A novel role for epidermal growth factor receptor tyrosine kinase and its downstream endoplasmic reticulum stress in cardiac damage and microvascular dysfunction in type 1 diabetes mellitus.

Abstract

Epidermal growth factor receptor tyrosine kinase (EGFRtk) and endoplasmic reticulum (ER) stress are important factors in cardiovascular complications. Understanding whether enhanced EGFRtk activity and ER stress induction are involved in cardiac damage, and microvascular dysfunction in type 1 diabetes mellitus is an important question that has remained unanswered. Cardiac fibrosis and microvascular function were determined in C57BL/6J mice injected with streptozotocin only or in combination with EGFRtk inhibitor (AG1478), ER stress inhibitor (Tudca), or insulin for 2 weeks. In diabetic mice, we observed an increase in EGFRtk phosphorylation and ER stress marker expression (CHOP, ATF4, ATF6, and phosphorylated-eIF2α) in heart and mesenteric resistance arteries, which were reduced with AG1478, Tudca, and insulin. Cardiac fibrosis, enhanced collagen type I, and plasminogen activator inhibitor 1 were decreased with AG1478, Tudca, and insulin treatments. The impaired endothelium-dependent relaxation and -independent relaxation responses were also restored after treatments. The inhibition of NO synthesis reduced endothelium-dependent relaxation in control and treated streptozotocin mice, whereas the inhibition of NADPH oxidase improved endothelium-dependent relaxation only in streptozotocin mice. Moreover, in mesenteric resistance arteries, the mRNA levels of Nox2 and Nox4 and the NADPH oxidase activity were augmented in streptozotocin mice and reduced with treatments. This study unveiled novel roles for enhanced EGFRtk phosphorylation and its downstream ER stress in cardiac fibrosis and microvascular endothelial dysfunction in type 1 diabetes mellitus.

Full text

Page 1

Henrion and Khalid Matrougui
Maria Galán, Modar Kassan, Soo-Kyoung Choi, Megan Partyka, Mohamed Trebak, Daniel
Dysfunction in Type 1 Diabetes Mellitus
Downstream Endoplasmic Reticulum Stress in Cardiac Damage and Microvascular
A Novel Role for Epidermal Growth Factor Receptor Tyrosine Kinase and Its
Print ISSN: 0194-911X. Online ISSN: 1524-4563
Copyright © 2012 American Heart Association, Inc. All rights reserved.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Hypertension
doi: 10.1161/HYPERTENSIONAHA.112.192500
2012;60:71-80; originally published online June 4, 2012;
Hypertension.

