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Klinikum der Johann Wolfgang Goethe Universität Frankfurt am Main. Antagomirs and angiogenesis Stefanie Dimmeler Institute for Cardiovascular Regeneration Center of Molecular Medicine. Supported by: - PowerPoint PPT Presentation
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Klinikum derJohann Wolfgang Goethe Universität
Frankfurt am Main
Antagomirs and angiogenesisAntagomirs and angiogenesis
Stefanie DimmelerStefanie DimmelerInstitute for Cardiovascular RegenerationInstitute for Cardiovascular Regeneration
Center of Molecular MedicineCenter of Molecular Medicine
Supported by:DFG (SFB 553, FOR 501), the European Network of Excellence(EVGN) and the Transatlantic Network of Excellence (Leducq Foundation)
Supported by:DFG (SFB 553, FOR 501), the European Network of Excellence(EVGN) and the Transatlantic Network of Excellence (Leducq Foundation)
Non-coding DNA & RNA and microRNAs
Non-coding DNA & RNA and microRNAs
microRNA
MicroRNA are encoded by introns or intergenic regions
microRNA
Gene 1 Gene 2
a) b)
Length: app. 22 bpmicroRNA Each microRNA binds
to and blocks up to hundreds of genes
Dicer/Drosha
Role of miRNA processing enzymes in angiogenesis
Role of miRNA processing enzymes in angiogenesis
Drosha
miRNAGene
Pri-miRNA
Pre-miRNA
Dicer
miRNAduplex
RISCassembly
Translationalrepression
mRNAcleavage
Nucleus
Because miRs interfere with patterns of targets, miRs may represent an attractive therapeutic target to interfere with complex processes such as
neovascularization and tissue repair.
8mer (7mer)
Watson-Crick and G:U pairing at position 15-17
AU-rich content/ conserved binding site
Target site close to poly(A) (position 989 – 996)
5' ...UAAACUCUGUUGCAAGUGCAAUA...
3' GUCCGGCCCUGUUCACGUUAU
3´UTR target
miR-92a
TARGET PREDICTION
MicroRNAs in the heartMicroRNAs in the heart
(Van Rooij et al., Circ Res 2008)
Expression profile of microRNAs in endothelial cells
Expression profile of microRNAs in endothelial cells
miR-17-92Cluster
Role of highly
expressed microRNAs?
c-kit
VCAM, PIK3R2, SPREDHox/Gax
TSP-1TSP-1
Kuehbacher et al, Circ Res 2007
•The miR-17-92 cluster is induced in myc-induced tumors (enhancement of The miR-17-92 cluster is induced in myc-induced tumors (enhancement of tumor angiogenesis by paracrine effects (miR-18:CFGF; miR-19: TSP1) tumor angiogenesis by paracrine effects (miR-18:CFGF; miR-19: TSP1) (Dews (Dews et al, Nature Genetics)et al, Nature Genetics)
•miR-17~92 deficient mice defect in B lymphopoiesis, lung development and miR-17~92 deficient mice defect in B lymphopoiesis, lung development and ventricular septal defects ventricular septal defects (Ventura et al, Cell 2008)(Ventura et al, Cell 2008)
•miR-92 has several interesting predicted targets involved in vessel miR-92 has several interesting predicted targets involved in vessel remodelling and angiogenesisremodelling and angiogenesis
•miR-92 is induced by ischemia and is up-regulated in patients with CADmiR-92 is induced by ischemia and is up-regulated in patients with CAD
Functions of the miR-17~92 cluster Functions of the miR-17~92 cluster
Mouse: Chromosome 14 qE4 Human: Chromosome 13
§
§
§
Expression of miR-92a by ischemia Expression of miR-92a by ischemia
Regulation of neovascularization by miR-92a?Hind limb ischemia
miR-92a regulates angiogenesis and miR-92a regulates angiogenesis and vessel patterningvessel patterning
Angiogenic sprouting & Angiogenic sprouting & Vessel formationVessel formation
Pre-miR-92
miR-92
Spheroid model Network formation Matrigel plug model
O-methyl-miR-92inhibitor
Zebra fish
Cu
mu
lati
ve
sp
rou
t le
ng
th p
er
sp
he
roid
(%
vs
. C
on
tro
l) *
(Bonauer et al, Science 2009)
Silencing of miR-92a in vivo using antagomirsSilencing of miR-92a in vivo using antagomirs
Antagomirs (Krutzfeldt,et al. Nature 2005)
• single-stranded RNA oligonucleotides
• complementary to specific miRNAs
• chemical modification for stability and cholesterol conjugation for better delivery
(Krutzfeldt, J. et al. Nature, 2005)
Krutzfeldt, J. et al. Nature Genetics (2006)
80 mg/kg i.v.
