculogenesis in Flk-1 deficient micerdquoNature vol 376 no 6535 pp 62ndash66 1995

[31] S Hiratsuka O Minowa J Kuno T Noda and M ShibuyaldquoFlt-1 lacking the tyrosine kinase domain is sufficient for normaldevelopment and angiogenesis in micerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 95 no 16 pp 9349ndash9354 1998

[32] H Fujisawa ldquoRoles of neuropilinneuropilin and plexin in thedevelopment of nervous systemrdquo Tanpakushitsu kakusan kosovol 42 no 3 supplement 1 pp 584ndash588 1997

[33] G Neufeld T Cohen N Shraga T Lange O Kessler andY Herzog ldquoThe neuropilins multifunctional semaphorin andVEGF receptors that modulate axon guidance and angiogene-sisrdquo Trends in Cardiovascular Medicine vol 12 no 1 pp 13ndash192002

[34] A-K Olsson A Dimberg J Kreuger and L Claesson-WelshldquoVEGF receptor signallingmdashin control of vascular functionrdquoNature Reviews Molecular Cell Biology vol 7 no 5 pp 359ndash3712006

[35] S Raab and K H Plate ldquoDifferent networks common growthfactors shared growth factors and receptors of the vascular andthe nervous systemrdquo Acta Neuropathologica vol 113 no 6 pp607ndash626 2007

[36] F Lennmyr K A Ata K Funa Y Olsson and A TerentldquoExpression of vascular endothelial growth factor (VEGF) and

its receptors (Flt-1 and Flk-1) following permanent and transientocclusion of the middle cerebral artery in the ratrdquo Journal ofNeuropathology and Experimental Neurology vol 57 no 9 pp874ndash882 1998

[37] X Yang andC L Cepko ldquoFlk-1 a receptor for vascular endothe-lial growth factor (VEGF) is expressed by retinal progenitorcellsrdquo Journal of Neuroscience vol 16 no 19 pp 6089ndash60991996

[38] J Iłzecka ldquoCerebrospinal fluid vascular endothelial growthfactor in patients with amyotrophic lateral sclerosisrdquo ClinicalNeurology and Neurosurgery vol 106 no 4 pp 289ndash293 2004

[39] D P McCloskey T M Hintz and H E Scharfman ldquoModula-tion of vascular endothelial growth factor (VEGF) expressionin motor neurons and its electrophysiological effectsrdquo BrainResearch Bulletin vol 76 no 1-2 pp 36ndash44 2008

[40] E Bogaert P van Damme K Poesen et al ldquoVEGF protectsmotor neurons against excitotoxicity by upregulation of GluR2rdquoNeurobiology of Aging vol 31 no 12 pp 2185ndash2191 2010

[41] DDevos CMoreau P Lassalle et al ldquoLow levels of the vascularendothelial growth factor in CSF from early ALS patientsrdquoNeurology vol 62 no 11 pp 2127ndash2129 2004

[42] C Moreau D Devos V Brunaud-Danel et al ldquoParadoxicalresponse ofVEGF expression to hypoxia in CSF of patients withALSrdquo Journal of Neurology Neurosurgery and Psychiatry vol 77no 2 pp 255ndash257 2006

[43] T Nagata I Nagano M Shiote et al ldquoElevation of MCP-1 andMCP-1VEGF ratio in cerebrospinal fluid of amyotrophic lateralsclerosis patientsrdquoNeurological Research vol 29 no 8 pp 772ndash776 2007

[44] M Suzuki T Watanabe H Mikami et al ldquoImmunohisto-chemical studies of vascular endothelial growth factor in skinof patients with amyotrophic lateral sclerosisrdquo Journal of theNeurological Sciences vol 285 no 1-2 pp 125ndash129 2009

[45] P K Gupta S Prabhakar C Abburi N K Sharma and AAnand ldquoVascular endothelial growth factor-A and chemokineligand (CCL2) genes are upregulated in peripheral bloodmononuclear cells in Indian amyotrophic lateral sclerosispatientsrdquo Journal of Neuroinflammation vol 8 article 114 2011

[46] P K Gupta S Prabhakar S Sharma and A Anand ldquoVascularendothelial growth factor-A (VEGF-A) and chemokine ligand-2(CCL2) in amyotrophic lateral sclerosis (ALS) patientsrdquo Journalof Neuroinflammation vol 8 article 97 2011

