The long non-coding RNA MIAT regulates smooth muscle cell function and macrophage activity in

advanced atherosclerotic lesions

Yuhuang Li (Daniel), MD, PhDVascular Biology Unit

Department of Vascular and Endovascular SurgeryKlinikum rechts der Isar Technischen Universität München

Munich Vascular Conference, Dec 1-3, 2016

Background: Long non-coding RNA (LncRNA) and its function

<3%

>70% >200 nt

T. Thum, et al, Cir Res.2015~22 nt

Lars Maegdefessel et al.J Cell Physiol. 2011J Clin Invest 2012;Sci Transl Med 2012;Nat Commun 2014ATVB, 2015

miR-26amiR-21miR-24miR-29b

AtherosclerosisAneurysm

R.A.Boon, et al, JACC.2016

Role of lncRNA-MIAT (Myocardial Infarction Associated Transcript) in atherogenesis?

RNA profiling reveals highly increased lncRNA-MIAT in human carotid plaques

NA: normal arteryCP: carotid plaque

RNA profiling

Biobank of Karolinska Endarterectomies (BiKE)

NA

CP

Upregulated MIAT expression correlates with SMC content in human carotid plaque

NA

C

P

SMA

MIAT

SMA

MIAT

NA CP

NA: normal artery CP: carotid plaque

Rel

. MIA

T R

NA

exp

ress

ion

(F

old

ch

ange

)

Validated by q-PCR on additional n=77 BiKE patients

MIAT expression correlates with fate of SMCs in mouse plaque

4 w

Ligation

+ 4 d

Cuff

SMA SMA

MIAT

SMA SMA

NA

CP

NA

CP

MIAT

MIATMIAT

Ki67

Casp3

Ki67

Casp3

NA CP

Overexppressing lncRNA-MIAT promotes proliferation, inhibits apoptosis of HctSMC

40

50

60

70

80

90

0 8 16 24 32 40 48 56 64 72

NC MIAT

0

2

4

6

8

10

0 8 16 24 32 40 48 56 64 72

NC MIAT

Rel

. MIA

T ex

pre

ssio

n

NC

MIATP

rolif

era

tion

(Confluen

ce %

)

hr

Ca

sp

3

A CA

pop

tosis

(Casp3+

/ m

m2)

C

hr

D

B

0

1

2

NC MIAT

900

1100

Knocking down lncRNA-MIAT suppresses proliferation, induces apoptosis of HctSMC

30

50

70

90

110

0 8 16 24 32 40 49 57 65

SCR MIAT KD

Rel

. MIA

T ex

pre

ssio

n

0

0,2

0,4

0,6

0,8

1

1,2

Pro

lifera

tion

(Confluen

ce %

)

hr

0

20

40

60

0 8 16 24 32 40 48 56 64 72

SCR MIAT KD

Apo

pto

sis

(Casp3+

/ m

m2)

C

hr

A CB

Michalik, et al, Circ Res. 2014

NC

MIAT KDCa

sp

3

D

A Transcriptional factor array (free energy <-30 kcal/mol )

lncRNA-MIAT regulates HctSMC functions via regulating transcription factor?

B MIAT effects upon modulation of TF→Abolish single effect on cell fates (IncuCyte)→EGR1 siRNA/GapmeRs?

LncRNA-MIAT

VSMCs

Proliferation

Apoptosis

Carotid plaque development

Via EGR1?

Rel

. mR

NA

exp

ress

ion

(Fo

ld)

