Prezentacja programu PowerPointbiotka.mol.uj.edu.pl/zbm/handouts/2006_03_angiogeneza.pdf · 2009....

71
Hypoxia and hypoxia inducible factor-1 - angiogenic and non-angiogenic functions Lecture III

Transcript of Prezentacja programu PowerPointbiotka.mol.uj.edu.pl/zbm/handouts/2006_03_angiogeneza.pdf · 2009....

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Hypoxia and hypoxia inducible factor-1- angiogenic and non-angiogenic functions

Lecture III

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VEGFVEGF

Required for developmental angiogenesis

Ferrara et al.. Nature 1996Carmeliet et al., Nature 1996

Induced at pathologicalInduced at pathologicalconditionsconditions

Hypoxia

Inflammatory cytokines & growth factors

IL-1βTNFαIL-6

MCP-1GM-CSF

vascular endothelial growth factor

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Regulation of Regulation of VEGF VEGF expression expression

1. Enhancement of VEGF mRNA expression

- activation of VEGF promoter - increased stability of VEGF mRNA

2. Enhanced translation through internal ribosome entry site (IRES)

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Half-life of endogenous VEGF mRNA is about 65 minstability increases ~ 3 times in hypoxia

HuR – a member of Elav-like family of binding proteins; binds todistal AU-rich region in the VEGF 3’UTR. Stabilizes VEGF mRNA

Hypoxia Hypoxia –– one one of the strongest inducers of of the strongest inducers of VEGF VEGF expression expression

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Tumor growth is dependent on angiogensis

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Hypoxic area is formed inside growing tumor Hypoxic area is formed inside growing tumor

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Hypoxic areas in bone marrow – EPCs home to this side

Ceradini et al., Nature Med. 2004

Pimonidazole - green

SDF-1 – red

DAPIBM – bone marrowC – cortical bone P – non-ischemicperiosteal tissue

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Causes of tissue hypoxia Causes of tissue hypoxia

1. Decrease in blood oxygenation (pulmonary disorders) 2. Altered oxygen release from hemoglobin3. Impaired blood delivery (ischemia) – cardiovascular diseases

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Coronary artery diseasePeripheral vascular disease

Impaired blood flow, hypoxic simulation of angiogenic factors,

JAMA & Archives Journals

Currents: Fall 2001

Ischemia in cardiovascular diseasesIschemia in cardiovascular diseases

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What is hypoxiaWhat is hypoxia??

Atomospheric 21% O2

Lung capillaries 13% O2

Healthy tissues 2.5-9% O2

Diseased tissue < 1% O2

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Protective physiological mechanisms against hypoxia Protective physiological mechanisms against hypoxia

1. Increased production of tyrosine hydroxylase – controls the ventilation through the carotid body

2. Increased expression of glycolytic enzymes 3. Increased synthesis of erytropoietin 4. Increased production of VEGF – stimulation of new blood

vessels

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How do cells sense the changes in the oxygen level?

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Acker, T. et al. J Exp Biol 2004;207:3171-3188

Dual role of HIF in regulating cell survival and cell death, depending on

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The studies of hypoxia response element of the erythropoietin gene lead to the discovery of HIF-1 by

Semenza and Wang in 1992. Semenza GL & Wang GL. (1992). Mol. Cell. Biol. 12: 5447-5454.

HIFHIF--1: 1: HHypoxia ypoxia IInducible nducible FFactor actor -- 11

HIF-1 is a protein with DNA binding activity. It is composed of two subunits: HIF-1α and HIF-1β.

