iron, hypoxia and rls · uses iron as a means to bind oxygen and transport it to the tissues. •...
TRANSCRIPT
JamesR.Connor,Ph.D.andStephanieM.Patton,Ph.D.
Iron,HypoxiaandRLS
©2016RLSFoundation
• IntroductionofanewparadigmforBBBtransportandRegulation
BrainIronAcquisitioninRLS:
©2016RLSFoundation
Iron
Transferrin
Mitochondria
Tf Receptor
KeyBlood
Brain
Transferrin(Tf)transcytosistheworkingmodelforBBBirontransport:theconduitmodel
? ?
? ?
?Ferritin
Endosome
Fe2+
TfR
DMT1pH
Fe3+
Fe2+
holoTf
Blood Brain
Astrocyte
Endothelial Cell
IronisreleasedinEC
apoTf
?
©2016RLSFoundation
Transwell collagen membrane
BREC
Apical Chamber for additionOf substances for transport
CSFfromnormalorIDmonkeys
CanIronReleasefromtheBBBcellsberegulated?
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59Fe(GammaCounts)
Baseline 1 2 3 4Time (hr)
IDA
IS*
BBBBioassayforCSF
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Iron
CSF
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/L)
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Ferr
itin
CSF(
mcg
/L)
Tran
sfer
rin C
SF (
mg/
L)RLSNormal
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. # # # # 2 #RLS NormalRLS Normal 0
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CSFironstatussuggestsbrainisirondeficient:ironlevelsintheCSFnotdecreased:problemorcompensatoryresponse??
©2016RLSFoundation
Transwell insert
Microporous membrane
ApicalChamber
BasalChamberforplacementofCSF
BrainMicrovascularEndothelialCell
TransportofTransferrinandIronacrossamodeloftheBBBinRLS
©2016RLSFoundation
• CSFfromRLSpatientsdidnotaltertransportorreleaseinaninvitroBBBmodel– IstheprobleminRLSafailuretosignalregardingbrainironstatus?– Isthisrelatedtothesetpointconcept?
• Hemoglobin,notserumferritin,mayfunctionassystemicpredictorforbrainironuptake– Ishypoxicstatusmoreimportantthanironstatusforbrainironuptake?
Summary
©2016RLSFoundation
• Redbloodcells(erythrocytes)arethecelltypeinthebloodthatcarryoxygenfromthelungsthroughoutthebody.
• Approximately35%ofthetotalcontentoftheredbloodcellsismadeupofaproteincalledhemoglobin.
• Hemoglobinisametalloprotein,thatusesironasameanstobindoxygenandtransportittothetissues.
• Whenirondeficiencyoccurs,thesynthesisofheme,amajorbuildingblockofhemoglobin,isdecreasedandlessoxygencanbeboundandtransported.
Whatistherelationshipbetweenironandoxygen?
©2016RLSFoundation
• TofurtherexploretheimpactofcellularirondeficiencyinRLSneuromelanin cells,weproposedthehypothesisthatthelossofcellularironhomeostasisincellscouldelicitasurvivalresponseandactivationofsurvivalpathways.
• Onesuchsurvivalpathwayisactivationofhypoxiainduciblefactor1(HIF-1),whichregulatesexpressionofanumberofproteinsincludingthoseinvolvedinironhomeostasissuchasTfR,vascularendothelialgrowthfactor,anderythropoietin.
• Ironisaco-factorforHIFdegradation,thereforeirondeficiencywillleadtostabilizationofHIF-1αandactivationoftheHIFpathway.(seeHypoxiaResponsePathwaySchematic)
SoWhyExamineHypoxiaPathway?
©2016RLSFoundation(http://dx.doi.org/10.14348/molcells.2014.0150)
HypoxiaResponsePathway
©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
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INDICATORSOFACTIVATIONOFTHEHYPOXICPATHWAYINTHESUBSTANTIANIGRA OFRLS
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ControlRLS
HIF-1αImmunostaininginSubstantiaNigra
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MICROVESSEL PERTURBATIONSINRLS
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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
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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
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PROTEOMICANALYSISOFTHECEREBROSPINALFLUIDOFPATIENTSWITHRESTLESSLEGSSYNDROME
©2016RLSFoundation
We sought to identify proteins that could prove valuable in understanding pathways affected in RLS/WED and identifying targets for treatment strategies.
ReasoningforCSFbiomarkeranalysispilotstudy
©2016RLSFoundation
ProteinsLevelsAlteredinRLSCSF
©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
©2016RLSFoundation
ALTERATIONSINHEARTRATE,MINUTEVENTILATIONANDFEMORALARTERYBLOODFLOWINRLS:APILOTSTUDY
©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
©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
©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
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•Activation of hypoxia pathway •Neuromelanin cells of the substantia nigra in RLS demonstrate:
• increased HIF
•Brain microvessels• increased HIF-2α • increased VEGF
DiscussionPoints– Hypoxiainthebrain
©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
©2016RLSFoundation
• Increased erythropoetin in lymphocytes suggests activation of the hypoxia pathway.
• Physiologicalalterationswithnormoxia andhypoxiademonstratechangesconsistentwithHIFactivationintheperiphery:
• Increasedfemoralarterybloodflow• Increasedheartrate• Increasedminuteventilation
DiscussionPoints-hypoxiaintheperiphery
©2016RLSFoundation
Acknowledgements
Questions & Answers
JointheRLSFoundationatwww.rls.org
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