[47] P K Gupta S Prabhakar S Sharma and A Anand ldquoA predic-tive model for amyotrophic lateral sclerosis (ALS) diagnosisrdquoJournal of the Neurological Sciences vol 312 no 1-2 pp 68ndash722012

[48] A Nalini K Thennarasu M Gourie-Devi S Shenoy and DKulshreshtha ldquoClinical characteristics and survival pattern of1153 patientswith amyotrophic lateral sclerosis experience over30 years from Indiardquo Journal of the Neurological Sciences vol272 no 1-2 pp 60ndash70 2008

[49] W G Bradley ldquoCommentary on Professor Stephen Hawkingrsquosdisability advicerdquo Annals of Neurosciences vol 16 pp 101ndash1022009

[50] A Anand P K Gupta N K Sharma and S Prabhakar ldquoSolubleVEGFR1 (sVEGFR1) as a novel marker of amyotrophic lateralsclerosis (ALS) in theNorth IndianALS patientsrdquoTheEuropeanJournal of Neurology vol 19 no 5 pp 788ndash792 2012

[51] J H P Skene and D W Cleveland ldquoHypoxia and lou gehrigrdquoNature Genetics vol 28 no 2 pp 107ndash108 2001


[52] E Storkebaum D Lambrechts and P Carmeliet ldquoVEGF onceregarded as a specific angiogenic factor now implicated inneuroprotectionrdquo BioEssays vol 26 no 9 pp 943ndash954 2004

[53] G AlievM A SmithM E Obrenovich J C de la Torre andGPerry ldquoRole of vascular hypoperfusion-induced oxidative stressand mitochondria failure in the pathogenesis of Alzheimerdiseaserdquo Neurotoxicity Research vol 5 no 7 pp 491ndash504 2003

[54] K Orino L Lehman Y Tsuji H Ayaki S V Torti and F MTorti ldquoFerritin and the response to oxidative stressrdquoBiochemicalJournal vol 357 no 1 pp 241ndash247 2001

[55] J Harned J Ferrell M M Lall et al ldquoAltered ferritin subunitcomposition change in iron metabolism in lens epithelialcells and downstream effects on glutathione levels and VEGFsecretionrdquo Investigative Ophthalmology and Visual Science vol51 no 9 pp 4437ndash4446 2010

[56] J S Henkel J I Engelhardt S L Siklos et al ldquoPresence ofdendritic cells MCP-1 and activated MicrogliaMacrophagesin amyotrophic Lateral sclerosis spinal cord tissuerdquo Annals ofNeurology vol 55 no 2 pp 221ndash235 2004

[57] C Maihofner S Probst-Cousin M Bergmann W NeuhuberB Neundorfer and D Heuss ldquoExpression and localization ofcyclooxygenase-1 and -2 in human sporadic amyotrophic lateralsclerosisrdquo The European Journal of Neuroscience vol 18 no 6pp 1527ndash1534 2003

[58] W G M Spliet E Aronica M Ramkema J Aten andD Troost ldquoIncreased expression of connective tissue growthfactor in amyotrophic lateral sclerosis human spinal cordrdquo ActaNeuropathologica vol 106 no 5 pp 449ndash457 2003

[59] W G M Spliet E Aronica M Ramkema et al ldquoImmunohis-tochemical localization of vascular endothelial growth factorreceptors-1 -2 and -3 in human spinal cord altered expressionin amyotrophic lateral sclerosisrdquo Neuropathology and AppliedNeurobiology vol 30 no 4 pp 351ndash359 2004

[60] SMalessa P N Leigh O Bertel E Sluga andOHornykiewiczldquoAmyotrophic lateral sclerosis glutamate dehydrogenase andtransmitter amino acids in the spinal cordrdquo Journal of NeurologyNeurosurgery and Psychiatry vol 54 no 11 pp 984ndash988 1991

[61] M V Sofroniew ldquoMolecular dissection of reactive astrogliosisand glial scar formationrdquo Trends in Neurosciences vol 32 no12 pp 638ndash647 2009

[62] B A Barres ldquoThe mystery and magic of Glia a perspective ontheir roles in health and diseaserdquoNeuron vol 60 no 3 pp 430ndash440 2008

[63] M V Sofroniew ldquoReactive astrocytes in neural repair andprotectionrdquo Neuroscientist vol 11 no 5 pp 400ndash407 2005

[64] M Martineau G Baux and J-P Mothet ldquoGliotransmission atcentral glutamatergic synapses D-serine on stagerdquo Journal ofPhysiology vol 1 pp 211ndash217 2006