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A Novel Role for Epidermal Growth Factor Receptor
Tyrosine Kinase and Its Downstream Endoplasmic
Reticulum Stress in Cardiac Damage and Microvascular
Dysfunction in Type 1 Diabetes Mellitus
Maria Galán, Modar Kassan, Soo-Kyoung Choi, Megan Partyka, Mohamed Trebak,
Daniel Henrion, Khalid Matrougui
See Editorial Commentary, pp 20–21
Abstract—Epidermal growth factor receptor tyrosine kinase (EGFRtk) and endoplasmic reticulum (ER) stress are important
factors in cardiovascular complications. Understanding whether enhanced EGFRtk activity and ER stress induction are
involved in cardiac damage, and microvascular dysfunction in type 1 diabetes mellitus is an important question that has
remained unanswered. Cardiac fibrosis and microvascular function were determined in C57BL/6J mice injected with
streptozotocin only or in combination with EGFRtk inhibitor (AG1478), ER stress inhibitor (Tudca), or insulin for 2 weeks.
Indiabeticmice,weobservedanincreaseinEGFRtkphosphorylationandERstressmarkerexpression(CHOP,ATF4,ATF6,
and phosphorylated-eIF2?) in heart and mesenteric resistance arteries, which were reduced with AG1478, Tudca, and insulin.
Cardiac fibrosis, enhanced collagen type I, and plasminogen activator inhibitor 1 were decreased with AG1478, Tudca, and
insulin treatments. The impaired endothelium-dependent relaxation and -independent relaxation responses were also restored
after treatments. The inhibition of NO synthesis reduced endothelium-dependent relaxation in control and treated
streptozotocin mice, whereas the inhibition of NADPH oxidase improved endothelium-dependent relaxation only in
streptozotocin mice. Moreover, in mesenteric resistance arteries, the mRNA levels of Nox2 and Nox4 and the NADPH
oxidase activity were augmented in streptozotocin mice and reduced with treatments. This study unveiled novel roles for
enhanced EGFRtk phosphorylation and its downstream ER stress in cardiac fibrosis and microvascular endothelial
dysfunction in type 1 diabetes mellitus. (Hypertension. 2012;60:71-80.) ● Online Data Supplement
Key Words: EGFRtk ? ER stress ? type 1 diabetes ? Tudca ? cardiac fibrosis ? resistance arteries
? endothelial function
D
Increasing evidence from experimental and clinical studies
indicates a higher prevalence of cardiac damage and micro-
vascular complications in diabetic patients.3–7Epidermal
growth factor receptor (EGFR) is a glycoprotein containing a
single transmembrane domain with intracellular portion har-
boring the tyrosine kinase domain. The EGFRtk is regulated
by glucose through EGFR-N-glycosylation,8and although
there is a plethora of information on the growth-promoting
iabetes mellitus is a major cause of morbidity and
mortality worldwide and is a threat to human health.1,2
effects of EGFR, its role in cardiovascular complications in
type 1 diabetes mellitus remains unknown. We and others
have demonstrated that increased EGFRtk phosphorylation
contributes to resistance artery dysfunction in type 2 diabetes
mellitus.9,10In addition, it has been reported that the inhibi-
tion of EGFR activity promotes vasodilatation and reduces
elevated arterial blood pressure in hypertensive animal mod-
els with or without insulin resistance.11,12
Recently, several studies in different cancer cell lines and
human tissues have reported a relationship between aberrant
Received February 5, 2012; first decision February 13, 2012; revision accepted May 7, 2012.
From the Department of Physiology, Hypertension, and Renal Center of Excellence (M.G., M.K., S.-K.C., M.P., K.M.), Tulane University, New
Orleans, LA; Center for Cardiovascular Sciences (M.T.), Albany Medical College, Albany, NY; Centre National de la Recherche Scientifique UMR 6214
(D.H.), Angers, France; Institut National de la Santé et de la Recherche Médicale U771 (D.H.), Angers, France, CHU d’Angers, Angers France, Université
d’Angers, Angers, France.
M.G. and M.K. are cofirst authors.
M.G. and M.K. researched data and wrote the article; S.-Y.C. and M.P. researched data. M.T. and D.H. contributed to the discussion and reviewed the
article. K.M. was the project director, contributed to the discussion, wrote the article, and was the guarantor.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.
112.192500/-/DC1.
Correspondence to Khalid Matrougui, Department of Physiology, Hypertension, and Renal Center of Excellence, Tulane University, 1430 Tulane Ave,
New Orleans, LA 70112. E-mail kmatroug@tulane.edu
© 2012 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.orgDOI: 10.1161/HYPERTENSIONAHA.112.192500
71
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EGFRtk expression-activation and endoplasmic reticulum
(ER) stress-related proteins.13,14ER stress plays a critical role
in the pathogenesis of diabetes mellitus and associated
cardiovascular complications.15,16Various cellular stresses,
including ischemia, hypoxia, gene mutation, oxidative stress,
and protein synthesis overload, lead to impairment of ER
function and create a state termed “ER stress” that leads to the
activation of a complex signaling network called the unfolded
protein response.17,18The unfolded protein response is regu-
lated in the cell by 3 ER membrane-associated proteins that
act as sensors of ER homeostasis. The 3 membrane-bound
protein are protein kinase-like ER eukaryotic initiation factor
2? kinase, inositol requiring ER-to-nucleus signaling protein-
1?, and activating transcription factor 6 (ATF6). The involve-
ment of ER stress in the development of diseases, such as
obesity, stroke, myocardial ischemia, and type 2 diabetes
mellitus, has been widely demonstrated and is considered a
key element in pancreatic ?-cell dysfunction and peripheral
insulin resistance.18–23
The significance and role of exacerbated EGFRtk and ER
stress in cardiac damage and microvascular dysfunction in
type 1 diabetes mellitus are important questions that have
remained unanswered. Thus, the aim of this study was to
determine the role of increased EGFRtk activity and its
downstream ER stress as important factors in cardiac damage
and microvascular dysfunction in the type 1 diabetic mouse
model.
Materials and Methods
See the online-only Data Supplement.
Results
Effect of EGFRtk Inhibition on Glucose Level,
Body Weight, Cardiac Fibrosis, and ER Stress
Markers in the Heart
The induction of type 1 diabetes mellitus with streptozotocin
(STZ) injection increased blood glucose levels to 408%
