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Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. •...

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James R. Connor, Ph.D. and Stephanie M. Patton, Ph.D. Iron, Hypoxia and RLS
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Page 1: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

JamesR.Connor,Ph.D.andStephanieM.Patton,Ph.D.

Iron,HypoxiaandRLS

Page 2: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

• IntroductionofanewparadigmforBBBtransportandRegulation

BrainIronAcquisitioninRLS:

Page 3: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

Iron

Transferrin

Mitochondria

Tf Receptor

KeyBlood

Brain

Transferrin(Tf)transcytosistheworkingmodelforBBBirontransport:theconduitmodel

? ?

? ?

?Ferritin

Page 4: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

Endosome

Fe2+

TfR

DMT1pH

Fe3+

Fe2+

holoTf

Blood Brain

Astrocyte

Endothelial Cell

IronisreleasedinEC

apoTf

?

Page 5: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

Transwell collagen membrane

BREC

Apical Chamber for additionOf substances for transport

CSFfromnormalorIDmonkeys

CanIronReleasefromtheBBBcellsberegulated?

Page 6: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

25

50

75

100

125

150

59Fe(GammaCounts)

Baseline 1 2 3 4Time (hr)

IDA

IS*

BBBBioassayforCSF

Page 7: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

Iron

CSF

(mcg

/L)

-

0

1

2

3

4

5

6

7

8

Ferr

itin

CSF(

mcg

/L)

Tran

sfer

rin C

SF (

mg/

L)RLSNormal

0

10

20

30

40

50

60

70

80

. # # # # 2 #RLS NormalRLS Normal 0

5

10

15

20

25

30

35

40

45

50

CSFironstatussuggestsbrainisirondeficient:ironlevelsintheCSFnotdecreased:problemorcompensatoryresponse??

Page 8: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

Transwell insert

Microporous membrane

ApicalChamber

BasalChamberforplacementofCSF

BrainMicrovascularEndothelialCell

TransportofTransferrinandIronacrossamodeloftheBBBinRLS

Page 9: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

• CSFfromRLSpatientsdidnotaltertransportorreleaseinaninvitroBBBmodel– IstheprobleminRLSafailuretosignalregardingbrainironstatus?– Isthisrelatedtothesetpointconcept?

• Hemoglobin,notserumferritin,mayfunctionassystemicpredictorforbrainironuptake– Ishypoxicstatusmoreimportantthanironstatusforbrainironuptake?

Summary

Page 10: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

• Redbloodcells(erythrocytes)arethecelltypeinthebloodthatcarryoxygenfromthelungsthroughoutthebody.

• Approximately35%ofthetotalcontentoftheredbloodcellsismadeupofaproteincalledhemoglobin.

• Hemoglobinisametalloprotein,thatusesironasameanstobindoxygenandtransportittothetissues.

• Whenirondeficiencyoccurs,thesynthesisofheme,amajorbuildingblockofhemoglobin,isdecreasedandlessoxygencanbeboundandtransported.

Whatistherelationshipbetweenironandoxygen?

Page 11: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

• TofurtherexploretheimpactofcellularirondeficiencyinRLSneuromelanin cells,weproposedthehypothesisthatthelossofcellularironhomeostasisincellscouldelicitasurvivalresponseandactivationofsurvivalpathways.

• Onesuchsurvivalpathwayisactivationofhypoxiainduciblefactor1(HIF-1),whichregulatesexpressionofanumberofproteinsincludingthoseinvolvedinironhomeostasissuchasTfR,vascularendothelialgrowthfactor,anderythropoietin.

• Ironisaco-factorforHIFdegradation,thereforeirondeficiencywillleadtostabilizationofHIF-1αandactivationoftheHIFpathway.(seeHypoxiaResponsePathwaySchematic)

SoWhyExamineHypoxiaPathway?

Page 12: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation(http://dx.doi.org/10.14348/molcells.2014.0150)

HypoxiaResponsePathway

Page 13: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

•DecreasedoxygenstatushasbeencorrelatedwithdecreasedIRP1levelsaswellasincreasedIRP2levels.(Hansonetal.,1999)

• EpidemiologicstudieshavedemonstratedahigherprevalenceofRLSinpatientsresidinginhigheraltitudes(Sevim etal.,2003andCastilloetal.,2006)

• SkeletalmusclemorphologyofpatientswithRLSdemonstratessignificantlylowerpredictedmaximaloxygenuptake(Larssonetal.,2007)

•IncreasedRLSprevalenceinpatientswithpulmonarydisease,includingchronicobstructivepulmonarydisease(LoCocoetal.,2009)andobstructivesleepapnea(Kapsimalis etal.,2009).

• Mostrecently,adirectclinicalmeasureofhypoxia,decreasedO2partialpressure,hasbeenreportedinthelegsofRLSpatients(Salminen etal.,2014)

PlausibilityoftheHypoxiaParadigm

Page 14: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

INDICATORSOFACTIVATIONOFTHEHYPOXICPATHWAYINTHESUBSTANTIANIGRA OFRLS

Page 15: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

ControlRLS

HIF-1αImmunostaininginSubstantiaNigra

Page 16: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

MICROVESSEL PERTURBATIONSINRLS

Page 17: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

RLS microvessel lysates contain significantly higher HIF-2α expression levels compared to control subjects (p <0.05).

