What happens when the matrix goes bad?

Dorothée Weihrauch DVM, PhDDepartment of AnesthesiologyMedical College of Wisconsin

Acknowledgements• John G. Krolikowski• Hao Xu• Omoshalewa Bamkole• Kirkwood A. Pritchard• Ulrich Broeckel

The Disease

Scleroderma

• Autoimmune, connective tissue disorder • Marked increases in fibrosis of the skin and internal

organs• Increases myocardial inflammation• Chronic states of oxidative stress and inflammation

impair heart function in other well-known diseases

thedoctorwillseeyounow.com

Immunofluorescence studies of Hearts of SSc Patients

Animal Model

The Tight Skin Mouse

• Tight skin (Tsk) mutation is a genomic duplication within the fibrillin-1 (Fbn 1) gene

• Synthesize and secrete normal fibrillin (~305 kD), mutant oversized Tsk fibrillin-1 (~405 kD)

• Increases in fibrosis of the skin and internal organ • Myocardial inflammation and fibrosis impair heart function• Tsk fibrillin-1 is stably incorporated into cell layers. • Defects in microtubule assembly and stability • Experience chronic increases in oxidative stress and inflammation

• Chronic vascular disease

C57/Bl6 Tsk-/+ Tsk-/+ +D4F

T15- oxidized Phosphorylcholine

Angiostatin

C57/Bl6 Tsk-/+ Tsk-/+ +D4F

Collagen XVIII/ EndostatinC57/Bl6 Tsk-/+ Tsk-/+ +D4F

In Vitro

# of

cel

ls/w

ell

30000

20000

15000

10000

5000

0

25000

A

Abs

orbe

nce

(450

nm)

B

0.8

0.6

0.4

0.2

0.0

1.0

M T S as s ayE C P r o lif e r a t ion

Tsk -/+ Tsk + D-4F

-/+C57Bl/6J

Tsk -/ + Tsk + D-4F

-/+C57Bl/6J

##

Endothelial Cells seeded on defective matrix have impaired proliferation

Rela

tive F

luores

cenc

e Unit

s

% o

f TUN

EL p

ositiv

e ce

lls/w

ell 500

400

300

200

100

0

Caspase Activity by FLICAApoptosis by TUNEL

Tsk -/+ Tsk + D-4F

-/+C57Bl/6J

Tsk -/+ Tsk + D-4F

-/+C57Bl/6J

Viable CellsTunnel Positive Cells #

80

60

40

20

0

100#

A B

Endothelial Cells seeded on defective matrix undergo more often apoptosis

C57Bl/6J

Tsk -/+

Tsk +D-4F-/+

FSP-1

CBA

Fluorescence In tensity

0 10 20 30 40 50

*

7060

C57Bl/6J

Tsk -/+

Tsk +D -4F-/+

Twist

0 10 20 30 40 50

*

60

*

VE-cadherin

0 10 20 3025 355 15

*

Endothelial Cells seeded on defective matrix express FSP-1

VE-Cadherin

FSP-1

twist

Tsk-/+ +D-4F Tsk-/+ C57Bl/6J

C57Bl/6J

Tsk-/+

Tsk +D-4F-/+

A B C

C57Bl/6J

Tsk -/+

Tsk +D-4F-/+

0 1 0 2 0 3 0 4 0 5 0 7 06 0

*

DDR2 Coll-1

Fluorescence In tensity

Hsp47

0 10 20 30 40 50 60

*

0 10 20 30 40 50

*

Endothelial Cells seeded on defective matrix express Collagen-I related proteins

Tsk-/+ +D-4F Tsk-/+ C57Bl/6J

Hsp47

DDR-2

Coll-1

b-Actin

IL-6 (Endothelial Cells on matrix)

Gelatin C57 Tsk/+720

740

760

780

800

820

840

860

c-fos expression in human car-diomyocytes on different matrices

Mea

n Gr

een

Fluo

resc

ence

C57 Tsk/+600

700

800

900

BNP expression in human cardiomyocytes on different matrices

Mea

n Gr

een

Fluo

resc

ence

Characterization of the Matrix

Tsk/+ C57

PDGF

TGFβ and PDGF are bound to ECM isolated from Tsk/+

Tsk/+ C57

TGFb

Advanced Oxidized Protein Products (matrix)

C57 Tsk0.38

0.39

0.4

0.41

0.42

0.43

0.44

0.45

0.46

0.47

0.48

Abso

rptio

n

*

*

4 - Hydroxynonenal (matrix)

Dens

ity

C57 Tsk0

200

400

600

800

1000

1200

C57 Tsk

Advanced Glycated End Products (matrix)

*

3 – Nitrotyrosine (matrix)

75 kDa

37 kDa

25 kDa

100 kDa

C57 Tsk

C57 Tsk0

40000

80000

120000

1600003-Nitrotyrosine

*

Oxidized adducts on Matrix • What else to look for• How to remove them

Oxidative Stress • How to better measure it

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