compared with control mice (100%; Figure 1A), decreased
the body weight to 40% (Figure 1B), and induced cardiac
fibrosis associated with increased plasminogen activator in-
hibitor (PAI) 1 (3.5-fold) and collagen type 1 expression
(3-fold; Figure 1C through 1E). In STZ mice, the inhibition of
EGFRtk reduced blood glucose level to 337% and increased
the body weight to 84% (Figure 1A and 1B). Fibrosis was
reduced by 50%, and collagen type 1 expression was reduced
to 2-fold compared with STZ mice, whereas PAI-1 was
blunted (Figure 1C through 1E). Interestingly, the mRNA
levels and phosphorylation of EGFRtk were augmented by
2.5- and 2.0-fold, respectively, in cardiac tissue from STZ
mice and were significantly reduced after EGFRtk inhibition
(Figure 1F and 1G). ER stress marker expression assessed by
real-time RT-PCR revealed an increase by 2.0- and 2.5-fold
for CHOP, ATF4, and ATF6, respectively, in cardiac tissue
from STZ mice and were reduced after EGFRtk inhibition,
with the exception of ATF6 (Figure 1H, 1J, and 1K). The
protein expression of CHOP was increased by 4-fold in STZ
mice and blunted after EGFRtk inhibition (Figure 1I).
Effect of ER Stress Inhibition on Glucose Level,
Body Weight, Insulin, Cardiac Fibrosis, and ER
Stress Markers in the Heart
The injection of STZ significantly increased blood glucose
concentration to 436% compared with control mice (100%)
and was significantly reduced to 326% after ER stress
inhibition and normalized with insulin injection (94%; Figure
2A). The ER stress inhibition increased body weight from
58% in STZ mice to 72%, whereas the treatment with insulin
completely restored the body weight to control mice value
(Figure 2B). Insulin level was not detectable in STZ mice
treated with or without Tudca (0.036?0.0 and 0.035?0.0
ng/L, respectively) but was restored with insulin treatment
(0.99?0.2 ng/L; Figure 2C).
We observed cardiac fibrosis induction in STZ mice
evidenced by an increase in collagen type I deposition and an
increase by 2.5- and 2.0-fold in PAI-1 and collagen type 1
expression, respectively, which were blunted with Tudca and
insulin treatments (Figure 2D through 2F). The cardiac
fibrosis in STZ mice was associated with ER stress evidenced
by augmented mRNA levels of ATF4 (2.5-fold), CHOP
(3.0-fold), and ATF6 (2.5-fold), which were reduced with
Tudca and insulin treatment to control levels (Figure 2G, 2I,
and 2J). The expression of CHOP was increased by 3.5-fold
in STZ mice and was blunted after insulin treatment and ER
stress inhibition (Figure 2H). In addition, Western blot
analysis in heart showed no changes in EGFRtk phosphory-
lation in the STZ group compared with the STZ?Tudca
group (Figure S1A).
Effect of EGFRtk Inhibition on Vascular
Relaxation and ER Stress Markers in Mesenteric
Resistance Arteries
The EGFRtk expression in mesenteric resistance arteries
(MRAs) assessed by Western blot and real-time RT-PCR
revealed no changes in all of the groups of mice (Figure 3A
and 3B), whereas phosphorylated EGFRtk was 2-fold in-
creased in MRA from STZ mice compared with control and
diabetic mice treated with the EGFRtk inhibitor (Figure 3B).
To determine the role of EGFRtk in microvascular
dysfunction in type 1 diabetes mellitus, we examined the
endothelium-dependent relaxation (EDR) response in MRA
from STZ mice infused with and without EGFRtk inhibitor.
EDR was significantly decreased in STZ mice (33.6%)
compared with control (84%; Figure 3C) and was associated
with reduced endothelial NO synthase (eNOS) expression
and phosphorylation and cGMP levels (Figure 3E and 3F).
Endothelium-independent relaxation was shifted to the right
in STZ mice (EC50?6.3?0.07) compared with control mice
(EC50?7.29?0.03; Figure 3D). Importantly, the inhibition of
EGFRtk in STZ mice improved EDR (76.6%), restored
endothelial independent sensitivity (EC50?7.47?0.04), in-
creased by 2-fold eNOS phosphorylation, and restored cGMP
levels to control in MRAs (Figure 3C through 3F).
The phosphorylation of eIF2? and mRNA levels of CHOP,
ATF4, and ATF6 were increased by 3.0-, 2.5-, 4.0-, and
7.0-fold, respectively, in MRAs from STZ mice compared
with control mice and were reduced after EGFRtk inhibition
by2.5-,1.6-,and2.8-fold,respectively,withtheexceptionofthe
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AB
02468
Time (Days)
10 12 14 16 18 20 22
0
100
200
300
400
500
600
STZ
STZ + AG 1478 Control
Glucose (mg/dl)
#
*
$
02468
Time (Days)
10 12 14 16 18 20 22
12.0
14.5
17.0
19.5
22.0
24.5
27.0
STZ
STZ + AG 1478
Control
BW (g)
*
#
$
ControlAG1478
0
5
10
15
20
25
*
Fibrosis (%)
$
#
E
D
C
ControlAG1478
0
1
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4
5
*
PAI-1/β actin
#
ControlAG1478
0
1
2
3
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*
Collagen-I / actin
$
#
β-actin
AG1478
STZ
PAI-1
β-actin
AG1478
STZ
Collagen-I
AG1478
STZ
Control
Control
Control
STZ
STZ
STZ
K
Control AG1478
0
1
2
3
*
ATF6 / r18S mRNA (Heart)
$
STZ
ControlAG1478
0
1
2
3
*
#
EGFR / r18S mRNA (Heart)
F
STZ
H
CHOP / r18S mRNA (Heart)
Control AG1478
0
1
2
3
*
$
#
STZ
J
Control AG1478
0
1
2
3
*
ATF4 / r18S mRNA (Heart)
#
STZ
Control AG1478
0
2
4
6
*
#
CHOP/
actin (Heart)
β
STZ
I
P-EGFR
T-EGFR
β-actin
G
STZ
AG1478
Control
0
1
2
3
0
1
2
3
*
#
P-EGFR/ actin (Heart)
T-EGFR/
β
actin (Heart)
Control
AG1478
STZ
CHOP
β-actin
AG1478
STZ
Control
β
β
Figure 1. Effect of epidermal growth factor receptor (EGFR) tyrosine kinase (EGFRtk) inhibition on plasma glucose, body weight, fibro-
sis, EGFRtk, and endoplasmic reticulum (ER) stress markers in heart from streptozotocin (STZ) mice. A and B, Glucose levels (mg/dL)
and body weight (BW) were determined in control, STZ, and STZ?AG1478 groups; n?7. The vertical dashed lines denote the start of
infusion of AG1478. ?, control; ?, STZ?AG1478; e, STZ. C, Representative histological sections from the heart stained with Sirius-
red; bars indicate the quantitative data, n?5. D and E, Representative Western blot analysis and quantitative data for plasminogen acti-
vator inhibitor (PAI) 1 and collagen I in heart in all groups, n?4 to 5. F and G, EGFR mRNA levels, n?5 and representative Western blot
analysis and quantitative data for phosphorylated EGFR and total EGFR in all groups; n?5. H and I, CHOP mRNA levels, n?5, and
CHOP representative Western blot and quantitative data in all groups, n?3. J and K, ATF-4 and ATF-6 mRNA levels, normalized to
