josé javier fuster ([email protected]) vascular biology...

José Javier Fuster

([email protected])

Vascular Biology Unit

Instituto de Biomedicina de Valencia

http://www.ibv.csic.es/en/ubv.htm

Deficient phosphorylation of p27 at serine 10

is associated with human atherosclerosis and

aggravates disease progression in

hypercholesterolemic mice

Atherosclerosis is a multifactorial

inflammatory disorder

Cardiovascular Risk Factors

Mechanical injury

Positive cell cycle regulators

Cyclin D1,2,3

CDK4,6

CDK1

Cyclin B

CDK1

Cyclin A

CDK2

Cyclin A CDK2

Cyclin E

p15Ink4b

p16Ink4a

p18Ink4c

p19Ink4d

Ink4

p21Cip1

p27Kip1

p57Kip2

Cip/Kip

CKIs

CDK3

Cyclin C

Growth

suppressors

apoE-/-

p27+/+

apoE-/-

p27+/-

apoE-/-

p27-/-

p27 expression

in aorta

Relative lesion area

*

**†

n=10 n=10

2

4

6

apoE-/-

p27+/+

apoE-/-

p27+/-

apoE-/-

p27-/-

n=12

GLOBAL p27 inactivation

leads to increased VSMC

and M proliferation and

aggravates atherosclerosis

Adapted from J. Vervoorts & B. Lüscher. Cell Mol. Life Sci. 2008

70%

Phosphorylation sites of p27 and known kinases

that modify individual sites

CK2-’

S8

3

CK2-’

Role of Serine 10 phosphorylation on p27

expression and function

Approximately 70% of p27 phosphorylation

occurs at Serine 10

CONTROVERSY: Does phosphorylation of p27

in Ser10 promotes its stability or its

degradation?

CONTROVERSY: Is phosphorylation of p27 in

Ser10 necessary for its nuclear export?

Role of p27 phosphorylation at Ser10 in

atherosclerosis: Animal studies

?apoE-/- versus

apoE-/-p27S10A

P<0.0001

Aortic arch

20

40

p<0.002

% l

es

ion

are

a

apoE-/-

(n=11)

apoE-/-

p27 S10A

(n=11)

apoE-/-

apoE-/-

p27S10A

apoE-/-

(n=11)

apoE-/-

p27 S10A

(n=11)

10

20

% l

es

ion

are

a

Thoracic aorta

p<0.0001

apoE-/-

p27S10AapoE-/-

% le

sio

n a

rea

Aortic arch

20

40

p<0.001

apoE-/-

(n=7)

apoE-/-

p27 S10A

(n=8)

p<0.001

Thoracic aorta

3

6

9

% le

sio

n a

rea

apoE-/-

(n=7)

apoE-/-

p27 S10A

(n=8)

Lack of p27 phosphorylation at Ser10 aggravates

diet-induced atherosclerosis in apoE-null mice

Male mice fed a high-fat diet

for three months

Female mice fed a high-fat diet

for two months

Male mice fed a high-fat diet for three months

Female mice fed a high-fat diet for two months

Lack of p27 phosphorylation at Ser10 aggravates


IIIII

I

Ascending

aorta

Cross-sections lesion analysis

Atherosclerotic lesions in the

aortic sinus and three different

zones in the ascending aorta

(separated aproximately by 30 m)

were analyzed by computer-

assisted planimetry

apoE-/-n=11

apoE-/-p27 S10A

n=11

2

4

AscendingAorta I

p=0.002

Inti

ma/M

ed

ia

2

4

Aorticsinus

Lesio

n A

rea

(mm

2)

apoE-/-n=8

apoE-/-p27 S10A

n=8

p=0.01

2

4

AscendingAorta II

Inti

ma/M

ed

ia

apoE-/-n=11

apoE-/-p27 S10A

n=11

p=0.004

2

4

AscendingAorta III

p=0.007

apoE-/-n=11

apoE-/-p27 S10A

n=11

Inti

ma/M

ed

ia

2

4

Aorticsinus

p=0.01

Lesio

n A

rea

(mm

2)

apoE-/-

n=7

apoE-/-

p27 S10A

n=7

1

2

3

AscendingAorta I

p=0.009

Inti

ma/M

ed

ia

apoE-/-

n=8

apoE-/-

p27 S10A

n=8

1

2

3

AscendingAorta II

p=0.0005

Inti

ma/m

ed

ia

apoE-/-

n=8

apoE-/-

p27 S10A

n=8

Inti

ma/M

ed

ia

1

2

3

AscendingAorta III

p=0.02

apoE-/-

n=7

apoE-/-

p27 S10A

n=7

Lack of p27 phosphorylation at Ser10 aggravates native

atherosclerosis in apoE-null mice (fed regular chow)