[65] Q Zhang and P G Haydon ldquoRoles for gliotransmission in thenervous systemrdquo Journal of Neural Transmission vol 112 no 1pp 121ndash125 2005

[66] P Li and M Zhuo ldquoCholinergic noradrenergic and seroton-ergic inhibition of fast synaptic transmission in spinal lumbardorsal horn of ratrdquo Brain Research Bulletin vol 54 no 6 pp639ndash647 2001

[67] G Karpati G Klassen and P Tanser ldquoThe effects of partialchronic denervation on forearm metabolismrdquo Canadian Jour-nal of Neurological Sciences vol 6 no 2 pp 105ndash112 1979

[68] T T Rissanen I Vajanto M O Hiltunen et al ldquoExpressionof vascular endothelial growth factor and vascular endothelialgrowth factor receptor-2 (KDRFik-1) in ischemic skeletal

muscle and its regenerationrdquoTheAmerican Journal of Pathologyvol 160 no 4 pp 1393ndash1403 2002

[69] I Nygren A Larsson A Johansson and H Askmark ldquoVEGFis increased in serum but not in spinal cord from patients withamyotrophic lateral sclerosisrdquo NeuroReport vol 13 no 17 pp2199ndash2201 2002

[70] K Vijayalakshmi P A Alladi T N Sathyaprabha J RSubramaniam A Nalini and T R Raju ldquoCerebrospinal fluidfrom sporadic amyotrophic lateral sclerosis patients inducesdegeneration of a cultured motor neuron cell linerdquo BrainResearch vol 1263 pp 122ndash133 2009

[71] V Crugnola C Lamperti V Lucchini et al ldquoMitochondrialrespiratory chain dysfunction in muscle from patients withamyotrophic lateral sclerosisrdquo Archives of Neurology vol 67 no7 pp 849ndash854 2010

[72] S Vielhaber D Kunz K Winkler et al ldquoMitochondrial DNAabnormalities in skeletal muscle of patients with sporadicamyotrophic lateral sclerosisrdquo Brain vol 123 no 7 pp 1339ndash1348 2000

[73] A Krasnianski M Deschauer S Neudecker et al ldquoMitochon-drial changes in skeletal muscle in amyotrophic lateral sclerosisand other neurogenic atrophiesrdquo Brain vol 128 no 8 pp 1870ndash1876 2005

[74] B M Kust J C W M Copray N Brouwer D Troost andH W G M Boddeke ldquoElevated levels of neurotrophins inhuman biceps brachii tissue of amyotrophic lateral sclerosisrdquoExperimental Neurology vol 177 no 2 pp 419ndash427 2002

[75] A Valavanidis T Vlachogianni and C Fiotakis ldquo8-hydroxy-21015840-deoxyguanosine (8-OHdG) a critical biomarker of oxidativestress and carcinogenesisrdquo Journal of Environmental Science andHealth C vol 27 no 2 pp 120ndash139 2009

[76] S-K Chen W A Hsieh M-H Tsai et al ldquoAge-associateddecrease of oxidative repair enzymes human 8-oxoguanineDNA glycosylases (hOGG1) in human agingrdquo Journal of Radi-ation Research vol 44 no 1 pp 31ndash35 2003

[77] F Coppede M Mancuso A L Gerfo et al ldquoAssociation of thehOGG1 Ser326Cys polymorphism with sporadic amyotrophiclateral sclerosisrdquo Neuroscience Letters vol 420 no 2 pp 163ndash168 2007

[78] A Brockington J Kirby D Eggitt et al ldquoScreening of theregulatory and coding regions of vascular endothelial growthfactor in amyotrophic lateral sclerosisrdquoNeurogenetics vol 6 no2 pp 101ndash104 2005

[79] F Gros-Louis S Laurent A A S Lopes et al ldquoAbsence ofmutations in the hypoxia response element of VEGF in ALSrdquoMuscle and Nerve vol 28 no 6 pp 774ndash775 2003

[80] W Chen M Saeed H Mao et al ldquoLack of association of VEGFpromoter polymorphisms with sporadic ALSrdquo Neurology vol67 no 3 pp 508ndash510 2006

[81] P W J van Vught N A Sutedja J H Veldink et al ldquoLack ofassociation betweenVEGF polymorphisms and ALS in a Dutchpopulationrdquo Neurology vol 65 no 10 pp 1643ndash1645 2005

[82] Y Zhang H Zhang Y Fu et al ldquoVEGF C2578A polymorphismdoes not contribute to amyotrophic lateral sclerosis susceptibil-ity in sporadic Chinese patientsrdquo Amyotrophic Lateral Sclerosisvol 7 no 2 pp 119ndash122 2006