miR-92a expression
(heart)
Antagomir-92a specifically inhibits miR-92aexpression
Antagomir-92a specifically inhibits miR-92aexpression
n.s.
p<0.05
p<0.05n.s.
8 mg/kg i.v. single injection48 h
miR-92a uauugcacuugucccggccugu
miR-92b uauugcacucgucccggccucc
Inhibition of miR-92a enhances cell invasionin matrigel plugs
Inhibition of miR-92a enhances cell invasionin matrigel plugs
Antagomir 92aPBS*
DapiLectin-FITC
DapiLectin-FITC
Antagomir-Co Antagomir-92a
Antagomir-CoAntagomir-
92aLectin-perfused vessels
23.7±5.5 48.6 ±7.9*
*
Inhibition of miR-92a stimulates recovery afterhind limb ischemia
Inhibition of miR-92a stimulates recovery afterhind limb ischemia
*PBS
Antagomir 92a
Ischemic leg
Ischemic leg
* * *# #
*Antagomir-Co
nuclei Lectin
SMA merge
Antagomir-92a
nuclei Lectin
SMA merge
Recovery of Recovery of
Blood flow Blood flow
HistologyHistology
14 days
C57/Bl6 mouse
Day 0,2,4,7, and 9
Antagomir 92a
Hind limb ischemia
Inhibition of miR-92a improves functionalrecovery after acute myocardial infarction Inhibition of miR-92a improves functionalrecovery after acute myocardial infarction
p<0.05
p<0.05
p<0.05
p<0.05p<0.05
p<0.05
sham AMI+PBS AMI+Antag.-92aAMI+ Antag.-co
Pre
ssu
re (
mm
Hg
)
Pre
ssu
re (
mm
Hg
)
Pre
ssu
re (
mm
Hg
)
Pre
ssu
re (
mm
Hg
)
Volume (µl) Volume (µl) Volume (µl) Volume (µl)
Recovery of Recovery of
heart heart
functionfunction
14 days
v
C57/Bl6 mouse
Day 0,2,4,7, and 9
Antagomir 92a
Acute myocardialinfarction W
MS
I
Antagomir- Co
Antagomir- 92a
Inhibition of miR-92a reduces infarct size and Inhibition of miR-92a reduces infarct size and perfusion after acute myocardial infarction perfusion after acute myocardial infarction
Lec
tin
-FIT
C
Antagomir-Co Antagomir-92a
Remote Border Infarct
#
#
Antagomir-Co Antagomir-92a
44+3% reduced infarct sizeIn antagomir-92a-treated mice
*
Inhibition of miR-92a improves recovery after ischemia
Inhibition of miR-92a improves recovery after ischemia
Antagomir-92a effectively and specifically reduces miR-92a expression
Antagomir-92a improves neovascularization of matrigel plugs and after hind limb ischemia
Antagomir-92a improves recovery after acute myocardial infarction:
Enhanced neovascularizationReduced infarct size
Is this beneficial effect due to a specific influence onendothelial cells / neovascularization?Expression and effect in other cardiac cells?Endogenous repair?
