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Post-ischemic neovascularization:
basic concept and new treatment options
Professor Costanza Emanueli, PhD
British Heart Foundation Senior Research Fellow
Professor and Chair of Vascular Pathology and Regeneration- Bristol
Visiting Professor -ICL
ESC Summer school, Nice 16 June 2013
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Index of this Lecture
•Introduction to ischemia and post-ischemic neovascularisation
•Research Models
• Therapeutic angiogenesis research: past, present and future
•microRNAs in post-ischaemic vascular regeneration
•Novel stem cell products: Empowered EPCs, Pericyte progenitor cells
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Ischemic Disease
•Tissue ischaemia is caused by poor blood flow supply, which impairs the delivery of oxygen (creating hypoxia) and nutrients (creating tissue starvation)
•Ischaemic disease is favoured by atherosclerosis, diabetes, aging and a series of cardiovascular risk factors
• Ischaemic disease is one of the biggest medical epidemic worldwide and its treatment is still an umet clinical need.
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Post-Ischaemic Vascular Regeneration: the Medical Need
MYOCARDIAL INFARCT
ISCHAEMIC STROKE
CRITICAL LIMB
ISCHAEMIA
LIMB ISCHAEMIA
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Vascular Regeneration: what can we try?
Substituting the diseased (large) vessel
By-pass grafting with pieces of patient own vessels (vena saphena and/or mammary artery)
By-pass grafting with “artificial” vesselscreated in the laboratory
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Therapeutic Neovascularization
VASCULOGENESISischemiaischemia
ischemia
SDF-1,
VEGF, IL-8, ....
SDF-1,
VEGF, IL-8, ....
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Strategies for Post-Ischemic Neovascularisation
• Increase the expression/activity of endogenous “pro-angiogenic” factors in ischemic tissues
• Stimulate endogenous stem and progenitor cells with various pro-angiogenic capacities
• Transplantation of vascular stem and progenitor cells with pro-angiogenic capacities
• Contrast “anti-angiogenic” molecular signatures induced by pathology and/or risks factors
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In Vivo Research Models
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Occlusion of LAD
Experimental models of myocardial infarction (MI) are routinely performed in Rodents.- Permanent ligation of LAD- Ischemia/reperfusion
Animal Models of Ischemia: MI
Mouse model of myocardial infarct
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Ligation of the (left) femoral
artery
Mouse ventral view
Experimental models of limb ischemia (LI) is used in mice to mimic a situation comparable to peripheral artery disease. Since peripheral artery disease is often associated with diabetes, this model is also performed in diabetic mice. LI is performed by permanent ligation of one femoral artery.
Animal Models of Ischemia: LI
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Intravenous infusion of stem cells/genes:
through the femoral vein
through the tail vein
through the left ventricular cavity
Limits of systemic delivery:
- Not possible to target a specific organ- (Loss of cell number)- (Loss of cell viability)
Systemic Gene/Cell Delivery
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Cardiac:
1. Endomyocardial needle injection
2. Catheter delivery (large animals)
3. Intracoronary infusion (large animals/humans)
4. Intrapericardial (large animals)
Lower limb
1. Direct intramuscular injection
2. Intra-arterial infusion
Cells/Genes are directly injected into the infarcted border zone
Injection is often performed immediately after MI
Cells/genes are directly injected into the adductor muscle often immediately after ischemia
Local Gene/Cell Delivery
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Body distribution of cells labelled with D-luciferin after intracardiac injection of different types of stem cells
Time after cell injection
Types of injected stem cells
BM-derived mononuclear cells
Skeletal myoblasts
Mesenchymal stem cells
Fibroblasts
Van der Bogt et al, Circulation 2008
In vivo optical bioluminescence imaging (by using luminescence protein)
Transgene/ Cell Tracking after Local Delivery
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New 3D bioluminescent IVIS system (Caliper)
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Non Invasive Colour Laser Doppler
Blood flow ratio (ischemic/contralateral foot)
Blood Flow Recovery
GroupA
Group B
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Histology /IHC for vessel analyzes
