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Modulating Angiogenesis inPeripheral Artery Disease
Christopher D. Kontos, M.D.Associate Professor of Medicine
Division of Cardiovascular MedicineOctober 10, 2008
Overview
• Overview of angiogenesis• Clinical trials of therapeutic
angiogenesis• VEGF delivery for PAD• Mechanisms of exercise-induced
angiogenesis
Endothelial RTKsIg Loops
EGF Domains
FN3 Repeats
Domain
KinaseDomains
FGF-R
PDGF-R
VEGF-RFlt-1, Flk-1
Tie1, Tie2
Transmembrane
Multiple Endothelial RTKs and TheirLigands Regulate Angiogenesis
Yancopoulos et al, Genes Dev 1999;13:1055
VEGF/VEGFR Angiopoietin/Tie Ephrin/Eph
Angiogenesis - Protease Production (BasementMembrane Breakdown)
Smooth MuscleCells
Endothelium
BasementMembrane
Proteases
AngiogenicStimulus
(hypoxia --> VEGF)
Ang2Ang1
Angiogenesis - Recruitment of Leukocytes and BoneMarrow-Derived Progenitor Cells
Smooth MuscleCells
Endothelium
BasementMembrane
VEGF
Ang2Ang1
Angiogenesis - Endothelial Cell Migration
Endothelium
NascentVascular Sprouts
Smooth MuscleCells
BasementMembrane
Ang2Ang1
VEGF
Angiogenesis - Endothelial Cell Proliferation
Endothelium
SproutElongationSmooth Muscle
Cells
BasementMembrane
Ang1 Ang2
VEGF
Angiogenesis - Capillary Morphogenesis
Endothelium
NewLumen
FormationSmooth MuscleCells
BasementMembrane
VEGF
Angiogenesis - Vascular Maturation
Endothelium
Vascular Pruning(apoptosis)
SMC, pericyterecruitment
Smooth MuscleCells
BasementMembrane
Ang1
Ang2
VEGF
Angiogenesis - Vascular Maturation
Endothelium
Endothelialcell-cell junctions
Smooth MuscleCells
BasementMembrane
NegativeFeedback
Ang1
VEGF
The Angiopoietins and VEGF Regulate the Transitionbetween Mature and Remodeling Vessels
SMC,pericyte
EC
lumen
BasementMembrane
Mature, StableBlood Vessel
Immature, UnstableBlood Vessel
Angiogenesis
Ang2
Ang1 Ang2+
VEGF
Ang2alone
VascularRegression
GGGGGTGACC GCTGGGGGCG 1 2-5 6 7 8+426 +509
1
1
1
2-5
2-5
2-5
8
7 8
7 86
VEGF120
VEGF164
VEGF188
VEGF-A geneHeparin-binding
Alternative Splicing Results in Expression ofMultiple VEGF-A Isoforms
Alternative Splicing
Promoter Elements
Delivery of Multiple VEGF Isoforms Induces"Better" Angiogenesis
Amano et al, Mol Ther 2004;12:716-24
GGGGGTGACC GCTGGGGGCG 1 2-5 6 7 8+426 +509
1
1
1
2-5
2-5
2-5
8
7 8
7 86
VEGF120
VEGF164
VEGF188
p65mVZ+509
VP16mVZ+426
VEGF-A gene
Heparin-binding
An Engineered Zinc Finger TranscriptionFactor Induces Multiple VEGF-A Splice
Variants
TAD
ZFP
Kontos and Annex, Curr Opin Mol Ther 2007;9:145
Rebar et al, Nat Med 2002;8:1427-32
VEGF-ZFP Induces FunctionalAngiogenesis and VEGF Gene
Expression
Intramuscular VEGF-ZFP EnhancesAngiogenesis in Hindlimb Ischemia
RT-PCR
VEGF-ZFPControl
Dai et al, Circulation 2004;110:2467
Alk Phos
VEGF121 VEGF165 VEGF189
VEGF-ZFPControl
Sham 1 injection 2 injections 4 injections
Multiple VEGF-ZFP Injections IncreaseSkeletal Muscle Capillary Density
CD31
Alk Phos
* p<0.05* *
Cap
illar
y / M
uscl
e Fi
ber
VEGF-ZFP Increases Bone Marrow-DerivedProgenitor Cell Number
Lineage markers-PETR
c-K
it - A
PC
Sca-1 FITC
c-K
it - A
PC
VEGFR2-PE
c-K
it - A
PC
20.2
41.9 6.76 %
1.3949.9
0.13
99.9%
00
20.2 %
*
Sham ZFP
HSCsc-Kit+/Sca-1+
Sca-
1+ c
ells
(% o
f c-K
it+)
*
Sham ZFP
EPCsc-Kit+/VEGFR2+
VEG
FR2+
cel
ls (%
of S
ca-1
+)
VEGF-ZFP Induces Angiogenesis and FiberType Switching at 14 days in Resting Muscle
CD31 Type IIa
Sham
ZFP
Gene Expression Analysis Suggests VEGF-ZFP May Have Off-Target Effects
• HEK-293 cells transfected with VEGF-ZFPplasmid or empty vector
