L.Sada
1 ,C.Savoia
1 ,M.Briani
1 ,E Arrabito 1 ,S.Michelini
1 ,L.Pucci
1 ,
T.Bucci
1 ,C.Nicoletti
2 ,E Candi 3 , EL Schiffrin, M Volpe 1 .
1 Clinical and Molecular Medicine Department, Sapienza University of Rome, Italy
2 DAHFMO-Unit of Histology and Med. Embr., Sapienza University of Rome, Italy
3 Dep. 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
Ca 2+
Covalent iso-peptide bond
TGs catalyze covalent cross-linking between reactive lysine and glutamine residues of protein polymers

TG2
(Tissue type TG)
TGase GTPase
• Cell growth and differentiation
• Wound healing
• Receptor- mediated endocytosis
• Apoptosis
• Activation of PLC
• Regulation of cell cycle progression

• 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

•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.

•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.

•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.

• 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
*
*
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
10.0
7.5
5.0
2.5
0.0
*
*
WT TG2-
K/O
WT
+AII
TG2-KO
+AII
15000
10000
5000
0
WT TG2-
K/O
WT
+AII
TG2-KO
+AII

Endothelium-dependent and -independent relaxation in mesenteric arteries from WT and TG2-K/O mice
100
WT
TG2-KO
WT+Ang II
TG2-KO+Ang II
*
50
0
-9 -8 -7 -6
Acetylcholine (log M)
-5 -4
100
WT
TG2-KO
WT+Ang II
TG2-KO+Ang II
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
100
75
50
25
0
WT_(Acetylcholine)
WT (Acetylcholine+LNAME)
-9 -8 -7 -6 -5
Acetylcholine (log M)
-4
*
100
75
50
25
0
WT+Angi II (Acetylcholine)
WT+Angi II (Acetylcholine+LNAME)
-9 -8 -7 -6 -5
Acetylcholine (log M)
-4
100
75
50
25
0
TG-2K/O (Acetylcholine)
TG-2K/O (Acetylcholine+LNAME)
100
75
50
25
0
-9 -8 -7 -6
Acetylcholine (log M)
-5 -4
TG2-KO+Ang II ( Acetylcholine)
TG2KO+Angi II (Acetyocholine+LNAME)
-
9
-
8
-
7
-
6
-
5
Acetylcholine (log M)
-
4
*
*

eNOS expression in aorta from WT and
TG2-K/O mice
200 * eNOS beta actin
100
0
WT TG2 -K/O WT
+AII
TG2-
K/O+AII

ROS production in aorta from WT and
TG2-K/O mice
WT
WT
+ Ang II
TG2 K/O
*
400
300
TG2-K/O
+ Ang II
200
100
0
*
WT TG2-K/O WT+A
II
TG2-
K/O+AII

• 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 II- induced vascular structural and functional alterations.