10/26/2013

1

Cholesterol accumulation in synovial lining

macrophages results in ectopic bone formation

during experimental osteoarthritis

W de Munter, AB Blom, MM Helsen, B Walgreen, PM van der Kraan, LAB Joosten,

WB van den Berg, PLEM van Lent

Department of Experimental Rheumatology,

Radboud university medical center

2013 ACR Annual Meeting

Disclosure

We have no competing interests

Osteoarthritis (OA)

Introduction – Experimental Design – Results – Conclusions

Not only a disease of the cartilage, also synovial involvement.

Synovium Joint cavity Cartilage

Fibroblast

Macrophage

Chondrocyte

Synovial macrophages and OA

• Essential for cartilage destruction during experimental OA.1

• Important players in driving inflammatory and destructive responses in OA.2

• Crucial for osteophyte formation (ectopic bone formation at cartilage margins) and enthesophyte formation (ectopic bone in tendons or

ligaments).3

1Blom et al., 2007 2 Bondeson et al., 2010 3 van Lent et al., 2004

Introduction – Experimental Design – Results – Conclusions

Modified LDL and macrophages

• In an inflammatory milieu, LDL can be modified.1

• Increased LDL levels will therefore result in enhanced oxLDL levels during inflammatory processes.2

• OxLDL is taken up by macrophages via SR-A and CD36.3-5

• OxLDL uptake can change the phenotype of macrophages.3-5

1Morel et al., 1983 2 Badimon et al., 2011 3 Groeneweg et al., 2006 4 van Tits et al., 2011 5 Jiang et al., 2012

Introduction – Experimental Design – Results – Conclusions

Hypothesis

Introduction – Experimental Design – Results – Conclusions

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2

Experimental design

Introduction – Experimental Design – Results – Conclusions

WT

CIOA CIOA

Histology Serum

Washouts

CIOA CIOA

84 days

35 days

LDLr-/-

Collagenase injection: ligament damage joint instability cartilage damage, osteophyte formation and synovial activation

Histology Serum

Washouts

Histology Serum

Washouts

Histology Serum

Washouts

LDLr deficiency and/or a cholesterol-rich diet leads to increased serum cholesterol levels

Introduction – Experimental Design – Results – Conclusions

***p<0.001 compared to WT normal ‡p<0.001 compared to all other groups

*** ***

*** ***

‡ ‡

Synovial lining cells take up ApoB during cholesterol-rich conditions, suggesting oxLDL accumulation

Introduction – Experimental Design – Results – Conclusions

WT

LDLr -/-

Normal diet Cholesterol-rich diet

Isotype control

* A p o B S t a i n i n g S y n o v i a l L i n i n g

0

1

2

3 N o r m a l d i e t

C h o l e s t e r o l - r i c h d i e t

W T L D L r - / -

A r

b i t

r a r

y s

c o

r e

*p<0.05

Norm. diet

Chol.-rich diet

LDLr deficiency or a cholesterol-rich diet does not affect synovial thickening

Introduction – Experimental Design – Results – Conclusions

Synovial thickening

0.0

0.5

1.0

1.5

2.0

2.5

Cholesterol-rich diet

Normal diet

WT LDLr-/-

Arb

itra

ry s

co

re

A cholesterol-rich diet enhances synovial S100A8 expression in WT mice (synovial activation)

Introduction – Experimental Design – Results – Conclusions

Alarmin S100A8 acts as an macrophage activation marker. van Lent et al., 2012 *p<0.05