[83] D Lambrechts K Poesen R Fernandez-Santiago et al ldquoMeta-analysis of vascular endothelial growth factor variations inamyotrophic lateral sclerosis increased susceptibility in malecarriers of the -2578AA genotyperdquo Journal of Medical Geneticsvol 46 no 12 pp 840ndash846 2009


[84] R Fernandez-SantiagoM Sharma J CMueller et al ldquoPossiblegender-dependent association of vascular endothelial growthfactor (VEGF) gene and ALSrdquo Neurology vol 66 no 12 pp1929ndash1931 2006

[85] N Oates and R Pamphlett ldquoAn epigenetic analysis of SOD1 andVEGF in ALSrdquo Amyotrophic Lateral Sclerosis vol 8 no 2 pp83ndash86 2007

[86] A Brockington B Wokke H Nixon J A Hartley and P JShaw ldquoScreening of the transcriptional regulatory regions ofvascular endothelial growth factor receptor 2 (VEGFR2) inamyotrophic lateral sclerosisrdquo BMC Medical Genetics vol 8article 23 2007

[87] L A Zilrsquober Z L Bajdakova A N Gardasjan N V KonovalovT L Bunina and E M Barabadze ldquoStudy of the etiologyof amyotrophic lateral sclerosisrdquo Bulletin of the World HealthOrganization vol 29 no 4 pp 449ndash456 1963

[88] R A Smith F M Balis K H Ott D D Elsberry M R Sher-man and M G P Saifer ldquoPharmacokinetics and tolerability ofventricularly administered superoxide dismutase in monkeysand preliminary clinical observations in familial ALSrdquo Journalof the Neurological Sciences vol 129 supplement 1 pp 13ndash181995

[89] E StorkebaumD LambrechtsM Dewerchin et al ldquoTreatmentof motoneuron degeneration by intracerebroventricular deliv-ery ofVEGF in a rat model of ALSrdquoNature Neuroscience vol 8no 1 pp 85ndash92 2005

[90] D H Hwang H J Lee I H Park et al ldquoIntrathecal transplan-tation of human neural stem cells overexpressingVEGF providebehavioral improvement disease onset delay and survivalextension in transgenic ALS micerdquo Gene Therapy vol 16 no10 pp 1234ndash1244 2009

[91] D Lambrechts E Storkebaum M Morimoto et al ldquoVEGFis a modifier of amyotrophic lateral sclerosis in mice andhumans and protects motoneurons against ischemic deathrdquoNature Genetics vol 34 no 4 pp 383ndash394 2003

[92] Y Sun K Jin L Xie et al ldquoVEGF-induced neuroprotectionneurogenesis and angiogenesis after focal cerebral ischemiardquoJournal of Clinical Investigation vol 111 no 12 pp 1843ndash18512003

[93] C Zheng I Nennesmo B Fadeel and J I Henter ldquoVascularendothelial growth factor prolongs survival in a transgenicmouse model of ALSrdquo Annals of Neurology vol 56 no 4 pp564ndash567 2004

[94] J Widenfalk A Lipson M Jubran et al ldquoVascular endothelialgrowth factor improves functional outcome and decreasessecondary degeneration in experimental spinal cord contusioninjuryrdquo Neuroscience vol 120 no 4 pp 951ndash960 2003

[95] M A Kliem B L Heeke C K Franz et al ldquoIntramuscularadministration of a VEGF zinc finger transcription factoractivator (VEGF-ZFP-TF) improves functional outcomes inSOD1 ratsrdquo Amyotrophic Lateral Sclerosis vol 12 no 5 pp 331ndash339 2011

[96] D F Silva D L Porto I G A Araujo et al ldquoEndothelium-derived nitric oxide is involved in the hypotensive and vasore-laxant effects induced by discretamine in ratsrdquo Pharmazie vol64 no 5 pp 327ndash331 2009

[97] M Kobari K Obara S Watanabe T Dembo and Y FukuuchildquoLocal cerebral blood flow inmotor neuron disease correlationwith clinical findingsrdquo Journal of the Neurological Sciences vol144 no 1-2 pp 64ndash69 1996

[98] A G Barbeito L Martinez-Palma M R Vargas et al ldquoLeadexposure stimulates VEGF expression in the spinal cord and

extends survival in a mouse model of ALSrdquo Neurobiology ofDisease vol 37 no 3 pp 574ndash580 2010