20

40

60

p<0.005

Les

ion

are

a (

%)

apoE-/-

(4♀ + 6♂)

apoE-/-

p27S10A

(5♀ + 6♂)

apoE-/-

apoE-/-

p27S10AAORTIC ARCH

Irradiated apoE-/- female mice

transplanted with bone marrow

obtained from apoE-/- or

apoE-/- p27S10A male mice

fed a high-fat diet for 2 months

Lack of p27 phosphorylation at Ser10 restricted to

hematopoietic cells aggravates


COLLABORATORS:

A. Bernad (CNIC, Spain)A. Díez (CNIC, Spain)

Role of phosphorylation of p27 at Ser10 and

atherosclerosis: animal studies

apoE-/- p27S10A

actin

pSer10-p27

apoE-/-

MURINE ASCENDING AORTALack of p27 phosphorylation at Ser10

accelerates murine atherosclerosisCan we extrapolate this to humans?

?

Defective p27 phosphorylation at Ser10 is

associated with mouse and human

atherosclerosis

Human non-atherosclerotic

internal mammary

arteries

Human atheroscleroticcoronary arteries

p-Ser10-p27

p27

-actin

-actin

COLLABORATION with J. Martínez (CSIC)

apoE-/- p27S10A

actin

pSer10-p27

apoE-/-

MURINE ASCENDING AORTA

Ki67

-SMA

Atheroma

Lumen

TO-PRO3

Ki67

-SMA

Atheroma

Lumen

TO-PRO3

-SMA

Atheroma

Lumen

Lack of p27 phosphorylation at Ser10 does not affect VSMC

proliferation in advanced atheromas (male, 3 mo ATHG diet)

apoE-/-

(n=11)

apoE-/-

p27S10A

(n=13)

5

10

% proliferating cells

ns

10

20

% VSMC

ns

10

% proliferating VSMC

ns

TO-PRO3

Ki67Ki67

Mac3

TO-PRO3

Atheroma

Lumen

5

10ns

25

50

75

ns

5

10

ns

Lack of p27 phosphorylation at Ser10 does not affect macrophage

proliferation in advanced atheromas (male, 3 mo ATHG diet)

apoE-/-

(n=10)

apoE-/-

p27S10A

(n=8)

% proliferating cells % M % proliferating M

Mac3

Mac3

Atheroma

Lumen

Atheroma

Lumen

MCP1 expression

1

2p=0.032

MC

P1 r

ela

tive m

RN

A e

xp

ressio

n

n=3 pools/genotype

(each pool 2 aortic archs from

female mice fed high-fat diet

for 2 months)

apoE-/-

apoE-/-

p27S10A

Lack of p27 phosphorylation at Ser10 increases MCP1

mRNA expression

LPS-induced MCP1 expression

10

20

p<0.001

BONE-MARROW DERIVED

MACROPHAGES

ATHEROSCLEROTIC

AORTIC ARCH

Three different experiments performed.

One representative experiment shown.

MC

P1 r

ela

tive m

RN

A e

xp

ressio

n

(rela

ted

to

un

treate

d c

on

tro

l)

No foam cell Foam cells

In vivo foam cells

Lack of p27 phosphorylation at Ser10 increases macrophage

AcLDL uptake and foam cell formation

In vitro AcLDL uptake

apoE-/-

(n=17)

apoE-/-

p27S10A

(n=14)

0.4

0.8

1.2p=0.0004

Rela

tive F

luo

rescen

ce

Med

ian

in

ten

sit

y

PERITONEAL MACROPHAGES

apoE-/-

(n=7)

apoE-/-

p27S10A

(n=7)

65

70

75

80

85

% m

acro

ph

ag

e f

oam

cells

p=0.0001

HYPOTHESIS:

Increased

atherogenesis

associated to lack of

p27 phosphorylation at

S10 is mediated by

excesive activity of the

RhoA/ROCK pathwayROCK1/2

Phosphorylation of downstream targets

(LCP, MLC, ERM, LIM-kinase, adducin, etc)