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Hammond Hammond et al., et al., Mol Mol Cell Biol Cell Biol 20022002

Stabilisation of Stabilisation of HIFHIF--11αα proteinprotein in hypoxia in hypoxia

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HypoxiaHypoxia inducibleinducible factorfactor ––a master regulator a master regulator of oxygen homeostasis of oxygen homeostasis

HIF

Erythropoiesis & iron metabolismErythropoietinTransferrinTransferrin receptorCeruloplasminHeme oxygenase-1 (rodents)

Vasomotor controlNOS II

Cell proliferation & viability IGF-1IGFBP-1&3TGFβ3NOS II

Energy metabolismGLUT1,2 & 3PEPCKLDH APGK3Aldolase A & C PFK L & C Pyruvate kinaseEnolase

AngiogenesisVEGFVEGFR-1PlGFPDGFTGFβ

O2

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Structure of Structure of VEGF VEGF human promoter human promoter

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HIFHIF--1 1 binds binds to to hypoxia responsive hypoxia responsive element element present in regulating present in regulating regions of many genesregions of many genes

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Zagórska & Dulak Acta Biochim Pol 2004

Various HRE

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Mole et al., IUBM, 2001

Activation and degradation of Activation and degradation of HIFHIF--11αα

HIF-1β &

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- Both α and β subunits are members of basic helix/loop/helix (Per/Arnt/Sim) (PAS) family

- there are three HIFα family members: HIF-1α, HIF-2α, HIF-3αand three HIFβ members: HIF-1β/ARNT1, HIF-2β/ARNT2, HIF-3β/ARNT3,

- IPAS3, a HIF-3α isoform, can act as inhibitor of HIF

- HIF-α members exclusively dimerize with HIF-β members, while the oppositeis not true

- over 100 HIF-dependent genes

- canonical HIF binding site: 5’-RCGTG

MoreMore on HIF on HIF transcription factorstranscription factors

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HIF-1 transcription factors

HIF-1α – knockouts die at E 9.0

HIF-2α – expressed in fetal type 2 pneumocytes

- knockouts die in utero – adrenal insufficiency

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Activation and degradation of Activation and degradation of HIFHIF--11αα

Zagórska & Dulak, Acta Biochimica Polonica, 2004

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Prolyl hydroxylases are ironProlyl hydroxylases are iron--dependent dependent dioxygenasesdioxygenasesinvolved ininvolved in HIFHIF--11αα degradation degradation

PHD1 PHD1 –– hydroxylateshydroxylates P 402 & P564P 402 & P564

PHD2 PHD2 -- hydroxylateshydroxylates P 402 & P564 P 402 & P564

PHD3 PHD3 -- hydroxylateshydroxylates P564 P564

Asparaginyl hydroxylase Asparaginyl hydroxylase (FIH)(FIH) modifiesmodifies HIFHIF--11αα and prevents binding and prevents binding ofof p300 cop300 co--activator activator

CoCo--factors of PHDsfactors of PHDs: : ironiron, , oxygenoxygen, 2, 2--oxoglutarate oxoglutarate , , ascorbic acidascorbic acid

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Hypoxia enhances Hypoxia enhances VEGF VEGF expression and activity in several ways expression and activity in several ways

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IRES A is located within the 300 nucleotides upstream from the AUG start codon. RNA secondary structure prediction and site-directed mutagenesis allowed the identification of a 49-nucleotide structural domain (D4) essential to IRES A activity. UV cross-linking experiments revealed that IRES A activity was correlated with binding of a 100-kDa protein to the D4 domain. IRES B is located in the first half of the 5' UTR. An element between nucleotides 379 and 483 is required for its activity.Immunoprecipitation experiments demonstrated that a main IRES B-bound protein was the polypyrimidine tract binding protein (PTB), a well-known regulator of picornavirus IRESs

VEGF VEGF mRNA has two mRNA has two IRESIRESHuez I et al., Mol Cell Biol, 1998

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IRES IRES initiates translation initiates translation independent independent of of capcap

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Hypoxia Hypoxia upup--regulatesregulates VEGF VEGF expressionexpressionin in humanhuman microvascular microvascular endothelialendothelial cellscells

(HMEC(HMEC--1)1)