[99] M Azzouz G S Ralph E Storkebaum et al ldquoVEGF deliverywith retrogradely transported lentivector prolongs survival in amouse ALS modelrdquo Nature vol 429 article 6990 pp 413ndash4172004

[100] I Ay JW Francis and R H Brown Jr ldquoVEGF increases blood-brain barrier permeability to Evans blue dye and tetanus toxinfragment C but not adeno-associated virus in ALS micerdquo BrainResearch vol 1234 pp 198ndash205 2008

[101] Z Zhong R Deane Z Ali et al ldquoALS-causing SOD1 mutantsgenerate vascular changes prior tomotor neuron degenerationrdquoNature Neuroscience vol 11 no 4 pp 420ndash422 2008

[102] V Silani M Braga A Ciammola V Cardin and G ScarlatoldquoMotor neurones in culture as a model to study ALSrdquo Journal ofNeurology vol 247 supplement 1 pp I28ndashI36 2000

[103] L Lu L Zheng L Viera et al ldquoMutant CuZn-superoxidedismutase associated with amyotrophic lateral sclerosis desta-bilizes vascular endothelial growth factor mRNA and downreg-ulates its expressionrdquo Journal of Neuroscience vol 27 no 30 pp7929ndash7938 2007

[104] A B Scandurro and B S Beckman ldquoCommon proteins bindmRNAs encoding erythropoietin tyrosine hydroxylase andvascular endothelial growth factorrdquoBiochemical and BiophysicalResearch Communications vol 246 no 2 pp 436ndash440 1998

[105] B Li W Xu C Luo D Gozal and R Liu ldquoVEGF-inducedactivation of the PI3-KAkt pathway reduces mutant SOD1-mediated motor neuron cell deathrdquo Molecular Brain Researchvol 111 no 1-2 pp 155ndash164 2003

[106] J S Lunn S A Sakowski B Kim A A Rosenberg and E LFeldman ldquoVascular endothelial growth factor prevents G93A-SOD1-induced motor neuron degenerationrdquo DevelopmentalNeurobiology vol 69 no 13 pp 871ndash884 2009

[107] M J During and L Cao ldquoVEGF a mediator of the effect ofexperience on hippocampal neurogenesisrdquo Current AlzheimerResearch vol 3 no 1 pp 29ndash33 2006

[108] H Meng Z Zhang R Zhang et al ldquoBiphasic effects of exoge-nous VEGF on VEGF expression of adult neural progenitorsrdquoNeuroscience Letters vol 393 no 2-3 pp 97ndash101 2006

[109] A A Rizvanov A P Kiyasov I M Gaziziov et al ldquoHumanumbilical cord blood cells transfected with VEGF and L1CAMdo not differentiate into neurons but transform into vascularendothelial cells and secrete neuro-trophic factors to supportneuro-genesis-a novel approach in stem cell therapyrdquo Neuro-chemistry International vol 53 no 6ndash8 pp 389ndash394 2008

[110] K Poesen D Lambrechts P van Damme et al ldquoNovel rolefor vascular endothelial growth factor (VEGF) receptor-1 andits ligand VEGF-B in motor neuron degenerationrdquo Journal ofNeuroscience vol 28 no 42 pp 10451ndash10459 2008

[111] Y Persidsky S H Ramirez J Haorah and G D KanmogneldquoBlood-brain barrier structural components and functionunder physiologic and pathologic conditionsrdquo Journal of Neu-roimmune Pharmacology vol 1 no 3 pp 223ndash236 2006

[112] H J Schluesener R A Sobel C Linington and H L WeinerldquoA monoclonal antibody against a myelin oligodendrocyteglycoprotein induces relapses and demyelination in centralnervous system autoimmune diseaserdquo Journal of Immunologyvol 139 no 12 pp 4016ndash4021 1987


[113] A Germano M Caffo F F Angileri et al ldquoNMDA receptorantagonist felbamate reduces behavioral deficits and blood-brain barrier permeability changes after experimental sub-arachnoid hemorrhage in the ratrdquo Journal of Neurotrauma vol24 no 4 pp 732ndash744 2007

[114] D Graesser A Solowiej M Bruckner et al ldquoAltered vascularpermeability and early onset of experimental autoimmuneencephalomyelitis in PECAM-1-deficient micerdquo Journal of Clin-ical Investigation vol 109 no 3 pp 383ndash392 2002