Cell

migrationCytokine

production

Endothelial

dysfunction

RhoAGDP

RhoAGTP

ATHEROSCLEROSIS

GAP GEF

p27

p27S10AX

Foam cell

formation

Enhanced AcLDL uptake in apoE-/- p27S10A peritoneal

macrophages is attenuated by ROCK1/2 inhibitors

Hydroxyfasudil

p < 0.05 vs. Untreated apoE-/-

Average from 3 experiments

apoE-/-p27S10A (n=9)

apoE-/- (n=11)

*

AcLDL uptake assay

5

10

15

20

Med

ian

flu

ore

sce

nc

e

inte

ns

ity

(x1

03A

U)

Untreated Y-27632

+ 30% + 9 % + 13 %

5

10

15

20

Med

ian

flu

ore

sce

nc

e

inte

ns

ity

(x1

03A

U) *

+ 30% + 9 %

ROCK1/2 inhibitors

ns ns

Lack of p27 phosphorylation at Ser10 accelerates

atherosclerosis development in apoE-null mice without

affecting cell proliferation within the atheroma

Human atherosclerosis is asociated with a marked

reduction in the amount of pS10-p27

Lack of p27 phosphorylation at Ser10 increases foam cell

formation and MCP1 expression in murine macrophages

The increased AcLDL uptake associated to lack of p27

phosphorylation at Ser10 is dependent on the Rho/ROCK

signalling pathway.

p27 phosphorilation at Ser10 and

atherosclerosis: CONCLUSIONS

H. GonzálezJ.J.Fuster

A. Vinué M.J. Andrés J. Cubells

IBV-CSIC

MEC/MCINN, CSIC,

ISC iii, Fondos FEDER,

Generalitat Valenciana,

FINA Biotech, Fundación

Ramón Areces

Financial Support

Collaborators

J. Martínez (Spain)

A. Bernad (Spain)

A. Díez (Spain)

KI. Nakayama (Japan)

V. Andrés

¡THANK YOU VERY

MUCH FOR YOUR

ATTENTION!

• p27S10A accelerates murine atherosclerosis

• Human atherosclerosis is associated to defective

p27 phosphorylation at Ser10

• Proliferation studies

• MCP1 expression

• Foam cell formation

• Rho/ROCK hypothesis

• Foam cell formation and Rho inhibitors





• p27S10A protein levels: VSMC and BMDM

• p27S10A protein levels: arteries

• Atheroma content

• Rho/ROCK ongoing studies

• Body weigth

• Plasma lipid profile

• In vitro proliferation: VSMC and BMDM





p27

Tub

pS10-p27

mVSMC

apoE-/-

apoE-/-

p27

S10

A

p27

Tub

pS10-p27

mVSMC

apoE-/-

apoE-/-

p27

S10

A

p27

Tub

pS10-p27

apoE-/-

apoE-/-

p27

S10

A

BMDMacs

p27

Tub

pS10-p27

apoE-/-

apoE-/-

p27

S10

A

BMDMacs

How does lack of phosphorylation at Ser10

affect p27 protein levels?

Actin

Cdk2

p27

pS10-p27

apoE-null mice

fed a high fat diet

p27

Tub

pS10-p27

mVSMC

apoE-/-

apoE-/-

p27

S10

A

p27

Tub

pS10-p27

mVSMC

apoE-/-

apoE-/-

p27

S10

A

p27

Tub

pS10-p27

apoE-/-

apoE-/-

p27

S10

A

BMDMacs

p27

Tub

pS10-p27

apoE-/-

apoE-/-

p27

S10

A

BMDMacs

Actin

p27

pS10-p27

apoE-null mice

fed regular chow

How does lack of phosphorylation at Ser10

affect p27 protein levels?

