"When Good Matrix Goes Bad" by Dorothee Weihrauch, DVM, PhD
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What happens when the matrix goes bad? Dorothée Weihrauch DVM, PhD Department of Anesthesiology Medical College of Wisconsin
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Transcript of "When Good Matrix Goes Bad" by Dorothee Weihrauch, DVM, PhD
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
Other Suggestions What comes to your mind