Normal diet Cholesterol-rich diet Isotype control

*

S100A8 staining synovial lining

0

1

2

3

4

WT LDLr- /-

Arb

itra

rysco

re

WT

LDLr-/-

* * Norm. diet

Chol.-rich diet

LDLr deficiency or a cholesterol-rich diet does not affect cartilage damage

Introduction – Experimental Design – Results – Conclusions

Cartilage damage

Later

al F

emur

Later

al T

ibia

Med

ial F

emur

Med

ial T

ibia

Mea

n

0

5

10

15

20

WT normale

WT cholesterol diete

LDLR-/-

normal

LDLR-/-

cholesterol diet

ns

ns

ns

ns

Arb

itra

ry s

co

re

ns = not significant

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3

ns = not significant

Introduction – Experimental Design – Results – Conclusions

WT with diet LDLr-/- with diet

LDLr deficiency or a cholesterol-rich diet does not affect cartilage damage

LDLr deficiency and a cholesterol-rich diet increase enthesophyte formation

Ectopic Bone Formation

0

2

4

6

8

10Ectopic bone formation

Normal bone formation

Cholesterol-rich diet - + - +

WT LDLr-/-

Nu

mb

er

of

mic

e

Total enthesophyte formation

0

500

1000

1500

2000

Cholesterol-rich diet - + - +

WT LDLr-/-

Cro

ss-s

ecti

on

al

siz

e (

m2)

WT

LDL Normal diet

r -/-

WT Cholesterol-rich diet

Introduction – Experimental Design – Results – Conclusions

Enthesophyte formation Normal bone formation

Normal diet Enthesophyte Formation

LDLr deficiency and a cholesterol-rich diet increase osteophyte formation at the tibial plateau

Introduction – Experimental Design – Results – Conclusions

LDLr-/-

WT

LDLr-/-

Normal Cholesterol-rich

Osteophyte Formation

Later

al T

ibia

Med

ial T

ibia

Mea

n

0

100

200

300

400*

*

**

**

***

LDLR-/-

cholesterol diet

WT normale

WT cholesterol diete

LDLR-/-

normal

Mean

siz

e (

x100

m2)

***p<0.05

***p<0.01

***p<0.001

Osteophyte Formation

Later

al T

ibia

Med

ial T

ibia

Mea

n

0

100

200

300

400*

*

**

**

***

LDLR-/-

cholesterol diet

WT normale

WT cholesterol diete

LDLR-/-

normal

Mean

siz

e (

x100

m2)

Introduction – Experimental Design – Results – Conclusions

LDLr-/-

WT

LDLr-/-

Normal Cholesterol-rich

***p<0.05

***p<0.01

***p<0.001

LDLr deficiency and a cholesterol-rich diet increase osteophyte formation at the tibial plateau

Ectopic bone formation due to growth factors

Growth factors capable of ectopic bone formation • TGF-β is secreted by many cell types, including macrophages, in a latent

form. • TGF-β is activated by proteinases.1

Active TGF-β can be detected using a Luciferase reporter gene assay, wich detects active TGF-β signaling (CAGA box in PAI-1 promotor).2

• Also Bone Morphogenetic Proteins (BMP) 2, 4 and 7 have shown to

induce ectopic bone formation.1

BMP signaling can be detected using a BMP Responsive Element (BRE) Luciferase reporter gene assay.3

Introduction – Experimental Design – Results – Conclusions

1Blany Davidson et al., 2007 2Dennler et al., 1998 3Korchynskyi et al., 2002

LDLr-/- mice on a cholesterol-rich diet have increased levels of active TGF-β in synovial washouts compared to WT mice on a cholesterol-rich diet

CAGA-LUC wash-outs

WT LDLr-/-

WT LDLr-/-

0

50

100

150

Day 7 Day 36

***

[TG

F-

]

(ng

/mL

/g s

yn

oviu

m)

Introduction – Experimental Design – Results – Conclusions

**p<0.05

**p<0.01

Active TGF-β in Synovial Washouts

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In vitro design

Differentiation into macrophages (4 days)

50 µg/mL oxLDL (24 hours) Control

RNA expression growth factors

and functional Luc-assay

Macrophages without oxLDL

Macrophages with oxLDL

M-CSF

Introduction – Experimental Design – Results – Conclusions

OxLDL-stimulation of macrophages increases anabolic processes by activation of TGFβ, rather than production of TGFβ and BMP