NGF
Cap
/mm
2
Small Arteriole densitySmall Arteriole density
2
†
0204060
10080
140120 **
**††
**
6000**††
5500
4000
45005000
3500
3000
***††
6000**††
5500
4000
45005000
3500
3000
*
30 days
††
14 days
MI/Ad.Null
MI/Ad.Null
MI/Ad.hNGF
00.10.20.30.40.50.60.70.80.9
****
†
00.10.20.30.40.50.60.70.80.9
(ml/m
in/g
)
****
†
30 days 14 days 30 days
MyocardialMyocardialBlood FlowBlood FlowA
rt/m
m2
Capillary densityCapillary density
Sham/NullSham/NullMI/NullMI/NullMI/NGFMI/NGF
14 days
IsolectinB4/CD45/BrdU
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Blo
od F
low
to L
V
(mL
min
-1 g
r-1)
2
4
6
8
0Ad.Null Ad.NGFSham
*§§
§§
Artificial Respiration
Microsphere injection in LV.After 2 minutes, collect tissues
Reference Blood FlowCollection (170ml/min)
BF (mL/min/gr) R x (fl tissues/ fl ref blood) gr of tissue
=
R = rate (mL/min) of ref blood withdrawal
Absolute Blood Flow Measurement in Tissues
MI
Meloni et al. Circ Res, 2010
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Mouse Echocardiography
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Day 0Day -7
Arrival
From
Farm
Myocardial
Infarction
(n=45)
Cath lab
Day 14 Day 15
Surgical injection
Randomization to**:
- Group A (n=13)
- Group B (n=13)
- Group C (n=13)
BASELINE
IMAGING
Day 28
intermediate
IMAGING
Day 56
FINAL IMAGING
(endpoint)
*Randomization will
be based on baseline
LVEF by MRI.
From Dr Borja Ibanez, CNIC-Madrid
Large animal models for translational research
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Spontaneous post-ischemic neovascularization process
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Copyright ©1999 American Heart Association
OLDRABBIT
YOUNG RABBIT
Aging impairs post-ischemic collateral formation and healing in limbs
MICE
Experimentallimb ischemia
Rivard & Isner, Circulation, 1999
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Van Belle, & Isner Circulation 1997
Hypercholesterolemia attenuates post-ischemic angiogenesis in limbs
NORMALRABBIT
ATHEROSCLEROTICRABBIT
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diabetic
diabetic
Diabetes impairs post-ischamic angiogenesis and blood flow recovery
Foot Blood Flow Recovery
Control mouse
Diabetic mouse
Rivard & Isner, Am J Pathol, 1999
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Emanueli & Madeddu. Hypertension 2001
High blood pressure impairs post-ischemic angiogenesis and blood flow recovery in rats
hypertensivehypertensive
No-ischemia
Ischemia
Foot Blood Flow RecoveryCapillary Density
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Ischemic patients have at least one of these aforementionedconditions and often all of them plus additional risk factors(obesity, “wrong” genetic and epigenetic backgrounds, smoke,
etc) to prevent spontaneous healing.
They need some help to fight ischaemia!
MYOCARDIAL MYOCARDIAL INFARCTINFARCT
ISCHAEMIC ISCHAEMIC STROKESTROKE
CRITICAL CRITICAL LIMB LIMB
ISCHAEMIAISCHAEMIA
LIMB LIMB ISCHAEMIAISCHAEMIA
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Therapeutic Neovascularisation ofCardiac and Peripheral Ischaemia
Jeff Isner
First Stretegic Mind to use Angiogenesis as a Therapy
• First attemptTo increase the expression of the prototypicalpro-angiogenic factor VEGF-A in ischaemic tissues using asupply-side approach, mainly by gene therapy viaplasmids and first generation adenoviruses
• Second attemptTransplantation of endothelial progenitor cells (EPCs) in ischaemic tissues.Done in collaboration with Prof Asahara then a fellow of Isner. This approach
was then clinically translated by others (mainly in Frankfurt and Japan)
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“Classical” pro-angiogenic factors in mono-therapies
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Vascular stabilisation vs vascular regression
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Options for Improving Traditional Post-Ischaemic Neovascularisation Therapeutics
• More basic science to understand all the players in the angiogenesis and anti-angiogensis orchestras
•Test different angiogenesis modulator factors (growth factors and more)
•Combinatory approaches
•More severity in preclinical testing (large animal models, risk factors associated to experimental ischemia, etc)
•Work at gene vectors
•Success in in vivo small vessel imaging
•Design better clinical trials
•Do not give up!
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microRNAs (miRs)
DicerThomas ThumBreak-out sessionTuesday 2PM
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Dicer generates short interfering RNAs (including miRNAs) from longer double-stranded RNAs.
