Functional and Biochemical Endothelial Profiling In Vivo in a Murine Model of Endothelial Dysfunction; Comparison of Effects of 1-Methylnicotinamide and Angiotensin-converting Enzyme Inhibitor

Abstract

Although it is known that 1-methylnicotinamide (MNA) displays vasoprotective activity in mice, as yet the effect of MNA on endothelial function has not been demonstrated in vivo. Here, using magnetic resonance imaging (MRI) we profile the effects of MNA on endothelial phenotype in mice with atherosclerosis (ApoE/LDLR^-/-) in vivo, in comparison to angiotensin (Ang) -converting enzyme (ACE) inhibitor (perindopril), with known vasoprotective activity. On a biochemical level, we analyzed whether MNA- or perindopril-induced improvement in endothelial function results in changes in ACE/Ang II-ACE2/Ang-(1-7) balance, and L-arginine/asymmetric dimethylarginine (ADMA) ratio. Endothelial function and permeability were evaluated in the brachiocephalic artery (BCA) in 4-month-old ApoE/LDLR^-/- mice that were non-treated or treated for 1 month or 2 months with either MNA (100 mg/kg/day) or perindopril (10 mg/kg/day). The 3D IntraGate^®FLASH sequence was used for evaluation of BCA volume changes following acetylcholine (Ach) administration, and for relaxation time (T₁) mapping around BCA to assess endothelial permeability using an intravascular contrast agent. Activity of ACE/Ang II and ACE2/Ang-(1-7) pathways as well as metabolites of L-arginine/ADMA pathway were measured using liquid chromatography/mass spectrometry-based methods. In non-treated 6-month-old ApoE/LDLR^-/- mice, Ach induced a vasoconstriction in BCA that amounted to -7.2%. 2-month treatment with either MNA or perindopril resulted in the reversal of impaired Ach-induced response to vasodilatation (4.5 and 5.5%, respectively) and a decrease in endothelial permeability (by about 60% for MNA-, as well as perindopril-treated mice). Improvement of endothelial function by MNA and perindopril was in both cases associated with the activation of ACE2/Ang-(1-7) and the inhibition of ACE/Ang II axes as evidenced by an approximately twofold increase in Ang-(1-9) and Ang-(1-7) and a proportional decrease in Ang II and its active metabolites. Finally, MNA and perindopril treatment resulted in an increase in L-arginine/ADMA ratio by 107% (MNA) and 140% (perindopril), as compared to non-treated mice. Functional and biochemical endothelial profiling in ApoE/LDLR^-/- mice in vivo revealed that 2-month treatment with MNA (100 mg/kg/day) displayed a similar profile of vasoprotective effect as 2-month treatment with perindopril (10 mg/kg/day): i.e., the improvement in endothelial function that was associated with the beneficial changes in ACE/Ang II-ACE2/Ang (1-7) balance and in L-arginine/ADMA ratio in plasma.

Keywords: 1-methylnicotinamide; L-Arg/ADMA ratio; MRI; atherosclerosis; endothelial function; perindopril; plasma angiotensin profile.

FIGURE 1

Methodology to assess endothelium-dependent response and changes in endothelial permeability in brachiocephalic artery (BCA) in vivo by MRI. (A) Coronal view of the heart. Image showing position of the imaging layer (IL) used for 3D imaging of the aortic arch. (B) 3D image of aortic arch acquired with the cine IntraGate^® FLASH 3D sequence. Endothelial function assessment, expressed as changes in the vessels volume (V) can be performed in vessels arising from aortic arch. The end-diastolic volumes of BCA were assessed prior to and 25 min after intraperitoneal Ach administration. (C) Representative images of vessels cross-sections, in which a number of pixels, for which T₁ had changed more than 50% after CA administration (Npx50), is marked in red. BCA is indicated as 1, left carotid artery (LCA) as 2, left subclavian artery (LSA) as 3. RV, right ventricle; LV, left ventricle.

FIGURE 2

Effects of MNA and ACE-I (perindopril) treatment on endothelial function and permeability in vivo in ApoE/LDLR^-/- mice. Changes in end-diastolic volume of BCA 25 min after Ach administration (A) and number of pixels around BCA, for which T₁ had changed more than 50% after CA administration (Npx50) (B) in non-treated ApoE/LDLR^-/- mice (columns with diagonal lines) and in ApoE/LDLR^-/- mice treated with MNA (black columns) or perindopril (columns with vertical lines) for one month (Untreated_5m n = 8, perindopril n = 5, MNA n = 6) or two months (Untreated_6m n = 8, perindopril n = 9, MNA n = 7). Statistics: Kruskal–Wallis test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 3

Effects of MNA and ACE-I (perindopril) treatment on plasma Ang profile in ApoE/LDLR^-/- mice. Plasma Ang profile in non-treated ApoE/LDLR^-/- mice (columns with diagonal lines) and in ApoE/LDLR^-/- mice treated with MNA (black columns) or perindopril (columns with vertical lines) for one month (A: Untreated_5m n = 6, perindopril n = 5, MNA n = 5) or two months (B: Untreated_6m n = 6, perindopril n = 8, MNA n = 7). Statistics: one-way ANOVA followed by Tukey’s post hoc test (normality was assessed using the Kolmogorov–Smirnov test); ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 4

Effects of MNA and ACE-I (perindopril) treatment on L-arginine/ADMA ratio in plasma in ApoE/LDLR^-/- mice. Plasma concentration of L-Arg, its precursors (L-Cit, L-Orn), ADMA, SDMA, Met, Hcy as well as L-Arg/ADMA ratio in non-treated ApoE/LDLR^-/- mice (columns with diagonal lines) and in ApoE/LDLR^-/- mice treated with MNA (black columns) or perindopril (columns with vertical lines) for one month (A: Untreated_5m n = 6, perindopril n = 5, MNA n = 5) or two months (B: Untreated_6m n = 6, perindopril n = 8, MNA n = 7). Statistics: one-way ANOVA followed by Tukey’s post hoc test (normality was assessed using the Kolmogorov–Smirnov test); ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.