00

11

22

33

44

normoxianormoxia 1% O1% O22

VEGF

mRN

AVE

GF m

RNA

[[ relrel .. e

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RealReal--timetime PCRPCR

ELISAELISA

00

100100

200200

300300

400400

500500

VEGF

pro

tein

VEGF

pro

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[%

[% o

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normoxianormoxia 1% O1% O22

RTRT--PCRPCR

normoxia normoxia

VEGFVEGF

EF2EF2

1% O1% O22

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VEGF promoter luciferase

HRE

HRE luciferase

Activation of Activation of VEGF VEGF promoter and promoter and HRE HRE part part by by hypoxia hypoxia

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Regulation of Regulation of VEGF VEGF expression expression by by hypoxia is cellhypoxia is cell--type type dependent dependent

hypoxianormoxia

0

40

80

120

160

Normoxia Hypoxia

% o

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trol

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Normoxia Hypoxia0

100

200

300

400

500

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GAPDH

hypoxianormoxia

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NIH 3T3 NIH 3T3 fibroblastsfibroblasts

HMECHMEC--11

Łoboda A et al., in preparation

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How to modulate HIFHow to modulate HIF--1 activity?1 activity?

• Hypoxia • Iron chelators: desferrioxamine, CoCL2

• Dominant positive/negative forms, siRNA, oligonucleotide decoys

• Chemical compounds: DMOG (dimethyloxallyl glycine)

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Other mechanisms of stabilisation of Other mechanisms of stabilisation of HIFHIF--1 1

1. Iron chelators – eg. desferroxiamine effect was reversed by ferrous amonium sulfate

- induction of VEGF synthesis by desferroxiamine

2. Heavy metal – cobalt chloride

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CoClCoCl2 2 is known is known to to potently activate potently activate HIFHIF--11

Increase in HIF- protein levels by cobalt stimulation in osteoblast-like cells.

Kim at al.,Cytokine. 2002 Jan 7;17(1):14-27.

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CoClCoCl22 activates activates VEGF VEGF promoter in its promoter in its HRE HRE site site

VEGF promoter luciferase

HRE

HRE luciferase

luci

fera

seac

tivity

[% o

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0

100

200

300

400

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*

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*

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DMOG (Dimethyloxallyl glycine)

• Competitive inhibitor of the oxygen-sensing enzymes - prolyl hydroxylases (PHDs), which destruct HIF-1 α when hydroxylated at a specific proline residues.

• Stabilizes HIF-1 α expression at normal oxygen tensions at concentrations between 0.1 and 1 mM

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DimethyloxallylDimethyloxallyl glycineglycine (DMOG)(DMOG)

HIFHIF--11 hydroxylationhydroxylation andand actionaction ofofDMOGDMOG

HIFHIF--11αα HIFHIF--11αα

22--OXOGLUTARATEOXOGLUTARATE

OO22 COCO22

PROLYL HYDROXYLASESPROLYL HYDROXYLASES

OH OHOH OHpVHLpVHL

FeFe2+2+

SUCCINATESUCCINATE

NORMOXIANORMOXIAHYPOXIAHYPOXIA

proteasomalproteasomaldegradationdegradation

stabilizationstabilization

e.g. VEGFe.g. VEGF

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• Potent VEGF stimulation both at mRNA and protein level

Effect of HIF-1 activation on VEGF synthesis in HMEC-1

EF 2EF 2

VEGFVEGF

12 h12 h 24 h24 h

controlcontrol controlcontrolDMOGDMOG DMOGDMOG

RTRT--PCRPCR

00

10001000

20002000

30003000

40004000

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Diseases caused by disturbances in HIF-1 signaling

-von Hippel Lindau disease

Diseases dependent on enhanced HIF-1 activity- cancer

- atherosclerosis

-wound healing

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VHL protein VHL protein

Von Hippel Lindau protein

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Von Von Hippel Lindau disease Hippel Lindau disease

Hemangioblastoma of the retina

Brigham and Women’s Hospital, Boston

-rare (1:36 000 live births) dominantly inherited cancer syndrome

- multiple hemangioblastomas of CNS and retina, renal cell carcinomaspancreatic islet cell tumors and others

