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 )