1. 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 of Histology and Med. Embr., Sapienza University of Rome, Italy
3Dep. of Exp. Medicine and Surgery, Fac. of Med. Unicers. of Rome Tor Vergata, Itay

2. Transglutaminases (TGs) in the Vascular system
• Type 1 (keratinocyte TG)
• Factor XIII (plasma TG)
• Type 2 (Tissue type TG)
Epidermal differentiation
Wound healing
SMC EC
Bakker et al., J Vasc Res, 2008

3. Enzymatic Reaction Catalyzed by TransglutaminaseTG
Ca2+
Covalent iso-peptide
bond
TGs catalyze covalent cross-linking between reactive
lysine and glutamine residues of protein polymers

4. Functions of TG 2 TG2 (Tissue type TG) TGase GTPase
• Cell growth and differentiation
• Wound healing
• Receptor- mediated endocytosis
• Apoptosis
• Activation of PLC
• Regulation of cell cycle
progression

5. 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.

6. AIM
To determine whether Ang II may induce vascular remodeling in part through TG2

7. 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.

8. 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.

9. Results
Endothelium-dependent relaxation was similarly preserved in untreated WT, TG2-K/O and Ang II- treated 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.

10. 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

11. Blood Pressure in WT and TG2-K/O mice treated or not with angiotensin II*
150 *
100
mm Hg 50 WT
TG2-K/O WT
+AngII
TG2-K/O
+Ang II

12. Media-to-Lumen Ratio and Cross-sectional area
of mesenteric arteries from WT and TG2-K/O mice
10.0 *
15000 *
7.5
10000
M/L ratio (%)
5.0
CSA (μm2)
5000
2.5
0.0 WT
TG2-K/O WT
+AII
TG2-KO
+AII WT
TG2-K/O WT
+AII
TG2-KO
+AII

13. Endothelium-dependent and -independent relaxation in mesenteric arteries from WT and TG2-K/O mice
100
WT+Ang II
TG2-KO+Ang II
% of relaxation * 50 WT
TG2-KO -9 -8 -7 -6 -5 -4
100
WT+Ang II
Acetylcholine (log M)
TG2-KO+Ang II
% of relaxation 50 -8 -7 -6 -5 -4 -3
SNP (log M)

14. Dose response curves to Acetilcholine ± LNAME
in mesenteric arteries from WT and TG2-K/O mice
treated or not with angiotensin II
100
100
TG-2K/O (Acetylcholine)
WT_(Acetylcholine)
% of relaxation
TG-2K/O (Acetylcholine+LNAME)
WT (Acetylcholine+LNAME)
% of relaxation 75 75 50 50 * * 25 25 -9 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4
Acetylcholine (log M)
Acetylcholine (log M)
100
100
TG2-KO+Ang II ( Acetylcholine)
WT+Angi II (Acetylcholine)
TG2KO+Angi II (Acetyocholine+LNAME)
% of relaxation
WT+Angi II (Acetylcholine+LNAME) 75
% of relaxation 75 * 50 50 25 25 -9 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4
Acetylcholine (log M)
Acetylcholine (log M)

15. eNOS expression in aorta from WT and TG2-K/O mice
beta actin *
200
eNOS/β-actin (% CTRL)
100 WT
TG2 -K/O WT
+AII
TG2-K/O+AII

16. ROS production in aorta from WT and TG2-K/O mice*
400 *
300
Arbitrary Units
TG2-K/O
+ Ang II WT
+ Ang II
200
100 WT
TG2-K/O
WT+AII
TG2-K/O+AII

17. 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 II-induced vascular structural and functional alterations.