Lack of p27 phosphorylation at Ser10 does not affect atherosclerotic plaque

composition in fat-fed apoE-KO mice

apoE-/-

(n=8)

apoE-/-

p27S10A

(n=8)

20

40

60

%

Ma

c3

are

a

Macrofage content

ns

20

40

60

% S

M-α

-acti

n a

rea

apoE-/-

(n=8)

apoE-/-

p27S10A

(n=8)

VSMC content

ns

20

40

60

% c

oll

ag

en

are

a

Collagen content

apoE-/-

(n=8)

apoE-/-

p27S10A

(n=8)

ns

SM--actin staining Collagen stainingMac3 staining

AORTIC SINUS

SM--actin staining Collagen staining

% C

oll

ag

en

are

a

20

40

60

apoE-/-

(n=10)

apoE-/-

p27S10A

(n=10)

Collagen content

Mac3 staining

apoE-/-

(n=10)

apoE-/-

p27S10A

(n=10)

20

40

60

%

Ma

c3

are

a

Macrofage content

Lack of p27 phosphorylation at Ser10 does not affect atherosclerotic

plaque composition in fat-fed apoE-KO mice

ns

% S

M-α

-ac

tin

are

a

20

40

60

apoE-/-

(n=10)

apoE-/-

p27S10A

(n=10)

VSMC content

ns ns

ASCENDING AORTA

ROCK1/2



Cell

migration

Foam cell

formation

Cytokine

production

Endothelial

dysfunction

RhoAGDP

RhoAGTP

ATHEROSCLEROSIS

GAP GEF

p27S10AXp27

Inhibition assays

Is the phenotype

associated to p27S10A

dependent on the

RhoA/ROCK1/2 pathway?

Correlation assays

Is RhoA/ROCK1/2 activity

higher in

cells/tissues/animals

expressing p27S10A?

?? ?

ONGOING STUDIES

IN VITRO & IN VIVO STUDIES

ROCK1/2



Cell

migration

Foam cell

formation

Cytokine

production

Endothelial

dysfunction

RhoAGDP

RhoAGTP

ATHEROSCLEROSIS

GAP GEF

p27S10A

p27

NF-kB

¿Ensayos de inhibición de

actividad NFkB?

¿Ensayos de correlación

actividad NFkB/genotipo

p27?

- EMSA de

extractos de aorta,

BMDMs,

VSMCs…

- IF anti-p65:

¿localización

nuclear?

- Wb anti-

fosfo IkB

- Parthenolide

- BAY 11-7082

- SN50 peptide

- DHMEQ

FUTURE STUDIES

X

Female mice

Male mice

weeks on atherogenic diet0 1 2 3 4 5 8 10 12

10

20

30

BW

(g

)

apoE-/- (n=14)

apoE-/-p27S10A (n=10)

BW

(g

)

10

20

apoE-/- (n=7)

apoE-/-p27S10A (n=15)

Body weight in fat-fed apoE-null mice is unaffected by lack of

p27 phosphorylation at Ser10

weeks on atherogenic diet0 1 2 3 4 5 8 10 12

Male mice on atherogenic diet for 3 months

Lip

ids (

mg

/dL

)

200

400

600

Pre-diet

Post-diet

*p<0.0001 vs prediet

n= 13apoE-/-

Total-Cholesterol

**

apoE-/-

p27S10A

n= 8

HDL-Cholesterol

n= 13apoE-/-

apoE-/-

p27S10A

n= 8

Triglycerides

**

n= 13apoE-/-

apoE-/-

p27S10A

n= 8

Plasma lipid profile in control and fat-fed apoE-null mice is

unaffected by lack of p27 phosphorylation at Ser10

G0/G1 phases

0 10 20 30 4040

50

60

70

80

90

100

110

p27 A/A

p27 S/S

Time after serum stimulation (h)

% c

ell

s

Lack of p27 phosphorylation at Ser10 reduces p27 expressionin cultured bone marrow-derived macrophages but does not

affect their cell cycle kinetics

Asinchronously-growing BMDM

1 0.42

Tubulin

p27

pSer10-p27

apoE-/-apoE-/-

p27S10A

% c

ell

s

S phase

0 10 20 30 400

10

20

30

40

p27 A/A

p27 S/S


G2/M phases

0 10 20 30 400.0

2.5

5.0

7.5

10.0

12.5


% c

ell

sp27 A/A

p27 S/S

* Time after MCSF stimulation

p27

Tubulin

0 3 6 12 18 3024 0 3 6 12 18 3024

Wild-type p27S10A

t (h) *

pSer10-p27

Hoescht BrdU

- Starvation (72h, 0.5% FBS)

- Restimulation (24 h, 20% FBS)

- n=500 cells/genotype

Lack of p27 phosphorylation at Ser10 does not affect

proliferation of cultured primary VSMCs

apoE-/- apoE-/-

p27S10A

25

50BrdU-positive

cells (%)

josé javier fuster ([email protected]) vascular biology...

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