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

Therapeutic Angiogenesis Trials

Circulation 2003;107:1359-65

Therapeutic Angiogenesis Trials

Circulation 2003;108:1933-8

Therapeutic Angiogenesis Trials

Lancet 2002;359:2053-8

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

VEGF-ZFP GFP

VEGF-ZFP Induces Angiogenesis

Rebar et al, Nat Med 2002;8:1427-32

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

* **

VEGF-ZFP increases Oxidative Metabolism inResting Muscle

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

Altered Skeletal Muscle Metabolism in theAbsence of Exercise

VEH AICAR

Cell 2008;134:405-15

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

Exercise Is a Recommended Therapy in PAD

ACC/AHA Guidelines, Hirsch et al Circulation 2005;113:e463

Mouse Voluntary Running Model

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 and sTie2 Both Inhibit Exercise-InducedAngiogenesis

*

sFlt, but not sTie2, Blocks Exercise-InducedFiber Type Switching

Sedentarycontrol

ExercisesTie2

ExercisesFlt

Exercisecontrol

Type IIa Fibers

* *

*NS

sFlt, but not sTie2, Blocks Exercise-InducedFiber Type Switching

Soluble Tie2 Does Not Prevent Exercise-Induced Changes in Oxidative Metabolism

**

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

• Can we effectively treat PAD withangiogenic and/or myocyte-specifictherapies?

Chronic Ulcer Improvement after IntramuscularVEGF-ZFP

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)

Acknowledgments

Greg LamCauveh EramiClarence FindleyJanet HartJulie RoyBalint OtvosMike PadgettJen Jackson

Brian AnnexRob MitchellSchuyler JonesBrian Duscha

Asif AhmedDeb MuoioJerry EuClaude Piantadosi

Sangamo Biosciences