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Report
Improved Vascular Remodeling and
Endothelial Function in Transglutaminase
2 Knock-Out Mice Infused with
Angiotensin II
L.Sada1,C.Savoia1,M.Briani1,E Arrabito1,S.Michelini1,L.Pucci1,
T.Bucci1,C.Nicoletti2,E Candi3, EL Schiffrin, M Volpe1.
1
Clinical and Molecular Medicine Department, Sapienza University of Rome, Italy
2DAHFMO-Unit
3Dep.
of Histology and Med. Embr., Sapienza University of Rome, Italy
of Exp. Medicine and Surgery, Fac. of Med. Unicers. of Rome Tor Vergata, Itay
Transglutaminases (TGs) in the Vascular system
• Type 1 (keratinocyte TG)
• Factor XIII (plasma TG)
• Type 2 (Tissue type TG)
Epidermal differentiation
Wound healing
EC
SMC
Bakker et al., J Vasc Res, 2008
Enzymatic Reaction Catalyzed by Transglutaminase
TG
Ca2+
Covalent iso-peptide
bond
TGs catalyze covalent cross-linking between reactive
lysine and glutamine residues of protein polymers
Functions of TG 2
TG2
(Tissue type TG)
TGase
•
•
•
•
Cell growth and differentiation
Wound healing
Receptor- mediated endocytosis
Apoptosis
GTPase
• Activation of PLC
• Regulation of cell cycle
progression
Background
• TGs are involved in flow-induced vascular remodeling in rat
cremaster arteries.
Bakker et al., Circ res, 2005
•TGs are involved in aldosterone-induced vascular remodeling in
mesenteric arteries and in aorta.
Yamada et al. Cardiovascular research,2008
•Tissue Transglutaminase is involved in endothelin 1-induced
hypertrophy in cultured neonatal rat cardiomyocytes.
Li et al. Hypertension, 2009
•We previously demonstrated that angiotensin II (Ang II) may
positively regulate TG2 expression in vascular smooth muscle cells from
SHR.
AIM
To determine whether Ang II may induce
vascular remodeling in part through TG2
Methods
•TG2 Knock-out mice (TG2-K/O, 8 weeks old) and age
matched wild type (WT) mice were treated or not with
Ang II (400 ng/Kg/min) for 14 days.
•Blood pressure (BP) was measured by tail-cuff method.
•Functional, structural and mechanical studies were
performed on segments of pressurized (45 mmHg)
mesenteric arteries.
•Vascular reactive oxygen species (ROS) level in the
aorta was avaluated by dihydroethidium (DHE) staining.
•The expression of eNOS in aorta was evaluated by
immunoblotting.
Results
•BP was higher in TG2-K/O mice compared to WT
(120.3±1.3 mmHg vs 88.3±1.9 mmHg, P<0.05).Ang II
infusion significantly increased BP only in WT (+28% vs
untreated WT, P<0.05), whereas BP was unchanged in
TG2-K/O after Ang II infusion.
•TG2-K/O presented reduced M/L as compared to WT
(4.8±0.3% vs 6.5±0.2%, P<0.05). Ang II infusion
increased M/L only in WT (+13% vs untreated WT,
P<0.05). M/L resulted unchanged in TG2-K/O after Ang
II infusion. CSA was similar in all groups.
Results
•Endothelium-dependent
relaxation
was
similarly
preserved in untreated WT, TG2-K/O and Ang IItreated
TG2-K/O.
Ang
II
infusion
impaired
acetylcholine-induced relaxation only in WT (-50% vs
untreated
WT,
P<0.05).
L-NAME
blunted
acetylcholine-induced relaxation in all groups except in
Ang II-treated WT
•SNP-dependent relaxation was similar in all groups.
Results
• eNOS expression was similar in untreated WT and
untreated TG2-K/O. eNOS significantly increased
only in TG2-K/O treated with Ang II
• ROS production was similar in untreated WT and
untreated TG2-K/O. Ang II significantly increased
ROS in WT (2-fold increase), and significantly
decreased ROS in TG2-K/O
Blood Pressure in WT and TG2-K/O mice
treated or not with angiotensin II
*
150
*
mm Hg
100
50
0
WT
TG2-K/O
WT
+AngII
TG2-K/O
+Ang II
Media-to-Lumen Ratio and Cross-sectional area
of mesenteric arteries from WT and TG2-K/O mice
15000
*
*
7.5
CSA (μm2)
M/L ratio (%)
10.0
5.0
10000
5000
2.5
0.0
WT
TG2K/O
WT
+AII
TG2-KO
+AII
0
WT
TG2K/O
WT
+AII
TG2-KO
+AII
Endothelium-dependent and -independent relaxation in
mesenteric arteries from WT and TG2-K/O mice
WT
TG2-KO
WT+Ang II
TG2-KO+Ang II
*
50
WT
0
-9
-8
-7
-6
-5
-4
100
Acetylcholine (log M)
TG2-KO
WT+Ang II
TG2-KO+Ang II
% of relaxation
% of relaxation
100
50
0
-8
-7
-6
-5
SNP (log M)
-4
-3
Dose response curves to Acetilcholine ± LNAME
in mesenteric arteries from WT and TG2-K/O mice
treated or not with angiotensin II
TG-2K/O (Acetylcholine+LNAME)
WT (Acetylcholine+LNAME)
75
50
*
25
0
TG-2K/O (Acetylcholine)
100
WT_(Acetylcholine)
% of relaxation
% of relaxation
100
75
50
*
25
0
-9
-8
-7
-6
-5
-9
-4
WT+Angi II (Acetylcholine+LNAME)
50
25
0
-9
-8
-7
-6
-6
-5
-4
TG2-KO+Ang II ( Acetylcholine)
TG2KO+Angi II (Acetyocholine+LNAME)
% of relaxation
% of relaxation
100
WT+Angi II (Acetylcholine)
75
-7
Acetylcholine (log M)
Acetylcholine (log M)
100
-8
-5
Acetylcholine (log M)
-4
75
*
50
25
0
9
8
7
6
5
4
Acetylcholine (log M)
eNOS expression in aorta from WT and
TG2-K/O mice
eNOS
beta actin
*
eNOS/β-actin (% CTRL)
200
100
0
WT
TG2 -K/O
WT
+AII
TG2K/O+AII
ROS production in aorta from WT and
TG2-K/O mice
WT
TG2 K/O
*
WT
+ Ang II
TG2-K/O
+ Ang II
Arbitrary Units
400
*
300
200
100
0
WT
TG2-K/O
WT+A
II
TG2K/O+AII
Conclusion and perspectives
• Despite the higher BP values, TG2-K/O
presented improved vascular remodeling
compared to WT.
• In TG2-K/O, Ang II failed to increase
ROS production and M/L; moreover it failed
to impair endothelial function in this group.
• Hence, TG2 may play a role in Ang IIinduced vascular structural and functional
alterations.

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