Vascular dysfunctions in the isolated aorta of double transgenic hypertensive mice developing aortic aneurysm
Ludovic Waeckel1,2, 1Cécile Badier-Commander, Thibaut Damery1, Ralf Köhler3,4, Patricia Sansilvestri-Morel1, Serge Simonet1, Christine Vayssettes-Courchay, Heike Wulff5 and Michel Félétou1
1Institut de Recherches Servier, 92150 Suresnes, France; 2Centre de Recherche des Cordeliers, Inserm UMRS 872, Paris, France, 3Cardiovascular and Renal Research, IMM, University of Southern and Denmark, Odense, Denmark, 4Aragon Institute of Health Sciences-I+CS and ARAID, Zaragoza, Spain, 5Department of Pharmacology, University of California, Davis, CA 95616, USA.
Legend of Supplementary Figures
Supplementary Figure 1: Histo-morphological characteristics of aortae from A) wild type (BL) and C) double transgenic mice (AR) on control diet or after 4 weeks on a diet with salt plus L-NAME, B) BLSL and D) ARSL.
Five µm aortic sections colored with Masson’s trichrome showing in ARSL aorta, an increase in wall thickness ①, medial deconstruction ② with protein deposition ③ and extensive adventitial fibrosis ④.
Supplementary Figure 2: Concentration-response curves of the thromboxane analogue U-46619-induced contractions (0.1 nM to 1 µM) in isolated aortic rings with (A, n = 4-5) and without endothelium (B, n = 4-5) of wild type (BL) and double transgenic mice (AR) on control diet or after 4 weeks on a diet with salt plus L-NAME (BLSL and ARSL, respectively).
Values are expressed as mean SEM and n indicates the number of animals from which tissues were taken (ANOVA-2 followed by a Bonferroni post-hoc test).
Supplementary Figure 3: Endothelium-dependent relaxations to acetylcholine- (1 nM to 100 µM) in isolated aortic rings from wild type on control diet (BL) or after 4 weeks on a diet with salt plus L-NAME (BLSL) and in that of double transgenic mice (AR).
Top panels: Effects of the NO-synthase inhibitor L-nitro-arginine (LNA: 100 µM, n = 15) in A) BL, B) BLSL and C) AR.
Middle panels: Effects of the NO scavenger hydroxocobalamin (200 µM, n = 4) in D) BL, E) BLSL and F) AR.
Lower panels: Effects of the guanylyl cyclase inhibitor ODQ (5 µM, n = 4) in G) BL, H) BLSL and I) AR.
Values are expressed as mean SEM and n indicates the number of animals from which tissues were taken. The asterisk indicates a statistically significant difference produced by LNA, hydroxocobalamin or ODQ (ANOVA-2 followed by a Bonferroni post-hoc test, p < 0.05).
Supplementary Figure 4: Endothelium-dependent relaxations to acetylcholine (1 nM to 100 µM) and sodium nitroprusside (0.1 nM to 1 µM) in isolated aortic rings from double transgenic mice after 4 weeks on a diet with salt plus L-NAME (ARSL; n = 6).
Top panel: A) Effects of the superoxide dismutase mimetic, tempol (1mM), and the broad reactive oxygen species scavenger N-acetyl-cysteine (1 mM) on acetylcholine-induced endothelium-dependent relaxations.
Lower panels: Sodium nitroprusside-induced endothelium-independent relaxations, effects of B) the H2O2 scavenger catalase (1500 U/ml) in control conditions as well as C) the superoxide dismutase mimetic, tempol (1mM), and the broad reactive oxygen species scavenger, N-acetyl-cysteine (1 mM), and their association (in the presence of LNA: 100 µM). Tempol, N-acetyl-cysteine and their association produced a small but significant inhibition of the relaxation to sodium-nitroprusside. In BL, the association of tempol plus N-acetyl-cysteine produced, to a similar extent, a significant inhibition of the relaxation to sodium-nitroprusside (data not shown).
Values are expressed as mean SEM and n indicates the number of animals from which tissues were taken. The asterisk indicates a statistically significant difference produced by the drugs (ANOVA-2 followed by a Bonferroni post-hoc test, p < 0.05).
Supplementary Figure 5: Relaxations to sodium nitroprusside (1 nM to 10 µM) in isolated aortic rings with endothelium of wild type (BL) and double transgenic mice (AR) on control diet or after 4 weeks on a diet with salt plus L-NAME (BLSL) and ARSL).
Upper panel: A) Effect in the absence of the NO-synthase inhibitor, N-nitro-L-arginine (LNA: 100 µM; n = 7)
Lower panels: Not contemporary experiments showing the effects of LNA in B) BL (n = 33-7); C) BLSL (n= 13-7), D) AR (n = 13-7) and E) ARSL (n = 42-7).
Values are expressed as mean SEM and n indicates the number of animals from which tissues were taken. The asterisk indicates a statistically significant difference produced by LNA (ANOVA-2 followed by a Bonferroni post-hoc test, p < 0.05).
Supplementary Figure 6: Relaxations to the soluble guanylyl cyclase stimulator, BAY 41-2272 (10 nM - 0.3 µM) and the soluble guanylyl cyclase activator, BAY 58-2667 (0.1 nM – 0.1 µM) in isolated aortic rings with endothelium of wild type (BL) and double transgenic mice after 4 weeks on a diet with salt plus L-NAME (and ARSL): effects of LNA (100 µM).
To panels: Effects of BAY 41-2272 in A) BL and B) ARSL aortic rings (n = 4)
Lower panels: Effects of BAY 58-2667 in C) BL and D) ARSL aortic rings (n = 4)
Values are expressed as mean SEM and n indicates the number of animals from which tissues were taken. The stimulator and the activator of soluble guanylyl cyclase, in the presence or not of ODQ (see Figure 11) or LNA were studied in contemporary studies (same controls). The asterisk indicates a statistically significant effect produced by LNA (ANOVA-2 followed by a Bonferroni post-hoc test, p < 0.05).
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