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Transglutaminases (TGs) in the Vascular system

Improved Vascular Remodeling and Endothelial Function in Transglutaminase 2 Knock-Out Mice Infused with Angiotensin II. 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 .

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Transglutaminases (TGs) in the Vascular system

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  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 EC SMC Bakker et al., J Vasc Res, 2008

  3. 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

  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 0 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 * 15000 10.0 * 7.5 10000 M/L ratio (%) CSA (μm2) 5.0 5000 2.5 0.0 0 TG2-KO +AII TG2-KO +AII WT TG2-K/O WT +AII WT TG2-K/O WT +AII

  13. Endothelium-dependent and -independent relaxation in mesenteric arteries from WT and TG2-K/O mice WT TG2-KO 100 WT+Ang II TG2-KO+Ang II % of relaxation * 50 WT 0 TG2-KO -9 -8 -7 -6 -5 -4 100 WT+Ang II Acetylcholine (log M) TG2-KO+Ang II % of relaxation 50 0 -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 TG-2K/O (Acetylcholine) 100 100 WT_(Acetylcholine) % of relaxation TG-2K/O (Acetylcholine+LNAME) WT (Acetylcholine+LNAME) 75 75 % of relaxation 50 50 * * 25 25 0 0 -9 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4 Acetylcholine (log M) Acetylcholine (log M) TG2-KO+Ang II ( Acetylcholine) 100 100 WT+Angi II (Acetylcholine) TG2KO+Angi II (Acetyocholine+LNAME) % of relaxation WT+Angi II (Acetylcholine+LNAME) 75 75 % of relaxation * 50 50 25 25 0 0 -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 eNOS beta actin * 200 eNOS/β-actin (% CTRL) 100 0 WT TG2 -K/O WT +AII TG2-K/O+AII

  16. ROS production in aorta from WT and TG2-K/O mice WT TG2 K/O * 400 * 300 Arbitrary Units TG2-K/O + Ang II WT + Ang II 200 100 0 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.

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