Mice homozygous for a hypomorphic allele of Dicer showed impaired developmental angiogenesis and died at days 12.5 - 14.5 of gestation
Yang W J et al. J. Biol. Chem. 2005;280:9330-9335
First evidence for a regulatory role of miRs in vascular development
LowDicer
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Spontaneous post-ischaemic neovascularisation
process
VASCULOGENESIS ischemiaischemia
ischemia
SDF-1,
VEGF, IL-8, ....
SDF-1,
VEGF, IL-8, ....
VASCULOGENESIS
ANGIOGENESIS
Spontaneous post-ischemic neovascularization process
Scientific Questions:
Do miRs modulate it? What miRs? At what levels?
Therapeutic exploitation of miRs?
Bone marrow-derived endothelial progenitor cells (EPCs)/proangiogenic circulating cells (PACs)
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Mouse limb ischaemia model
Grundmann & Moser, Circ , 2011
Hypoxia
Kulshreshtha &, Ivan, CDD, 2008.
miR expression is regulated by hypoxia and ischemia
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Systemic miR-92a inhibition stimulates recovery after hind limb ischaemia
Day 0,2,4,7, and 9Antagomir 92a or Co
Hind limb ischemia
Blood flowMicrovessel density
Antagomir-Co
nuclei Lectin
SMA merge
Antagomir-92a
nuclei Lectin
SMA merge
Ischemic leg Ischemic leg
Antagomir-Co Antagomir-92a
Slide kindly provided by Stefanie Dimmeler
Bonauer& Dimmeler, Science, 2009
miR-92a: first example of mIR targeting for post-ischemic angiogenesis
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Example for miR targeting and contrasting anti-angiogenic factors
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2011
miR-503: how we joined the miR club!
Andrea Caporali
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Finnerty JR et al. JMB 2010
Member of miR-15/107 group: AGCAGC seed sequence
Chr X; intergenicCluster: miR-424-503
Examination of the mir-424 and mir-503 locishowed that they are separated by 383 bases onthe genome and derived from the same primarytranscript
miR-503
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NG
0
1
2
3
4
5
miR
-503
rela
tive
expr
essi
onHG
LGF
HG-LGF
*
miR-503 expression in HUVECs
miR-503 expression in ECs is increased by culture conditions mimicking hyperglycaemia and ischemia
NG HG
LGF HG-LGF
NG= normal glucose, 5 mM
HG= high glucose, 25 mM
LGF = reduced growth factors
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0
20
40
60
80
100
G0/G1 S G2/M
miR503GFP
% c
ells
gat
ed
*
*0
20
40
60
80
100
G0/G1 S G2/M
miR503GFPmiR503GFP
% c
ells
gat
ed
*
*
GFP miR503miR503GFP
Impaired EC migrationImpaired EC network formation
Impaired EC cycle miR-503 target genes
Consequences of miR-503 overexpression in endothelial cells
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Local gene therapy with a decoy for miR-503 improves post-ischaemic angiogenesis in diabetic mice
0
2
4
6
miR
-503
rela
tive
expr
essi
on
++#
Non diabetic Diabetic
IschNo Isch IschNo Isch
††
miR
-503
rela
tive
expr
essi
on
IschNon Diab
0
1
2
3
IschDiab
CD146pos ECs
†
Adductor Muscle
1
3
5
Foot
BF
Rec
over
y
30 min 7d 14d0.0
0.2
0.4
0.6
0.8
Time post-ischemia21d
***
§ §§
decoy503Null
Non Diab Isch Diab Isch Diab Isch
1.0
miR-503 expression
Non Diab Isch + Null Diab Isch + decoy503Diab Isch + Null
Limb microvessels
Blood flow recovery
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New cell therapy products
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# E
ven
ts
Fluorescence intensity
SS
C-A
FSC-A
PACs
99%
AcLDLUEAI
98%#
Even
ts
Fluorescence intensity
Negativecontrol
Specific marker
KDR
59±7%
CD34
11±2%
CXCR4
68±8%
CD45
82±5% 88±8% 90±6%
CD11bCD14
Phenotypic characterization
4 DAYS CULTURE IN EC MEDIUM
ADHERENCE ENRICHMENTON FIBRONECTIN
HISTOPAQUE MNC SEPARATION
Plasma
MNCs
erythrocytesFLOATING
ADHERENT CELLSIsolation and culture
PACs
Human circulating pro-angiogenic cells: PACs
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Circ Res, 2013
Gaia Spinetti
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miR screening in blood PACs of CLI Patients with/out Diabetes
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PAC miR-15a and -16 expression can be manipulated ex vivo
Pre-miR Anti-miR
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miR-15a and -16 overexpression increasesapoptosis of “healthy” PACs
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miR-15a and -16 inhibition improves survival of patient-derived PACs
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Overexpressing miR-15a and miR-16 inhibitsthe migratory capacity of healthy PACs
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Inhibition of miR-15a together with miR-16improves the migratory capacity of diseased PACs
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miR-15a and miR-16 target VEGF-A and AKT-3
3’UTR luciferase activity assays
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Ex-vivo miR-15a and miR-16 inhibition in PACs increases their regenerative potential:
PACs+SCR
PACs+pre-miR15a/16 PACs+anti-miR-15a/16
Medium
Foot Blood flow....