miR-92a targets relevant to neovascularization
miR-92a targets relevant to neovascularization
Target prediction using TargetScanS database
Predicted target Function Phenotype Reference
Integrin Cell-matrix interaction Embryonic lethal Yang et al., Development 1993
SIRT1 Histone deacetylase Lethal just before or after birth
Cheng et al., PNAS 2003
Rap1b GTP-binding protein Embryonic lethal Chzanowska-Wodnicka et al., Blood 2008
MKK4 Mitogen activated map kinase
Embryonic lethal Yang et al., PNAS 1997
EDG1 sphingosin-1-phosphate receptor
Embryonic lethal Liu et al., JCI 2000
Targets downregulated by miR-92Targets downregulated by miR-92
cont
rol
pre9
2
Integrin 5
eNOS
MKK 4
Tubulin
*
*
*
*
*
ProteinExpressionWestern blot
mRNA expression Microarray
Function of integrin 5 in vascular biology
Function of integrin 5 in vascular biology
Integrin subunit 5 (Fibronectin
receptor) plays a crucical role in vascular biology:
•Integrin 5 -/- mice: Embryonic lethal vessel patterning defect
• Shear - regulated (mechanotransduction)
• Protects endothelial cells from apoptosis
• Essential for endothelial cell migration and adhesion
(Yang / Hynes Development 1993; Mol Biol Cell 1996; Francis, ATVB; Urbich et al, ATVB 2000)
Matrigelplug
Hind limb ischemia
miR-92 regulates Integrin 5 by binding the 3`UTR
miR-92 regulates Integrin 5 by binding the 3`UTR
AAACUCUGUUGCAAGUGCAAUAAAUCUGACCCAGUGAAACUCUGUUGCAAGUGCAAUAAACCCCACCCACUGAAACUCUGUUGCAAGUGCAAUAAACCCCACUCACUGAAACUCUGUUGCAAGUGCAAUAAACCCGGCCCGGUG
HumanMouseRatDog
GUCCGGCCCUGUUCACGUUAU hsa-miR-92a
ITGA5 3`UTRITGA5 3`UTRITGA5 3`UTRITGA5 3`UTR
Seed sequence
CMV Luciferase 3`UTR
0.2 kb 0.4 kb 0.6 kb 0.8 kb 1 kb
Integrin 5 3`UTR
miR-92
*
Wild typeMutated
Sequence
•Integrin a5 is increased by antagomir-92a treatment in vivo•Integrin a5 lacking the endogenous 3ÚTR partially rescues the anti-angiogenic activity of miR-92a•Integrin a5 siRNA reduced the proangiogenic activity of antagomir-92a
miR-92a
SIRT1
others
Integrinv
FBXW7
Mkk4
EDG-1
eNOS
Integrin 5
microRNA StudiesmicroRNA Studies
A. Kühbacher / BonauerA. Kühbacher / BonauerC. DoebeleC. DoebeleH. FoxH. Fox
G. CarmonaG. CarmonaC. UrbichC. UrbichJ. BurchfieldJ. BurchfieldM. IwasakiM. IwasakiM. KoyanagiM. KoyanagiM. PotenteM. PotenteE. ChavakisE. Chavakis
A. FischerA. FischerT. RöxeT. RöxeM. Muhly-ReinholzM. Muhly-Reinholz
COLLABORATORSCOLLABORATORS
A.A. ZeiherZeiher
M. Tjwa (Leibniz Group)M. Tjwa (Leibniz Group)
M. Mione (Milano)M. Mione (Milano)
Support:Alfried Krupp StiftungLeibniz Preis der DFGDeutsche Forschungsgemeinschaft (SFB553, TR-SFB23)Leducq Foundation: Transatlantik Network of ExcellenceExcellence Cluster ECCPSEuropean Union: IP Heart RepairERC Advanced Grant
microRNA Team
Dr. Angelika Bonauer(Kühbacher)
DapiLectinCy3
d1
Distribution of Cy3-conjugated antagomir-92
Distribution of Cy3-conjugated antagomir-92
Dapi Lectin-FITC
Cy3-Antagomir 92a Merge
Infusion of Cy3-conjugated antagomir-92, 8 mg/kg b.w.
miR-92a expression pattern: effect of antagomirs-92a
miR-92a expression pattern: effect of antagomirs-92a
miR-92a
Antagomir-Colectin
DAPI Merge
miR-92a
Antagomir-92alectin
MergeDAPI
U6, Lectin, DAPI
In situ U6 (control)
miR-92a is expressed in endothelial cells,but also in cardiomyocyte/interstitial cell
In situ hybridisation miR-92a
miR-92a
Neovascularization & Functional recovery after ischemia
Matrigel plug model
Hindlimbischemia
Acute myocardial infarction
Targets?