[115] V Bartanusz D Jezova B Alajajian andM Digicaylioglu ldquoTheblood-spinal cord barrier morphology and clinical implica-tionsrdquo Annals of Neurology vol 70 no 2 pp 194ndash206 2011

[116] S Garbuzova-Davis S Saporta E Haller et al ldquoEvidenceof compromised blood-spinal cord barrier in early and latesymptomatic SOD1 mice modeling ALSrdquo PLoS ONE vol 2 no11 article e1205 2007

[117] H Wolburg K Wolburg-Buchholz J Kraus et al ldquoLocal-ization of claudin-3 in tight junctions of the blood-brainbarrier is selectively lost during experimental autoimmuneencephalomyelitis and human glioblastoma multiformerdquo ActaNeuropathologica vol 105 no 6 pp 586ndash592 2003

[118] E Ambrosini M E Remoli E Giacomini et al ldquoAstrocytesproduce dendritic cell-attracting chemokines in vitro and inmultiple sclerosis lesionsrdquo Journal of Neuropathology and Exper-imental Neurology vol 64 no 8 pp 706ndash715 2005

[119] A T Argaw L Asp J Zhang et al ldquoAstrocyte derived VEGF-A drives blood brain barrier disruption in CNS inflammatorydiseaserdquo Journal of Clinical Investigation vol 122 no 7 pp2454ndash2468 2012

[120] S Esser M G Lampugnani M Corada E Dejana andW Risau ldquoVascular endothelial growth factor induces VE-cadherin tyrosinerdquo Journal of Cell Science vol 111 no 13 pp1853ndash1865 1998

[121] K Zhang J Lu T Mori et al ldquoBaicalin increases VEGFexpression and angiogenesis by activating the ERR120572PGC-1120572pathwayrdquo Cardiovascular Research vol 89 no 2 pp 426ndash4352011

[122] R B Vega J M Huss and D P Kelly ldquoThe coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor120572 in transcriptional control of nuclear genes encoding mito-chondrial fatty acid oxidation enzymesrdquoMolecular and CellularBiology vol 20 no 5 pp 1868ndash1876 2000

[123] Z Arany ldquoPGC-1 coactivators and skeletal muscle adaptationsin health and diseaserdquo Current Opinion in Genetics and Devel-opment vol 18 no 5 pp 426ndash434 2008

[124] Z Arany S Y Foo Y Ma et al ldquoHIF-independent regulation ofVEGF and angiogenesis by the transcriptional coactivator PGC-1120572rdquo Nature vol 451 article 7181 pp 1008ndash1012 2008

[125] R P Singh A K Tyagi S Dhanalakshmi R Agarwal and C NAgarwal ldquoGrape seed extract inhibits advanced human prostatetumor growth and angiogenesis and upregulates insulin-likegrowth factor binding protein-3rdquo International Journal of Can-cer vol 108 no 5 pp 733ndash740 2004

[126] MKaur R P SinghMGu RAgarwal andCAgarwal ldquoGrapeseed extract inhibits in vitro and in vivo growth of humancolorectal carcinoma cellsrdquoClinical Cancer Research vol 12 no20 pp 6194ndash6202 2006

[127] M Arii ldquoChemopreventive effect of grape seed extract onintestinal carcinogenesis in the APCMinmouserdquo Proceedings ofthe American Association for Cancer Research vol 39 article 201998

[128] J Lu K Zhang S Chen and W Wen ldquoGrape seed extractinhibits VEGF expression via reducing HIF-1120572 protein expres-sionrdquo Carcinogenesis vol 30 no 4 pp 636ndash644 2009

[129] C Desnuelle M Dib C Garrel and A Favier ldquoA double-blindplaceho-controlled randomized clinical trial of 120572-tocopherol(vitamin E) in the treatment of amyotrophic lateral sclerosisALS riluzole-tocopherol Study Grouprdquo Amyotrophic LateralSclerosis and Other Motor Neuron Disorders vol 2 no 1 pp 9ndash18 2001

[130] M Graf D Ecker R Horowski et al ldquoHigh dose vitamin Etherapy in amyotrophic lateral sclerosis as add-on therapy toriluzole results of a placebo-controlled double-blind studyrdquoJournal of Neural Transmission vol 112 no 5 pp 649ndash6602005

[131] RW Orrell R J M Lane andM Ross ldquoA systematic review ofantioxidant treatment for amyotrophic lateral sclerosismotorneuron diseaserdquo Amyotrophic Lateral Sclerosis vol 9 no 4 pp195ndash211 2008