18S rRNA, in all of the groups, n?5. *P?0.05 for STZ vs control; #P?0.05 for STZ vs STZ?AG1478; $P?0.05 for STZ?AG1478 vs
control.
Galán et al EGFR/ER Stress in Diabetes Mellitus
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02468
Time (Days)
10 12 14 16 18 20 22
0
100
200
300
400
500
600
STZ
STZ + Tudca
STZ + insulin
Control
Glucose (mg/dl)
02468
Time (Days)
10 12 14 16 18 20 22
12
17
22
27
32
STZ
STZ + Tudca
STZ + insulin
Control
BW (g)
A
B
ControlInsulin
STZ
Tudca
0.0
0.5
1.0
1.5
*
Insulin ( g/l)
$
C
*
*
Control
Insulin
Tudca
ControlInsulin
STZ
Tudca
*
#
$
Fibrosis (%)
PAI-1
β-actin
Control
Insulin Tudca
Control Insulin
STZ
STZ
Tudca
0
1
2
3
4
*
#
PAI-1 / actin (Heart)
STZ
β-actin
Collagen-I
#
β
Collagen-I /
STZ
Control
Insulin Tudca
STZ
D
E
F
STZ
#
#
$
$
J
G
I
Control Insulin
STZ
Tudca
0
1
2
3
*#
ATF4/r18S mRNA (Heart)
Control Insulin
STZ
Tudca
0
1
2
3
4
*#
CHOP / r18S mRNA (Heart)
ControlInsulin
STZ
Tudca
0
1
2
3
*#
ATF6/r18S mRNA (Heart)
H
Control Insulin
STZ
Tudca
0.0
1.5
3.0
4.5
*#
CHOP/ actin (Heart)
CHOP
β-actin
Control
Insulin
Tudca
actin (Heart)
ControlInsulinTudca
0
1
2
3
*
β
0
10
20
30
β
n
Figure 2. Effect of endoplasmic reticulum (ER) stress inhibition on plasma glucose, body weight, plasma insulin, cardiac fibrosis, and
ER stress markers expression in heart from streptozotocin (STZ) mice. A and B, Glucose levels (mg/dL) and body weight (BW) were
determined in control, STZ, STZ?insulin, and STZ?Tudca groups; n?10. The vertical dashed lines denote the start of the injection of
insulin or Tudca. ?, control; Œ, STZ?insulin; e, STZ; Œ, STZ?Tudca. C, Plasma insulin levels (ng/dL) in all groups, n?10. D, Repre-
sentative histological sections from the heart stained with Sirius-red, bars indicate the quantitative data, n?5. E and F, Representative
Western blot analysis and quantitative data for plasminogen activator inhibitor (PAI) 1 and collagen I in heart from all of the groups,
n?4 to 5. G and H, CHOP mRNA levels, n?5, and CHOP representative Western blot and quantitative data in all groups, n?3. I and J,
ATF-4 and ATF-6 mRNA levels, normalized to 18S rRNA, in all groups, n?5 to 6. *P?0.05 for STZ vs control or STZ?insulin, #P?0.05
for STZ vs STZ?Tudca, $P?0.05 for STZ?Tudca vs control.
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Control AG1478
0
1
2
3
EGFR / r18S mRNA (MRA)
STZ
A
D
-8-7-6-5-4
0
25
50
75
100
STZ
log ACh (M)
STZ + AG 1478
Control
MRA relaxation (%)
*
#
C
-10-9-8 -7 -6-5 -4-3
0
25
50
75
100
125
STZ
STZ + AG 1478
Control
log SNP (M)
MRA relaxation (%)
*
#
E
STZ
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
*
$
#
P-eNOS /β -actin (MRA)
T-eNOS /β -actin (MRA)
ControlAG1478
P-eNOS
T-eNOS
β-actin
AG1478
STZ
Control
F
ControlAG1478
0.00
0.05
0.10
0.15
cGMP (pmol / mg)
STZ
STZ
P-EGFR
T-EGFR
β-actin
AG1478
STZ
Control
Control
AG1478
T -EGFR /β -actin (MRA)
P-EGFR / β -actin (MRA)
B
*
*
Control AG1478
0
2
4
6
8
*
#
$
ATF4 / r18S mRNA (MRA)
STZ
I
ControlAG1478
0
3
6
9
12
*
$
ATF6 / r18S mRNA (MRA)
STZ
J
0
1
2
3
4
0
1
2
3
4
*
$
#
P-eIF2 α / β actin (MRA)
T-eIF2α / β actin (MRA)
STZ
G
P-eIF2α
Τ-eIF2α
β-actin
AG1478
STZ
Control
AG1478
Control AG1478
0
1
2
3
*
#
$
CHOP / r18S mRNA (MRA)
STZ
H
0
1
2
0
1
2
#
Figure 3. Effect of epidermal growth fac-
tor receptor (EGFR) kinase inhibition on
EGFR expression, endothelial function,
and endoplasmic reticulum (ER) stress
markers in mesenteric resistance arteries
(MRAs) from streptozotocin (STZ) mice. A,
EGFR mRNA levels in all groups, n?5. B,
Representative Western blot analysis and
quantitative data for phosphorylated
EGFR and total EGFR in all groups, n?3.
C, Endothelium-dependent relaxation in
response to acetylcholine (ACh) in MRA
from control, STZ?AG1478, and STZ
groups, n?5. D, Endothelium-independent
relaxation in response to single nucleotide
polymorphism in MRAs from all groups,
n?5. ?, control; ?, STZ?AG1478; e,
STZ. E, Representative Western blot anal-
ysis and quantitative data showing the
phosphorylated endothelial NO synthase
eNOS (P-eNOS) and total eNOS
(T-eNOS), normalized to ?-actin, in MRAs
from all groups, n?4. F, Cyclic GMP lev-
els in all the groups, n?4. G through J,
Representative Western blot analysis
showing the expression of phosphorylated
eIF2-?, normalized to ?-actin; total
eIF2-?, n?4; and ATF-4, CHOP, and
ATF-6 mRNA levels, normalized to 18S
rRNA, in all groups, n?5. *P?0.05 for STZ
vs control, #P?0.05 for STZ vs
STZ?AG1478, and $P?0.05 for
STZ?AG1478 vs control.
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C
D
P-eNOS
T-eNOS
β-actin
Control
Insulin Tudca
STZ
ControlInsulin
STZ
Tudca
0.000
0.025
0.050
0.075
0.100
0.125
*
cGMP (pmol/mg)
#
-8-7 -6-5-4
0
25
50
75
100
STZ
log ACh (M)
STZ+Tudca
Control
STZ+Insulin
MRA relaxation (%)
#
A
$
*
-10-9-8-7-6 -5-4 -3
0
25
50
75
100
125
STZ
STZ+Tudca
Control
STZ+Insulin
log SNP (M)
MRA relaxation (%)
#
*
B
$
STZ
Control
Insulin
Tudca
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
*
#
PeNOS /β actin (MRA)
T-eNOS /β actin (MRA)
ControlInsulin
STZ
Tudca
0
3
6
9
12
*#
ATF6 / r18S mRNA (MRA)
ControlInsulin
STZ
Tudca
0
2
4
6
*#
ATF-4/r18S mRNA (MRA)
G
ControlInsulin
STZ
Tudca
0
1
2
3
4
*#
CHOP/r18S mRNA (MRA)
F
H
P- eIFα / β actin (MRA)
T-eIF2 α / β actin (MRA)
E
P-eIF2α
T-eIF2α
β-actin
Control
Insulin Tudca
STZ
STZ
ControlInsulin Tudca
0
1
2
3
4
5
0
1
2
3
4
5
*#
Figure 4. Effect of endoplasmic reticulum (ER) stress inhibition on endothelial function and ER stress markers expression in mesenteric
resistance arteries (MRAs) from streptozotocin (STZ) mice. A and B, Endothelium-dependent and independent relaxation in response to
acetylcholine (ACh) and single nucleotide polymorphism (SNP), respectively, in MRAs from control, STZ, STZ?insulin, and STZ?Tudca
groups, n?5. ?, control; e, STZ; Œ, STZ?insulin; Œ, STZ?Tudca. C, Representative Western blot analysis and quantitative data
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mRNA level of ATF6 (Figure 3F through 3I). The total eIF2?
protein expression was similar in all of the groups of mice.
Effect of ER Stress Inhibition on Microvascular
Function in MRAs
To delineate the role of ER stress in microvascular dysfunc-
tion in type 1 diabetes mellitus, we first examined EDR
response in MRAs from STZ mice with or without Tudca or
insulin. EDR was significantly improved in STZ mice treated
with Tudca or insulin (61.3% and 84.3%, respectively; Figure
4A). Endothelium-independent relaxation was shifted to the
right in STZ mice (EC50?6.27?0.05) and was normalized
after ER stress inhibition (EC50?6.94?0.05) or insulin in-