Antagomir-92a increases Integrin 5 levels in vivo
Antagomir-92a increases Integrin 5 levels in vivo
Control Antagomir-92a
Dapi Integrin 5 Dapi Integrin 5§ #
miR-92a regulates Integrin a5 expression in vitro and in vivo
Day 2 Day 2
qRT-PCRHind limb ischemia, day 2
Involvement of Integrin 5 Involvement of Integrin 5
AngiogenesisAngiogenesis
Antagomir-92a
miR-92
Integrin 5 ?scrambled siRNA +
-
-+
-+
-+
+-
+-
-+
+-
p<0.05
p<0.05
Integrin 5 siRNAPBSAntagomir-92a
Antagomir-92a increases Integrin 5 levels in vivo
Antagomir-92a increases Integrin 5 levels in vivo
Control Antagomir-92a
Dapi Integrin 5 Dapi Integrin 5§ #
miR-92a regulates Integrin a5 expression in vitro and in vivo
Day 2 Day 2
qRT-PCRHind limb ischemia, day 2
Involvement of Integrin 5 Involvement of Integrin 5
AngiogenesisAngiogenesis
Antagomir-92a
miR-92
Integrin 5
Integrin 5 contributes to antagomir-92a-mediatedstimulation of angiogenesis in vitro
Pre-miR-92
Integrin a5(lacking3’ UTR)
Plasmid: pcDNA ITGA5 pcDNA ITGA5miR-92a: - - + +
*
Network formation (matrigel assay)
MiR-92a is upregulated by ischemiaMiR-92a is upregulated by ischemia
miR-92a uauugcacuugucccggccugu
miR-92b uauugcacucgucccggccucc
Expression of miR-92aExpression of miR-92a
cardiomyocytes non-cardiomyocytes
miR
-92
expr
essi
on (
% c
ontr
ol)
MiR-92 overexpression reduces in vitro angiogenesis
MiR-92 overexpression reduces in vitro angiogenesis
Co
ntr
ol
mir-92
U6
pre
92
Ma
trig
el
ne
two
rk f
orm
ati
on
(%
vs
. C
on
tro
l)
Cu
mu
lati
ve
sp
rou
t le
ng
th p
er
sp
he
roid
(%
vs
. C
on
tro
l)*
Control pre92
sp
rou
t fo
rma
tio
n v
as
cu
lar
ne
two
rk
form
ati
on
miR-92 miR-92 AngiogenesisIn vitro vessel
formation
Overexpression of pre-miR-92a
Pre-miR-92a: Inhibition of migrationNo effect on apoptosis/proliferation (48 h)
miR-92 overexpression reduces invasion of endothelialcells and reperfusion of matrigel plugs
miR-92 overexpression reduces invasion of endothelialcells and reperfusion of matrigel plugs
Hemoglobin (Hb) content
Perfusion
s.c. injection
6 days
H&E staining
Invaded cells
500 µl Basal
Membrane MatriGel
Transfection of HUVEC withCo or pre92 in
vitro
*
*
Matrigel plugs implanted with:
Lectin-FITCDapi
Lectin-FITCDapi
HUVEC control HUVEC pre92
HUVEC control
HUVEC pre92
Lectin-perfused vessels
44.6 ±16.1 3.3 ±1.2*
Control Control
pre92 pre92
pre92pre92
Number of ISV
evaluated
Control pre miR
(n=7)
Pre miR92a
(n=7)12 12 412 11 512 12 612 12 412 12 812 12 512 11 6
miR-92 induces a vascular patterning defect inmiR-92 induces a vascular patterning defect inzebrafishzebrafish
Genbank Common ProductPre92/control
Predicted miR-92a target
Down-regulated by Integrin 5
NM_002205 CD49e Integrin 5 0.39 yes yes
NM_001400 EDG1 Endothelial differentiation, sphingolipid G-protein-coupled receptor, 1
0.54 yes no
NM_003010 MEK4 Mitogen-activated protein kinase kinase 4 0.59 yes no
