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

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

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 GTPase • Cell growth and differentiation • Wound healing • Receptor- mediated endocytosis • Apoptosis • 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 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.

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 * 100 mm Hg 50 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 * 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

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)

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)

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

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

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.