jection (EC50?7.43?0.06; Figure 4B). These results were
supported with the measurements of eNOS phosphorylation
and expression and cGMP level, which were decreased by
4.0-, 3.0-, and 2.5-fold, respectively, in STZ mice and were
normalized with Tudca and insulin treatments (Figure 4C and
4D). Microvascular endothelial dysfunction in STZ mice was
associated with ER stress induction, as evidenced by en-
hanced phosphorylated eIF2? (4-fold) and the increase in the
mRNA levels of ATF4 (4.5-fold), CHOP (3.0-fold), and
ATF6 (9.0-fold; Figure 4E through 4H). Interestingly, insulin
and ER stress inhibition (Tudca) were able to significantly
reduce ER stress marker expression in MRAs from STZ mice
(Figure 4E through 4H). The total eIF2? protein expression
was similar in all of the groups of mice. In addition, EGFRtk
phosphorylation in MRAs was similar in the STZ and
STZ?Tudca groups (Figure S1B). The inhibition of NO-
synthesis (NG-nitro-L-arginine methyl ester; 100 ?mol/L)
reduced EDR by 17.3% in STZ mice, whereas a great
reduction was observed in control and STZ mice treated with
AG1478 (40%), Tudca (40%), or insulin (64%; Figure 5A
through 5E).
To determine the link between ER stress and reactive
oxygen species in microvascular endothelial dysfunction, we
incubated MRAs with apocynin (100 ?mol/L). The results
revealed that apocynin significantly improved EDR in STZ
mice (25%), whereas no effect was observed in control or
STZ mice treated with AG1478, Tudca, or insulin (Figure 5A
through 5E). The enhanced mRNA levels of Nox2 and Nox4
isoforms by 4- and 6-fold, respectively, in MRA from STZ
mice supported these findings, which were blunted after
inhibition of EGFRtk, ER stress, or insulin injection (Figure
5F through 5I). Moreover, the NADPH oxidase activity,
determined in heart and MRA lysates, was increased in STZ
mice by 3- and 4-fold, respectively, compared with control
mice. The inhibition of EGFRtk and ER stress significantly
decreased this activity (Figure S1C and S1D).
Effect of ER Stress Induction by Tunicamycin on
Phosphorylated EGFR Expression in Heart
and MRAs
The EGFRtk phosphorylation in heart and MRAs was similar
in control and mice injected with Tunicamycin with and
without Tudca (Figure S2A and S2B). In addition, the phosphor-
ylated EGFRtk expression, in MRAs, remained unchanged in all
of the groups (Figure S2C).
Discussion
In the present study, we found that EGFRtk phosphorylation
and expression were upregulated in heart and microvessels of
diabetic mice and were associated with ER stress induction,
cardiac fibrosis, and microvascular endothelial dysfunction.
Our results are supported by previous studies reporting that
EGFRtk inhibition improved microvascular function in type 2
diabetes mellitus.9,10Interestingly, the inhibition of EGFRtk
improved glucose levels, body weight, and microvascular
function and reduced cardiac fibrosis and ER stress markers,
with the exception of ATF6. These results suggest that
exacerbated EGFRtk phosphorylation regulates cardiovascu-
lar dysfunction and metabolic alteration in type 1 diabetes
mellitus, likely through phosphorylated protein kinase–like
ER eukaryotic initiation factor 2? kinase-ATF4-derived ER
stress branch but independent of the ATF6 branch.
Emerging evidence from experimental and clinical studies
indicate that ER stress plays an important role in cardiovas-
cular diseases17and diabetes mellitus, as evidenced by
peripheral insulin resistance and pancreatic ?-cell dysfunc-
tion22,23related to ER stress.15,19In addition, ER stress has
been demonstrated to be involved in the development of
diabetes mellitus affecting different organs like liver, kidney,
and skeletal muscle in several models of diabetic ani-
mals15,24,25; however, the role and mechanisms of ER stress
in cardiac fibrosis and microvascular endothelial dysfunction
in type 1 diabetes mellitus remain unclear. Previous studies
have shown that ER stress is associated with heart failure and
cardiomyopathy in nondiabetic and diabetic animals support-
ing the potential role of ER stress in cardiac damage.26–28In
the present work, we found that ER stress induction and
cardiac fibrosis were associated with enhanced collagen type
1 and PAI-1 expression in diabetic mice. The inhibition of
EGFRtk and ER stress reduced ER stress markers, suggesting
that EGFRtk is upstream to ER stress activation. These data
are supported by a recent publication showing that overex-
pression of aberrant EGFRtk in several cancers induces the
expression of CHOP.14In addition, chemical inhibition of
EGFRtk and ER stress reduced cardiac fibrosis, collagen type
1, and PAI-1. Although the reduction on myocardial fibrosis
appears pronounced in these animals, the effect of AG1487
and Tudca on blood glucose levels is modest, indicating that
these drugs may act by mechanisms independent of their
hypoglycemic effects. These results suggest that cardiac fibrosis
in type 1 diabetes mellitus is regulated by an ER stress-
dependent mechanism. However, it is unclear how ER stress
controls collagen type 1 turnover, and additional studies are
needed to delineate the mechanism.
Figure 4 (Continued). showing the phosphorylated eNOS (P-eNOS) and total eNOS (T-eNOS), normalized to ?-actin, in all groups,
n?4. D, Cyclic GMP levels in all the groups, n?4. E through H, Representative Western blot and quantitative data showing the expres-
sion of phosphorylated eIF2-?, normalized to ?-actin, and total eIF2-? protein in all groups, n?4, and CHOP, ATF-4, and ATF-6 mRNA
levels, normalized to 18S rRNA, in all groups, n?5 to 6. *P?0.05 for STZ vs control, STZ?insulin; $P?0.05 for STZ?Tudca vs
STZ?insulin or control; #P?0.05 for STZ vs STZ?Tudca.
Galán et al EGFR/ER Stress in Diabetes Mellitus
77
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It is well established that diabetes mellitus impairs micro-
vascular function.29It is known that hyperglycemia causes an
enhancement in advanced glycation end products, oxidative
stress levels, and increases EGFR tyrosine kinase activity
leading to microvascular endothelial dysfunction, in part
through the loss of NO bioavailability.9,30–32The impaired
EDR was also associated with a decrease in eNOS phosphor-
ylation and expression in diabetic mice. These results are
supported by previous studies showing a decrease in eNOS
mRNA levels in KKAy mice33and reduced eNOS phosphor-
ylation in diabetic mice.9Importantly, inhibition of EGFRtk