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.... Microvascular Density
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Spinetti G et al. Circ Res, 2013
Blood circulatingmiR-15a and -16
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CLI patients with DM undergoing PTA
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Association between serum miR-15 or miR-16 at revascularization and restenosis/amputation at follow up (1 year)
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miRNA Summary and Perspective
•miRs modulate the post-ischemic neovascularization responses at multiple levels
•miR therapeutic can directly target ischemic tissues in vivo and be used for ex-vivo enhancement of the proangiogenic capacities of stem and progenitor cells
•Circulating miRs might be novel predictive and pognostic markers in ischemic patients
•There is a huge potential for miR translational research
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Bristol Vena Saphena derived-Pericyte Progenitor Cells (SVPs)
Paolo Madeddu-Wednesday AM
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Isolectin/NG2
2 weeks post-ischemia
Limb muscle, basal conditions
Pericyte coverage of is essential for microvessel maturation and stabilisation, including during post-ischemic vascular repair
NG2pos pericytes around neovessels
59
Isolectin/NG2
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The saphenous vein: a convenient source of pericyte progenitor cells
Vein leftover
60
CABGCampagnolo et al, Circulation, 2010
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CD34 vWF
DAPI CD34 vWF DAPICD34 vWF DAPI
DiDii Diii
Div Dv
E F
CD34 vWF
DAPI CD34 vWF DAPI
Dii Diii
Div Dv
Campagnolo et al. Circulation 2010
CD34 vWF
DAPI CD34 vWF DAPICD34 vWF DAPI
DiDii Diii
Div Dv
E F
Tunica media
Adventitia
Vasa vasorum
Identification of pericytes in human saphenous vein vasavasorum
61
CD34vWFDAPI
CD34
CD34 CD31NG2 DAPI
CD34 CD31PDGF-R bDAPI
vWF
CD34 vWF DAPI
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Conclusions Myocardial and limb ischaemia are still unmet medical
needs
Novel options and revisited approaches hold therapeutic potential to treat limb and myocardial ischaemia
This area of research can deliver disappointments as well as huge satisfaction
Basic science and translational approaches must progress together to the ultimate clinical goal.
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Main collaborators
Emanueli Lab BristolMarco MeloniBetty DescampsTijana MiticLynsey HowardSaran ShantikumarIlaria FlorisMicol MarchettiSobia MushtaqAnran Li
Andrea CaporaliAudrey Nailor
Angelini & Emanueli Lab ImperialAbas H LaftahMore to be appointed...
BristolPaolo Madeddu & LabAndrew MumfordRaimondo AscioneChiara Bucciarelli-DucciGianni AngeliniGraciela Sala-NewbyBarney Reeves BRU unit
ImperialSian HardingAnna RandiMark Perry
GlasgowAndy BakerAshley Miller
KCLQingbo Xu
EdinburghDavid NewbyBruno PeaultRoslin Cells
Sheffield Tim Chico
MilanGaia SpinettiGianluigi Condorelli Maurizio CapogrossiDiego PasiniFabio Martelli
SassariGianfranco Pintus
UdineAntonio Beltrami
HannoverThomas Thum
FrankfurtStefanie DimmelerCarlo Gaetano
YaleBill SessaCarlos Fernandez
UmassNathan Lawson
MadridIntegromics
ViennaKlemens Verlienger
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Emanueli & Madeddu Labs in Bristol
We are currently recruiting and always happy to host visiting PhD students and sponsor excellent candidates for
fellowship [email protected]@yahoo.com