MIAT expression correlates with Macrophages in human advanced plaques

Stable Rupture

MIATMIAT

CD68CD68

MIATMIAT

CD68CD68

MRI BiobankDr. Jaroslav Pelisek

MIAT expression correlates with Macrophages in mouse advanced plaques

F4/80

MIAT

F4/80

MIAT

Stable Rupture

Knocking down lncRNA-MIAT impaires oxLDL uptakeof human Macrophage

Rel. M

IAT

expre

ssio

n

oxL

DL

up

take

hr

NC MIAT KD

0

200

400

600

800

1000

1200

0 4 8 12 16 20 24

NC MIAT KD

0

0,2

0,4

0,6

0,8

1

1,2

NC MIAT KD

FIT

C-o

xLD

L

Knocking down lncRNA-MIAT impaires oxLDL uptakeof mouse Macrophage

0

50

100

150

200

250

0 5 10 15 20

NC MIAT KDo

xL

DL

up

take

hr

NC MIAT KD

Rel. M

IAT

expre

ssio

n

0

0,2

0,4

0,6

0,8

1

1,2

NC MIAT KD

FIT

C-o

xLD

L

A

B1. IF staining/WB: NF-KB activation upon MIAT knockdown with/without oxLDL2.MIAT effects upon pathway specific inhibitor

C

QPCR: MIAT level in subtype of microphages (PMA-THP1)

Pro-inflammatory properties of lncRNA-MIAT via regulating Macrophage

LncRNA-MIAT

Macrophage

oxLDL uptake

Inflammatory potent

Carotid plaque progression & unstability

Cytokines array

0

2

4

NC LPS

0

0,5

1

1,5

NC IL4

Rel. M

IAT

expre

ssio

n

M1 M2

A

B

lncRNA-MIAT regulates SMC → Macrophage?

C Effects of MIAT after modulation of KLF4Expression: SM22a, MYH11, ACTA2; Mac2, CD68; Cytokines; Collagens

Rel

. mR

NA

exp

ress

ion

(Fo

ld)

KLF4

pELK-1

G-C Repressor

SM22α Promoter

HDAC2

Phenotypic Switching

KLF

4

NC MIAT MIAT KDSCR

Circ Res. 2012Nat Med. 2015

Summary:LncRNA-MIAT promotes atherosclerotic lesions via regulating

smooth muscle cell functions and macrophage activity

VSMCs

Proliferation

Apoptosis

Carotid plaque development

LncRNA-MIAT

Macrophage

oxLDL uptake

Inflammatory potent

Carotid plaque progression & unstability

Phenotypic Switching

Vascular Surgery, KIUlf HedinClaes BergmarkLjubica PerisicSilvia Aldi

Molecular Vascular Medicine, KIKatja ChernogubovaHong JinAlexandra BäcklundSuzanne M. EkenAlbert BuschChangyan SunNancy SimonAnna OlofssonGreg KorzunowiczLars Maegdefessel

Philip S. TsaoRonald L. DalmanJoshua M. SpinNicholas J. Leeper

Atherosclerosis Research Unit, KIPer ErikssonPeter GustafssonOlivera WerngrenEwa EhrenborgXintong JiangGabrielle Paulsson- BerneGöran K. Hansson

Vascular Biology Unit, TUMLars MaegdefesselJaroslav PelisekShengliang LiuRenate Hegenloh

Genomic structure and evolutionary conservation

Rapicavoli et al. BMC Developmental Biology 2010, 10:49

Gene location and interaction

Human Ch22

Mouse Ch5

lncRNA-MIAT interacts with transcriptional factors

EGR1

NF-KB

Treatment of NFKB inhibitor impaires oxLDL uptakeof mouse Macrophage

oxL

DL

up

take

hr

NFKBi-

oxLDL

Macrophage

0

1

2

3

4

5

0 2 4 6 9 11 13 15

NFKBi-

NFKBi+

NFKBi+

LncRNADisease database

lncRNA-myocardial infarction-associated transcript (MIAT)

lncRNA-MIAT regulates microvascular dysfunction by functioning as a competing endogenous RNA (miR-150, VEGF) (Circ Res. 2015;116:1143-1156.)

Mild but significantly positive correlations of MIAT with markers of T-lymphocytes, inflammation, apoptosis and matrix degradation in carotid plaques. All correlations with other typical markers of cells and pathways were ns.

Correlations with markers in BiKE

CASP9 IL17FTGFB1BCL2

MMP2

MMP24

MMP25

FOXP3

ADAMTS