Introduction – Experimental Design – Results – Conclusions

**p<0.05

**p<0.01

UD = undetectable

ns = not significant

Gene expression Growth factors

CAGA-LUC

F

Supunstim

. MF

SupoxLDL

MoxLDL

TGF

0

500

1000

1500

2000

2500

500010000

*

*

Lum

ines

cenc

e

TGF BMP2 BMP4 BMP7 IL-12p40-3

-2

-1

0

1

2

UD

DD

Ct

(co

mp

are

dto

un

stim

ula

ted

co

ntr

ol)

**

ns

Unstimulatedmacrophages

OxLDL-stimulatedmacrophages

BRE-Luc

F

Supunstim

. MF

SupoxLDL

MoxLDL

BMP2

0

5000

10000

15000

100000120000

*

Lum

ines

cenc

e

*

(Pos control)

OxLDL-stimulation of macrophages increases anabolic processes by activation of TGFβ, rather than production of TGFβ and BMP

Introduction – Experimental Design – Results – Conclusions

CAGA-LUC

F

Supunstim

. MF

SupoxLDL

MoxLDL

TGF

0

500

1000

1500

2000

2500

500010000

*

*

Lu

min

esce

nce

TGF BMP2 BMP4 BMP7 IL-12p40-3

-2

-1

0

1

2

UD

DD

Ct

(co

mp

are

dto

un

sti

mu

late

dc

on

tro

l)

**

ns

Unstimulatedmacrophages

OxLDL-stimulatedmacrophages

BRE-Luc

F

Supunstim

. MF

SupoxLDL

MoxLDL

BMP2

0

5000

10000

15000

100000120000

*

Lu

min

esce

nce

*Active TGF-β Active BMP

Gene expression Growth factors

CAGA-LUC

F

Supunstim

. MF

SupoxLDL

MoxLDL

TGF

0

500

1000

1500

2000

2500

500010000

*

*

Lum

ines

cenc

e

TGF BMP2 BMP4 BMP7 IL-12p40-3

-2

-1

0

1

2

UD

DD

Ct

(co

mp

are

dto

un

stim

ula

ted

co

ntr

ol)

**

ns

Unstimulatedmacrophages

OxLDL-stimulatedmacrophages

BRE-Luc

F

Supunstim

. MF

SupoxLDL

MoxLDL

BMP2

0

5000

10000

15000

100000120000

*

Lum

ines

cenc

e

*

(Pos control)

**p<0.05

**p<0.01

UD = undetectable

ns = not significant

Conclusions

Introduction – Experimental Design – Results – Conclusions

• Increased serum LDL levels result in enhanced ApoB accumulation in synovium oxLDL accumulation in LDLr deficient mice.

• OxLDL accumulation results in increased activation of synovial macrophages

in WT mice.

• OxLDL accumulation during experimental OA results in ectopic bone formation in the murine knee joint.

• Cholesterol-rich models show higher levels of active TGF-β in synovial washouts than models with less cholesterol accumulation.

• OxLDL accumulation leads to activation of TGF-β and, to a lesser extent,

activation of BMP.

LDL cholesterol accumulation during experimental OA aggravates pathology by activation of synovial resident cells and anabolic pathways

Introduction – Experimental Design – Results – Conclusions

Conclusions

Grant number 10-1-410

Acknowledgements Radboud university medical center Department of Experimental Rheumatology Therapeutics: Peter van Lent, PhD Arjen Blom, PhD Peter van der Kraan, PhD Annet Slöetjes, BSc Birgitte Walgreen, BSc Monique Helsen, BSc Wim van den Berg, PhD Department of Medicine and Nijmegen Institute for Infection, Inflammation and Immunity: Leo Joosten, PhD Leiden University Medical Center Department of Molecular Cell Biology:

Peter ten Dijke, PhD University of Muenster (DEU) Intitute of Immunology: Thomas Vogl, PhD Johannes Roth, PhD Wouter.deMunter@radboudumc.nl

Department of Experimental Rheumatology