[132] PKlivenyi R J Ferrante R TMatthews et al ldquoNeuroprotectiveeffects of creatine in a transgenic animal model of amyotrophiclateral sclerosisrdquo Nature Medicine vol 5 no 3 pp 347ndash3501999

[133] G J Groeneveld J H Veldink I van der Tweel et al ldquoArandomized sequential trial of creatine in amyotrophic lateralsclerosisrdquo Annals of Neurology vol 53 no 4 pp 437ndash445 2003

[134] C Raman S D McAllister G Rizvi S G Patel D H Mooreand M E Abood ldquoAmyotrophic lateral sclerosis delayeddisease progression in mice by treatment with a cannabinoidrdquoAmyotrophic Lateral Sclerosis and Other Motor Neuron Disor-ders vol 5 no 1 pp 33ndash39 2004

[135] PWeydt S Hong AWitting T Moller N Stella andM KliotldquoCannabinol delays symptom onset in SOD1 (G93A) transgenicmice without affecting survivalrdquo Amyotrophic Lateral Sclerosisand Other Motor Neuron Disorders vol 6 no 3 pp 182ndash1842005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


ANGANG

ANGNucleus

Endothelial Cell

rRNA

AngiogenesisEvents leading to angiogenesis in cell

Endocytosis

Nuclear ANG

VEGF expression regulated by nuclear ANG

ROS

NO

RNS

Mitochondria

VEGF

ROSHIF-1 alpha

HIF-1 alpha

O2

NF-120581B

Figure 1The role of hypoxia in stimulating theVEGF through an activation ofHIF-1 alpha element HIF-1 alpha gets activated in deficiency ofoxygen in mitochondria leading to creation of oxidative stress This involves the formation of reactive oxygen species which on reaction withfree nitrogen forms NO ultimately leading to reactive nitrogen species (RNS) This RNS further activates NF-120581B pathway which ultimatelyleads to activation of HIF-1 alpha factor The activated form of HIF-1 alpha further leads to VEGF activation thus leading to angiogenesis

or without dysarthria and dysphagia Fasciculation or muscletwitching is also an important clinical finding [7]

3 ALS Contributing Factors

ALS occurs in both sporadic and familial form at an incidencevarying between 04 and 26 for every 100000 individualsand a prevalence rate of 4ndash6 per 100000 population per year[8] The etiology of ALS has been elusive and believed tobe multifactorial Though causes of most cases of ALS areunknown major factors include genetic factors like pointmutations in superoxide dismutase 1 (SOD1) gene accountingfor around 20 of familial ALS (fALS) cases [9] The purelylower motor neuron (LMN) degeneration variant of ALSshows missense mutations in CHMP2B (charged multivesic-ular protein 2B involved in cellular transport) In 10 casesof ALS patients with CHMP2Bmutations are shown to havelower motor neuron degeneration Apart from this othergenes like vesicle-associated membrane protein B (VAPB)(which is involved in providing unfolded protein responseto endoplasmic reticulum) senataxin (SETX) (gene presentin central nervous system involving brain and spinal cord aswell as muscle and play major role in DNA repair to maintainintegrity of cell) and dynactin 1 (involved in cellular transportduring cell division and specially in axonal transport ofnerve cells) with mutations have been shown to play role inaggregate formation andhampering the normal activity of themotor neurons thus contributing to the pathogenesis of ALS

overall in subjectrsquos body [10ndash14] Genes encoding angiogenin(ANG) havingmissense mutations have also been involved inthe pathogenesis of ALS Angiogenin likeVEGF is producedin response to hypoxia and plays a role in neovascularisationas shown in Figure 1 Its importance further stems fromthe fact that it can regulate the expression of VEGF [1516] Hypoxia takes place when oxygen availability is low incell due to which the mitochondria produces ROS specieswhich in turn reacts with nitric oxide (NO) to producereactive nitrogen species RNS and activates HIF-120572 pathwaythrough NF-120581B pathway resulting in stimulation of VEGFThe expression of this VEGF is dependent on the nucleolarANG which directly helps in stimulating the proliferation ofepithelial cells and helps in angiogenesis [17] However thishypothesis raises a question whether angiogenin crosses theblood brain barrier or is retained in cerebrospinal fluid [18]

Apart from genetic factors the presence of insolubleintracellular protein aggregates in motor neurons and reac-tive astrocytes are considered as the hallmarks for the disease(Figure 3) [19] The other factors include glutamate toxicity[20] lack of trophic growth factors [6 21] autoimmunity[22] toxin [23] and susceptibility ofmotor neurons to neuro-degeneration because of their large size and high energydemands [24]