and ER stress restored eNOS phosphorylation and expression
in MRAs of diabetic mice indicating that eNOS expression
and activity are regulated by EGFRtk and ER stress-dependent
mechanisms. Although ER stress inhibition restored eNOS
phosphorylation and expression to the control levels, EDR was
partially improved, suggesting that other factors contribute to
EDR impairment in MRAs.
Previous reports provided evidence that ER stress increases
oxidative stress levels, which represent another mechanism
regulating eNOS activity and NO bioavailability.34,35
NADPH oxidase seems to be the main source of oxidative
stress in animal models of diabetes mellitus.36Our data
demonstrated that microvascular EDR was improved after the
AB
-8 -7-6 -5 -4
0
25
50
75
100
log ACh (M)
STZ+Insulin
STZ+Insulin +L-NAME
STZ+Insulin +APO
MRA relaxation (%)
D
^
-8 -7 -6-5 -4
0
25
50
75
100
log ACh (M)
Control
Control + L-NAME
Control + APO
MRA relaxation (%)
E
^
-8-7-6-5-4
0
25
50
75
100
log ACh (M)
STZ+Tudca
STZ+ Tudca+ L-NAME
STZ+Tudca+APO
MRA relaxation (%)
^
-8-7-6-5-4
0
25
50
75
100
STZ
STZ+ L-NAME
STZ+ APO
log ACh (M)
MRA relaxation (%)
&^
-8-7-6 -5-4
0
25
50
75
100
log ACh (M)
STZ + AG 1478
STZ + AG 1478 + LNAME
STZ + AG 1478 + APO
MRA relaxation (%)
&
C
ControlAG1478
0
2
4
6
8
#*
NOX4/r18S mRNA (MRA)
ControlAG1478
0
1
2
3
4
5
*
#
NOX2/r18S mRNA (MRA)
STZ
STZ
ControlInsulin
STZ
Tudca
0
1
2
3
4
5
*
Nox2/r18S (MRA)
#
G
ControlInsulin
STZ
Tudca
0.0
2.5
5.0
7.5
10.0
*
Nox4/r18S (MRA)
#
I
F
H
Figure 5. Endothelial function in mesenteric resistance arteries (MRAs) incubated with apocynin (APO) or NG-nitro-L-arginine methyl
ester (l-NAME) from streptozotocin (STZ) mice treated with or without insulin or Tudca and Nox2/4 mRNA levels. A, Endothelium-
dependent relaxation in response to acetylcholine (ACh) with and without apocynin (APO) or l-NAME treatments in STZ group, n?5. ?,
STZ; e, STZ?l-NAME; s, STZ?APO. B, Endothelium-dependent relaxation in response to ACh in the presence of apocynin or l-NAME
in MRAs from STZ?AG1478 group, n?5. ?, STZ?AG1478; ?, STZ?AG1478?l-NAME; ?STZ?AG1478?APO. C, Endothelium-
dependent relaxation in response to ACh with and without apocynin or l-NAME in STZ?Tudca group, n?5. Œ, STZ?Tudca; o,
STZ?Tudca?l-NAME; grey triangle, STZ?Tudca?APO. D, Endothelium-dependent relaxation in response to ACh with and without
apocynin or l-NAME in the STZ?insulin group, n?5. ?, STZ?insulin; Œ, STZ?insulin?l-NAME; grey circle, STZ?insulin?APO. E,
Endothelium-dependent relaxation in response to ACh with and without apocynin or l-NAME treatment in the control group, n?5. ?,
control; ?, control?l-NAME; grey diamond, control?APO. F through I, Nox2 and Nox4 mRNA levels, normalized to 18S rRNA, in all
groups, n?4 to 6. &P?0.05 for STZ?APO versus STZ.ˆP?0.05 for STZ, STZ?AG1478, STZ?Tudca, STZ?insulin, and control vs
STZ?l-NAME, STZ?AG1478?l-NAME, STZ?Tudca?l-NAME, STZ?insulin?l-NAME and control?l-NAME. *P?0.05 for STZ vs control
or STZ?insulin. #P?0.05 for STZ vs STZ?Tudca or STZ?insulin.
78 Hypertension
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inhibition of NADPH oxidase activity in diabetic mice,
whereas no effect was observed in control and diabetic mice
treated with EGFRtk and ER stress inhibitors. These data
indicate that EGFRtk and ER stress-dependent mechanisms
regulate NADPH oxidase activity. These results are sup-
ported by the reduction in NADPH oxidase activity and
Nox-2 and Nox-4 expression in diabetic mice after EGFRtk
and ER stress inhibition. In addition, Nox2 and Nox4 have
been shown to be predominantly located in the perinuclear
and/or ER membranes, suggesting a relationship between
reactive oxygen species generation and ER stress in diabetes
mellitus.37
In conclusion, we demonstrated that, in type 1 diabetes
mellitus, the exacerbation in EGFRtk signaling contributes to
ER stress induction as a mechanism in part responsible for
cardiac fibrosis and microvascular dysfunction. Thus,
EGFRtk and ER stress could be potential targets for novel
therapeutic strategies to improve cardiovascular function in
diabetes mellitus.
Perspectives
Diabetes mellitus is a metabolic disease associated with
cardiovascular complications, including cardiac damage and
impaired microvascular EDR. Most of clinical studies indi-
cate that diabetic patients are at high risk for cardiovascular
diseases. Despite the fact that treatments have progressed, the
development of novel effective treatments for diabetic pa-
tients with vascular complications remains a major research
goal. Therefore, there is a significant medical need to develop
novel therapies to restore microvascular endothelial function
in these patients. Our results indicate that exacerbated
EGFRtk activity and ER stress play key roles in heart damage
and vascular dysfunction in type 1 diabetic mice. Interest-
ingly, inhibition of EGFRtk activity decreases ER stress
markers, suggesting that ER stress is downstream of the
EGFRtk pathway. The inhibition of EGFRtk and ER stress
reduces cardiac fibrosis and improves microvascular function
associated with enhance in eNOS phosphorylation, cGMP
levels, and reduction in NADPH oxidase activity. Therefore,
EGFRtk and ER stress could be potential targets for novel
therapeutic strategies to improve cardiovascular function in
diabetes mellitus.
Sources of Funding
We acknowledge grant support from the National Institutes of Health
(HL095566 to K.M. and HL097111 to M.T.). Grant P20RR017659-
COBRE to Dr LG Navar paid 50% of M.K.’s salary.
Disclosures
None.
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Novelty and Significance
What Is New?
● This is the first study to demonstrate that the exacerbation in EGFRtk
signaling contributes to ER stress induction as a mechanism responsible
for cardiac fibrosis and microvascular dysfunction in type 1 diabetes
mellitus.
What Is Relevant?
● This study unveils novel roles for enhanced EGFRtk phosphorylation and
its downstream ER stress in cardiac fibrosis and microvascular endo-
thelial dysfunction in type 1 diabetes mellitus.
Summary
EGFRtkphosphorylationandexpressionwereupregulatedinheartand
microvessels of diabetic type 1 mice and were associated with ER
stress induction, cardiac fibrosis, and microvascular endothelial dys-
function. Interestingly, the inhibition of EGFRtk and ER stress improved
body parameters, cardiac fibrosis, and microvascular function.
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Supplemental Materials

