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.