NM_012238.3 Sirt1 Sirtuin 1 0.77 yes no
NM_002428 MT2-MMP Matrix metalloproteinase 15 0.24 no yes
NM_004995 MT1-MMP Matrix metalloproteinase 14 0.44 no yes
NM_000603 eNOS Nitric oxide synthase 3 (endothelial cell) 0.54 no yes
M37780 CD31 Platelet/endothelial cell adhesion molecule 0.63 no yes
NM_000201 ICAM1 Intercellular adhesion molecule 1 (CD54) 0.44 no no
Overexpression of miR-92 decreases a large subset of pro-angiogenic genes
Overexpression of miR-92 decreases a large subset of pro-angiogenic genes
Predicted target and directly downregulated by miR-92a
Secondarily downregulated by Integrin 5
No predicted target and not regulated by Integrin 5
miR-106-363cluster
**
Sham PBS Antagomir 92a
Age (weeks) 12 12 12
Weight (g) 21.4±1.7 20.9±1.2 21.6±0.6
HR (bpm) 455±110 478±38 482±45
LVESP (mmHg) 98±1 82±8 91±11
LVEDP (mmHg) 5.6±1.0 18.0±6.0 7.6±2.0
positive dp/dt (mmHg/sec) 10418±2314 6376±1419 9022±1542
negative dp/dt (mmHg/sec) 8482±1252 5867±1095 8019.81±810
HR: heart rate, LVESP: left ventricular end systolic pressure, LVEDP: left ventricular end diastolic pressure
Antagomir-92a improves cardiac functionAntagomir-92a improves cardiac function
Importance of vascularizationImportance of vascularization
• Neovascularization is essential for functional recovery after ischemia
• Vascularization is important for cardiac repair and regeneration (whatever cells are used)
• Microvascular dysfunction after reperfusion therapy or during heart failure might be a target for therapy
Integrinα5 and Delta/Notch Signaling Have Complementary Spatiotemporal Requirements during Zebrafish Somitogenesis Dörthe Ju¨lich1, Robert Geisler2, Tu¨bingen 2000 Screen Consortium3, 4 and Scott A. Holley1,
,
1Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 065202Max Planck-Institut fu¨r Entwicklungsbiologie, Tu¨bingen, D-72076, Germany
Expression of miR-92a Expression of miR-92a
N=6
N=15
Mir-92 expression in bone marrow-derived cells isolated from healthy controls
or patients with CAD
miR
-92a
exp
ress
ion
(Del
ta C
T)
Risk factors for coronary artery disease impair circulating and bone marrow-derived stem/progenitor cells
(Dimmeler & Leri, Circ Res 2008)
**
Inhibition of miR-92a enhances cell invasionin matrigel plugs
Inhibition of miR-92a enhances cell invasionin matrigel plugs
Antagomir 92aPBS *
DapiLectin-FITC
DapiLectin-FITC
Antagomir-Co Antagomir-92a
Antagomir-CoAntagomir-
92aLectin-perfused vessels
23.7±5.5 48.6 ±7.9* Ves
sels
( %
Ant
ago
mir-
Co
)
miR-92a: effect on cardiac myocytes?miR-92a: effect on cardiac myocytes?
Antagomir-92a - - + - +
0 % serum
H2O2
Apoptosis of cardiac myocytes in vitro
Cell death in vivo
miR-92 has a minimal effect on hypertrophy (Sucharov et al, JMCC 2008)
Antagomir-92a reduce apoptosis in vivo (by an indirect effect )