Desrcibed in 1911 by Eugene von Hippel and further studied in 1926 by Arvid Lindau

Defect in Defect in VHL VHL tumor suppresor gene tumor suppresor gene in chromosome in chromosome 3p253p25--p26p26

Mutations in VHL occur also in the majority of kidney cancers

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Is it not known which HIF-1αresidues are ubiquitinated

Mutation in VHL gene causes disruption in HIF-1 degradation process

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Nature Genetics, 2001: 28: 131-138

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Generation of VEGFGeneration of VEGFδδ/δ/δ micemice

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Deletion of Deletion of HRE region HRE region of of VEGF VEGF promoter causes promoter causes ALS ALS in in mice mice

-Cre/Lox deletion of HRE – VEGFδ/δ

- reduced hypoxic expression of VEGF in neural tissues, but not in cultured fibroblast

- no changes in other gene

real-time RT-PCR analysis

TissueTissue--specific regulation of specific regulation of VEGF VEGF expression in hypoxia expression in hypoxia

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Muscle weakness in VEGFMuscle weakness in VEGFδδ/δ/δ micemice

Oosthuyse et al., Nature Genetics, 28: 131-138, 2001

atrophic muscles

-~ 60% VEGFVEGFδδ/δ /δ died beforedied before or around birth or around birth - remaining – at around 5 months of age developed symptoms of motor neuron disease

Impaired motor performance

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Muscle and neuronal atrophy in VEGFMuscle and neuronal atrophy in VEGFδδ/δ/δ micemice

Axonal spheroid swelling

- muscle atrophy - motor neuron degeneration - axon degeneration - abnormal neural vascular perfusion – reduced blood flow

in the spinal cord

Vacuolized mitochondria

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Which human disease does this mouse Which human disease does this mouse model model resembleresemble? ?

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Amyotrophic lateral sclerosisAmyotrophic lateral sclerosis –– Lou Gehring diseaseLou Gehring disease

Spinal cord – the atrophy is apparent in ALS

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Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis

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AmyotrophicAmyotrophic lateral sclerosis lateral sclerosis (ALS) (1) (ALS) (1)

1869 – Jean Martin Charcot

Famous patients:

-Lou Gehring- Dimitir Shostakovich - Mao Zedong (infamous patient...) - Stephen Hawking – suffers from unusually slowly progressing

form of a disease

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1903-19411942 –

Diagnosed at age of 21

Famous Famous AML AML patientspatientsLou Gehring Stephen Hawking

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AmyotrophicAmyotrophic lateral sclerosis lateral sclerosis (ALS) (1) (ALS) (1)

Incidence – 2-3:100 000

Onset at 50-60 years

Sporadic (SALS) – most instances (90-95%)Familial (FALS) – 5-10% - of these 20-25% are mapped to CuZnSOD

gene

Degeneration of motor neurons – progressive loss of the ability to move, speak,

Usually fatal within 1-5 years of onset

No treatment available

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Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS) (3)(ALS) (3)90-95 % of cases – no apparent genetic linkage 5-10 % - familial ALS – mutation in SOD1 gene are responsible

for 10-20 % of cases of FALS

-about 100 different mutations in SOD1 gene

-mutations do not cause the lost of functions, but rather gaining of a toxic phenotype

-among others, the formation of hydroxyl radicals and peroxy-nitrite has been suggested

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Yuan J & Yankner BA, Nature 407, 802 - 809 (2000)

SOD1 and AML

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How does How does VEGF VEGF protect nervesprotect nerves??