Lines reflect the mean valuesData points are in triplicate

HIF-2αImmunoblotAnalysisofMicrovessel Lysates

Page 18: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

RLS microvessel lysates contain significantly higher vascular endothelial growth factor (VEGF) expression levels compared to control (p <0.00005).

Lines reflect the mean valuesData points are in triplicate

VEGFImmunoblotAnalysisofMicrovesselLysates

Page 19: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

PROTEOMICANALYSISOFTHECEREBROSPINALFLUIDOFPATIENTSWITHRESTLESSLEGSSYNDROME

Page 20: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

We sought to identify proteins that could prove valuable in understanding pathways affected in RLS/WED and identifying targets for treatment strategies.

ReasoningforCSFbiomarkeranalysispilotstudy

Page 21: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

ProteinsLevelsAlteredinRLSCSF

Page 22: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

• ThisstudyprovidedaCSFproteinprofileofRLSinwhichnumerousidentifiedproteins(VitaminDbindingprotein,ProstaglandinD2synthase,cystatinCandβ-hemoglobin)canbetiedtoactivationofthehypoxiaresponsepathway,thusfurthersupportingourconceptofhypoxiaactivationinRLS

• L-PGDSisupregulatedinthebrainsofsubjectswithhypoxic/ischemicencephalopathy(HIE).Also,inamousemodelofHIEthatL-PGDSandHIF-1αexpressionareco-localizedimplyingL-PGDSmayprotectneuronsfromhypoxicinsultbylocalsecretionofPGD2(Jordanetal.,2002).

• Astudyinwhichplasmaproteomesofhumanslivingatsealevelwerecomparedtothoselivingathighaltitude,VitaminDbindingproteinandβ-hemoglobinwerefoundtobeupregulatedinserumasaresultofhypoxia(Ahmadetal.,2013).

• CystatinChasbeendemonstratedtobeproducedinincreasedamountsbythechoroidplexusfollowinghypoxic/ischemicinsult(Palmetal.,1995).

Relationshipofbiomarkerproteinstohypoxia

Page 23: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

ALTERATIONSINHEARTRATE,MINUTEVENTILATIONANDFEMORALARTERYBLOODFLOWINRLS:APILOTSTUDY

Page 24: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

Alterations in Femoral Artery Blood Flow. This figure demonstrates the femoral artery blood flow in WED (blue) and control (orange) subjects. Error bars are standard error of the mean. n=18 (9 WED; 9 age- and gender-matched controls).

•Baseline femoral artery blood flow is increased 22.5% in RLS/WED subjects.

•RLS/WED subjects demonstrate only a 1.7% increase in blood flow following exposure to hypoxia.

•Control subjects demonstrate a 7.2% increase in blood flow following hypoxia.

FemoralArteryBloodFlowwithHypoxia

Page 25: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

Alterations in Heart Rate. This figure demonstrates the heart rate measurements in RLS/WED (blue) and control (orange) subjects. As shown in this figure, baseline heart rate is increased slightly, but not significantly in RLS/WED subjects as compared to their age-matched controls. Error bars are standard error of the mean. n=14 (7 WED; 7 age-matched controls).

HeartRateAlterationwithHypoxia

Page 26: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

Alterations in Minute Ventilation. This figure demonstrates the minute ventilation measurements in RLS/WED (blue) and control (orange) subjects. Error bars are standard error of the mean. n=14 (7 WED; 7 age-matched controls).

•Male RLS/WED subjects had an slightly elevated minute ventilation rate at baseline.

•Male RLS/WED subjects had a 65% increase in minute ventilation with hypoxic insult.

MinuteVentilationAlterationswithHypoxia

Page 27: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

•Activation of hypoxia pathway •Neuromelanin cells of the substantia nigra in RLS demonstrate:

• increased HIF

•Brain microvessels• increased HIF-2α • increased VEGF

DiscussionPoints– Hypoxiainthebrain

Page 28: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

•Elevated proteins in the CSF of patients with RLS also occur with hypoxia:

•Vitamin D binding protein, •Prostaglandin D2 synthase, •cystatin C •β-hemoglobin

•Decreased myelination and increased vascularization have been reported with constitutive HIF expression:

•Decreased CNPase, MBP, and PLP•Reduced volume by MRI •Elevated VEGF expression in brain microvessels

DiscussionPoints-hypoxiainthebrain

Page 29: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

• Increased erythropoetin in lymphocytes suggests activation of the hypoxia pathway.

• Physiologicalalterationswithnormoxia andhypoxiademonstratechangesconsistentwithHIFactivationintheperiphery:

• Increasedfemoralarterybloodflow• Increasedheartrate• Increasedminuteventilation

DiscussionPoints-hypoxiaintheperiphery

Page 30: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

©2016RLSFoundation

Acknowledgements

Page 31: Iron, Hypoxia and RLS · uses iron as a means to bind oxygen and transport it to the tissues. • When iron deficiency occurs, the synthesis of heme, a major building block of hemoglobin,

Questions & Answers

JointheRLSFoundationatwww.rls.org

RegisterforournextwebinarpresentationonNovember9- “ResearchReview:Then&Now”withDr.DavidRye


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