• Affymetrix gene arrays used to analyzed changesin gene expression
• In addition to VEGF-A, 15 genes upregulated, 13downregulated
• Upregulated genes include:– NFATC1 - regulator of slow myofiber phenotype– MSX1 - muscle homeobox transcription factor– IGF2 - muscle growth factor– c-Jun - transcription factor
PAD Patients Have Evidence of SkeletalMuscle "Myopathy"
• Altered expression of mitochondrialenzymes
• Accumulation of metabolic intermediates• Altered regulation of mitochondrial
respiration• Increased oxidative stress• Somatic mutations in mitochondrial
genome
Brass and Hiatt, Vasc Med 2000;5:55-9
Adaptations to Exercise Training in MiceSedentary Exercise
CD31
Type IIafibers
Waters et al,Am J Physiol
2004;287:C1342
Angiogenesis Precedes Fiber Type Switchingin Exercise
Capi
llary
/Fib
er R
atio
Running Time (days)
Type
IIa
Fibe
rs (%
)
Waters et al, Am J Physiol 2004;287:C1342
Angiogenesis Occurs Primarily AroundGlycolytic Fibers Following Exercise
EnduranceExercise
Type IIa fiber Type IIb, IId/x fiber Capillary
Waters et al, Am J Physiol 2004;287:C1342
Questions
• Is angiogenesis required for metabolicadaptation in skeletal muscle?
• What are the mechanisms of cross-talkbetween endothelial cells and skeletalmyocytes?
• Does VEGF have direct effects on skeletalmyocytes (are VEGFRs expressed onmyocytes)?
P P
SignalingInactive Inactive Inactive
Mechanisms of RTK Signaling and Inhibition
DominantNeg
SolubleReceptor
Approach• Inhibit angiogenesis through 2 distinct pathways:
– Soluble VEGFR1 (sFlt)– Soluble Tie2 (sTie2)
• Systemic adenovirus delivery - measure serumprotein concentrations
• Subject mice to voluntary running on exercisewheel - 21 days
• Quantify:– time/distance run– capillary density– oxidative fiber type– oxidative metabolism
Voluntary Exercise Performance
1.95.38.2AdsFlt
2.05.48.4AdsTie2
1.85.910.1Controlvirus
Speed(km/h)
Time Run(h/day)
Distance Run(km/day)
ExerciseGroup
sFlt, but not sTie2, Blocks Exercise-InducedFiber Type Switching
Sedentarycontrol
ExercisesTie2
ExercisesFlt
Exercisecontrol
Type IIa Fibers
Summary
• Tie2 signaling is required for exercise-induced angiogenesis but not changes infiber type or oxidative metabolism
• VEGF signaling is required for bothangiogenesis and metabolic changes inexercise
• VEGF may act directly on myocytes toinfluence fiber type and metabolism
Mobilization of Peripheral CD34+/ALDH+ ProgenitorCells
From Day 0 to 30, there was a 1.3 fold increase (p<0.09) in ALDH+ cells
From Day 0 to 90, there was a 1.9 fold increase (p<0.05) in ALDH+ cells
0 . 5
1
1 . 5
2
0 3 0 6 0 9 0 1 2 0 1 5 0 1 8 0
CD34+ / ALDH+ cell data obtained in 11 patients (Duke)
0 30 60 90 120 150 180 Days After Initial Dosing
Fold
Cha
nge
in A
LDH+
cel
ls
2.0
1.5
1.0
0.5
Summary
• Targeting multiple angiogenic factors/isoformsmay provide improved therapeutic angiogenesis
• Different angiogenic pathways have distincteffects on blood vessels and muscle metabolism
• Exercise in conjunction with therapeuticangiogenesis may enhance outcomes
• Targeting both blood vessels and muscle willlikely yield greater functional improvements inPAD
• Skeletal muscle-targeted gene delivery mayprovide decrease toxicity vs. systemic therapies
Future Directions
• Determine specificity of VEGF-ZFP forVEGF-A vs. muscle-specific genes
• Determine effects of VEGF-ZFP on musclefatigability
• Determine long-term effects of VEGF-ZFP -i.e., durability of angiogenesis
• Investigate alternative approaches todeliver multiple VEGF isoforms (AAV)