A novel role for EGFR tyrosine kinase and its downstream endoplasmic
reticulum stress in cardiac damage and microvascular dysfunction in type
1 diabetes

Maria Galán, PhD1#; Modar Kassan, PhD1#; Sookyoung Choi, PhD1; Megan Partyka,
BS1; Mohamed Trebak, PhD2; Daniel Henrion, PhD3; Khalid Matrougui, Ph.D1*

1Department of Physiology, Hypertension and Renal Center of Excellence, Tulane
University, 1430 Tulane Ave, New Orleans LA-70112; 2Center for Cardiovascular
Sciences, Mail code 8, Albany Medical College, Albany 12208, New York; 3Centre
National de la Recherche Scientifique UMR 6214, Angers, France; INSERM U771,
Angers, France, CHU d'Angers, Angers France, Université d'Angers, Angers, France;

# Dr. Galán and Dr. Kassan are co-first authors
Running title: EGFR/ER stress and cardiovascular complication in diabetes

*Corresponding Author:
Khalid Matrougui, Ph.D., Department of Physiology, Hypertension and Renal Center
of Excellence, Tulane University. 1430 Tulane Ave, New Orleans LA-70112.
E-mail: kmatroug@tulane.edu, Phone-(504)-889-2588, Fax-(504)-988-2675.  