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Neurotrophic functions of VEGF

Oosthuyse et al., Nature Genetics, 28: 131-138, 2001

VEGF165 (but not VEGF121) prevents neuronal apoptosis

survival effect of VEGF165 is blocked by antibodies against Kdr and Nrp

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Neuroprotective functions of Neuroprotective functions of VEGF VEGF

Oosthuyse et al., Nature Genetics, 28: 131-138, 2001

Storkebaum E & Carmeliet P, JCI 2004

Both Kdr and Nrp-1 are required

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Neuroprotective functions of Neuroprotective functions of VEGF VEGF

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Neuroprotective functions of Neuroprotective functions of VEGF VEGF –– other other data data - increased axonal outgrowth and cell survival in peripheral neuronal culture

- protects immortalized hippocampal cell culture from serumwithdrawal and hypoxia in vitro – mediated by VEGFR2, PI3K, Akt, NF-kB

- reduced hypoxic death of cultured cereberal cortical neurons –due to activation of VEGR2, PI3K, decrease in caspase 3

- protects hippocampal neurons from glutamate toxicity

- Stimulate the birth of new neurons (neurogenesis)

- Neuroprotective at in vivo ischemia

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Questions created Questions created by by seminal paper of Oosthuyse seminal paper of Oosthuyse et al. et al.

- what is the level of VEGF in patients with ALS? – search for VEGF gene polymorphism

- if SOD1 mutation causes the neuronal damage, what will be the result of a combined effect of SOD1 mutation and VEGFδ/δ ?

- will VEGF prevent ALS development?

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Single nucleotide polymorphisms or SNPs (pronounced "snips") areDNA sequence variations that occur when a single nucleotide (A,T,C,orG) in the genome sequence is altered. For example a SNP might change the DNA sequence AAGGCTAA to ATGGCTAA. For a variation to beconsidered a SNP, it must occur in at least 1% of the population. SNPs,which make up about 90% of all human genetic variation, occur every100 to 300 bases along the 3-billion-base human genome. Two of every three SNPs involve the replacement of cytosine (C) with thymine (T).SNPs can occur in both coding (gene) and noncoding regions of the genome. Many SNPs have no effect on cell function, but scientists believe others could predispose people to disease or influence their response to a drug.

Single Single nucleotide polymorphisms and diseasesnucleotide polymorphisms and diseases

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A common polymorphism in 5’UTR of VEGF gene is associated with diabetic retinopathy in type 2 diabetes

Awata T et al., Diabetes, 51: 1635-1639, 2002

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Proliferative diabetic retinopathy

Hemorrhages in non-proliferative diabetic retinopathy

New blood vessels around optic nerve in proliferative diabetic retinopathy

Hemorrhage from new blood vessel growth in proliferative diabetic retinopathy

Normal macula

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Polymorphism in 5’UTR changes the structure of mRNA

VEGF VEGF promoter polymorphism and promoter polymorphism and ALS ALS

-no sequence alterations in HRE in individuals with ALS

-three VEGF promoter and 5’UTR polymorphism and ALS

VEGF plasma level was the lowest in ALS patient with AAG/AAG and AGG/AGG haplotypes

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Correlation of Correlation of median serum VEGF median serum VEGF values with the different values with the different genotypes in Belgian control population genotypes in Belgian control population

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Effect of combined mutation in Effect of combined mutation in SOD SOD and and VEGFVEGF

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Shorter survival of Shorter survival of SOD1SOD1G93A G93A VEGFVEGFdd/d/d mice mice relative relative to SOD1to SOD1G93A G93A VEGFVEGF+/++/+

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score 1 score 4Unable to walkLimbs immobile when lifted by tail

Administration of VEGF improves motor recovery after acute spinal cord ischemia

PGF was not effective

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Gene therapy with Gene therapy with VEGF VEGF can prolong survival in can prolong survival in a a mouse mouse model model of of ALSALS

Azzouz et al, Nature 27 May 2004

single injection of VEGF-expressing lentiviral vector into various muscles delayed onset and slowed progression of of ALS in mice engineered to overexpress the G93A mutated form of SOD1, even when treatment was only initiated at the onset of paralysis

Survival of Survival of mice mice injected at three weeks of age and at injected at three weeks of age and at 90 90 days of agedays of age

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Multitasking Multitasking VEGFVEGF

Velde & Cleveland, Nature Neuroscience 2005