Currently there is no treatment that could substantiallyalleviate the disease burden because of incomplete under-standing of ALS etiology Food and Drug Administration(FDA) has approved only single drug for the treatment of





121119881119864119866119865

145119881119864119866119865

165119881119864119866119865

183119881119864119866119865

189119881119864119866119865

206)








Hypoperfusion


Glucose


No glycolysis


Ferritin

Ferritin



oxidative stress

ROSROS

ROSROS

Oxidative stress

Fe2+

Fe2+ Fe2+ Fe2+

Fe2+

Fe2+Fe2+

Fe3+

Fe3+


Acetyl CoA


ADP

ATP

NAD+










Na+K+

Na+

K+

Astrocyte


homeostasis

Hypoxia

DNA damage

Motor neuron

Motor neuron

Blocking of synapse

formation





cell

Na+

K+K+

K+

K+

K+

K+

K+

K+

K+

K+ K+

K+

K+

K+

Na+K+



































Hypoxia

Astrocytes

PP

P

P

PP

P

P

Phosphorylation


BBBBSCB


HIF

-alp

ha

VEGF-A








Acknowledgments


References




















































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















121119881119864119866119865

145119881119864119866119865

165119881119864119866119865

183119881119864119866119865

189119881119864119866119865

206)








Hypoperfusion


Glucose


No glycolysis


Ferritin

Ferritin



oxidative stress

ROSROS

ROSROS

Oxidative stress

Fe2+

Fe2+ Fe2+ Fe2+

Fe2+

Fe2+Fe2+

Fe3+

Fe3+


Acetyl CoA


ADP

ATP

NAD+










Na+K+

Na+

K+

Astrocyte


homeostasis

Hypoxia

DNA damage

Motor neuron

Motor neuron

Blocking of synapse

formation





cell

Na+

K+K+

K+

K+

K+

K+

K+

K+

K+

K+ K+

K+

K+

K+

Na+K+



































Hypoxia

Astrocytes

PP

P

P

PP

P

P

Phosphorylation


BBBBSCB


HIF

-alp

ha

VEGF-A








Acknowledgments


References




















































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Hypoperfusion


Glucose


No glycolysis


Ferritin

Ferritin



oxidative stress

ROSROS

ROSROS

Oxidative stress

Fe2+

Fe2+ Fe2+ Fe2+

Fe2+

Fe2+Fe2+

Fe3+

Fe3+


Acetyl CoA


ADP

ATP

NAD+










Na+K+

Na+

K+

Astrocyte


homeostasis

Hypoxia

DNA damage

Motor neuron

Motor neuron

Blocking of synapse

formation





cell

Na+

K+K+

K+

K+

K+

K+

K+

K+

K+

K+ K+

K+

K+

K+

Na+K+



































Hypoxia

Astrocytes

PP

P

P

PP

P

P

Phosphorylation


BBBBSCB


HIF

-alp

ha

VEGF-A








Acknowledgments


References




















































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Na+K+

Na+

K+

Astrocyte


homeostasis

Hypoxia

DNA damage

Motor neuron

Motor neuron

Blocking of synapse

formation





cell

Na+

K+K+

K+

K+

K+

K+

K+

K+

K+

K+ K+

K+

K+

K+

Na+K+



































Hypoxia

Astrocytes

PP

P

P

PP

P

P

Phosphorylation


BBBBSCB


HIF

-alp

ha

VEGF-A








Acknowledgments


References




















































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































Hypoxia

Astrocytes

PP

P

P

PP

P

P

Phosphorylation


BBBBSCB


HIF

-alp

ha

VEGF-A








Acknowledgments


References




















































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































Hypoxia

Astrocytes

PP

P

P

PP

P

P

Phosphorylation


BBBBSCB


HIF

-alp

ha

VEGF-A








Acknowledgments


References




















































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Hypoxia

Astrocytes

PP

P

P

PP

P

P

Phosphorylation


BBBBSCB


HIF

-alp

ha

VEGF-A








Acknowledgments


References




















































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Hypoxia

Astrocytes

PP

P

P

PP

P

P

Phosphorylation


BBBBSCB


HIF

-alp

ha

VEGF-A








Acknowledgments


References




















































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References




















































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of















































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Review Article ALS and Oxidative Stress: The Neurovascular ...downloads.hindawi.com/journals/omcl/2013/635831.pdf · ect lower motor neuron (progressive muscular atrophy) or upper