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Page 13

MATERIALS AND METHODS

General protocol in mice
All experiments were performed according to the American Guidelines for the
Ethical Care of Animals and were approved by Tulane University Animal Care and
Use Committee. Mice (C57BL/6J, 8 weeks-old males) were purchased from Jackson
Laboratories (Bar Harbor, ME) housed in groups of five and maintained at a
temperature of 23ºC with 12 hours light/dark cycle. Mice were fed on a solid standard
diet (Na+ content 0.4%) and water.
Diabetes was induced by a single intra-peritoneal injection of streptozotocin
(STZ, 200 mg/kg, dissolved freshly in citrate buffer, pH 4.5) to fasted mice for 12
hours.1 Hyperglycemia was confirmed by measuring tail vein blood glucose levels
with glucometer (Accu-Chek, Roche Diagnostic, Germany). Mice with blood glucose
levels ≥ 300 mg/dl were considered as type 1 diabetic.
One week after the induction of diabetes, mice were divided into five groups:
1) Diabetic group (STZ); 2) Diabetic group treated with AG1478 (10 mg/Kg/day)2
(STZ + AG1478); 3) Diabetic group treated with Tudca (150 mg/kg/day)3 (STZ +
Tudca); 4) Diabetic group treated with Insulin (0.1 U/day) (STZ + Insulin) using
insulin-implants placed underneath of the skin (Linshin, Canada); 5) Control group
(Control). Mice were treated for 2 weeks.
Body weight and blood glucose levels were measured during the treatment
period. At the end of treatment period, mice were sacrificed and blood samples were
collected to determine the plasma concentration of insulin by the ELISA kit
(Mercodia Ultrasensitive Mouse Insulin ELISA, USA). Heart and MRA were
harvested immediately, placed in PSS solution (composition in mmol/L: NaCl 118;
KCl 4.7; CaCl2 2.5; KH2PO4 1.2; MgSO4x7H2O 1.2; NaHCO3 25 and glucose 11),
pH=7.4 and processed appropriately for further studies.
In another set of experiments, we used 8 weeks-old C57BL/6J male mice
divided into three groups: 1) Control group; 2) control group that received intra-
peritoneal injection of Tunicamycin (Tunica, 1 mg/kg, 2 injections/week for 2 weeks,
Control + Tunica); 3) control group that received Tunicamycin and Tudca (150
mg/kg/day) for 2 weeks (Control + Tunica + Tudca). At the end of treatment, mice
were anaesthetized with isoflurane and then heart and mesenteric resistance arteries
were immediately harvested and processed for further experiments.
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Cardiac fibrosis
A transverse section of heart was fixed in 4% of formalin, embedded in
paraffin and cut into 4 µm thick sections. Slices were stained with the collagen-
specific stain Sirius-red (Sigma-Aldrich, USA). At least eight areas from each heart
were captured using a high-resolution digital camera (Olympus DP50, Japan). The
collagen was quantified using Adobe Photoshop CS2 (Microsoft). For each image, the
percentage of interstitial fibrosis was determined as the ratio of collagen surface area
to myocardial surface area.

Mesenteric Resistance Arteries Reactivity
Microvascular responses to acetylcholine (ACh) and sodium nitroprusside
(SNP) were performed as previously described.40 To determine the role of NADPH
oxidase in the impaired endothelium-dependent relaxation in diabetic mice, MRA
were incubated with apocynin (100 µmol/L) for 30 minutes and then EDR responses
were performed after pre-contraction with phenylephrine.

Western blot analysis
Mice were sacrificed, heart and MRA were immediately harvested and frozen
in liquid nitrogen and then stored at -80°C. Western blot analysis for eNOS, PAI-1,
eIF2-α, CHOP and EGFRtk (1:1000 dilution, Cell Signaling Technology, Inc, USA),
collagen-1 and β-actin (1:500 dilution, Santa Cruz Biotechnology, Inc) was performed
using specific antibodies as previously described.4

RT-PCR real-time assay
EGFRtk, Nox, CHOP, ATF4 and ATF6 mRNA levels were determined in
MRA and heart samples from all groups as previously described.4 Assays-on-Demand
(Applied Biosystems) of TaqMan fluorescent real time PCR primers and probes were
used for Egfr (Mm00433023_m1), Chop (Mm00492097_m1), Atf4
(Mm00515324_m1), Atf6 (Mm01295317_m1), Nox-2 (Mm01287743_m1), Nox-4
(Mm00479246_m1) and 18S rRNA (Hs99999901_s1), which was used as
endogenous control to normalize results. Quantitative RT-PCR was carried out in an
ABI PRISM 7000 Sequence Detection System (Applied Biosystems) using the
following conditions: 2 min at 50°C, 10 min at 95°C followed by 40 cycles of 15 s at
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95°C and 1 min at 60°C. Relative mRNA levels were determined using the 2-∆∆Ct
method. Results are expressed as the relative expression of mRNA in treated mice
compared with untreated mice.

Colorimetric Determination of cGMP
The cGMP levels were measured in MRA lysates in all groups of mice.
Measurements were performed using a sandwich enzyme-linked immunosorbent
assay (ELISA; Cayman Chemical, Ann Arbor, MI) according to the manufacturer
instructions.

Immunohistochemistry
Hearts and MRA were fixed in 4% of paraformaldehyde followed by zinc-
saturated formalin and paraffin-embedded for immunoperoxidase staining using the
Vectastain ABC Kit (USA). The sections were incubated overnight with anti-p-EGFR
antibody (1:200, sc-101668, Santa Cruz Biotechnology, USA). At least eight sections
from each heart and MRA were captured using a high-resolution digital camera
(Olympus DP50, Japan).

NADPH oxidase activity assay
Superoxide anion levels generated by NADPH oxidase activity were measured
in lysates of heart and MRA using lucigenin chemiluminescence. Briefly, lysates were
prepared in a sucrose buffer containing KH2PO4 50 mM, EGTA 1 mM, sucrose 150
mM; pH=7.0 and protease inhibitor cocktail (complete mini, Roche Diagnostics, IN,
USA) in a Tissue Dounce homogenizer on ice, and aliquots of the homogenates were
used immediately. To start the assay, a volume of 100 µL of each lysate was used in a
total volume of 1 mL PBS buffer preheated at 37ºC, containing lucigenin (5 µM) and
NADPH (100 µM). Blank samples were prepared using 100 µL of sucrose buffer.
Lucigenin activity was measured every 30 seconds during about 10 min in a
luminometer (Turner biosystem 20/20, single tube luminometer) till enzymatic
activity is reaching the plateau. Data are expressed as area under the curve of relative
light units (RLU) normalized to protein content (μg protein).



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