A Novel α-Calcitonin Gene-Related Peptide Analogue Protects Against End-Organ Damage in Experimental Hypertension, Cardiac Hypertrophy, and Heart Failure

Figures

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  Figure 1.. α-CGRP analogue (αAnalogue) increases vascular blood flow via CGRP receptors. Blood flow monitored using Full-field Laser Perfusion Imager in the ear vasculature of mice pretreated with control (saline) or CGRP receptor antagonist BIBN4096 (0.3 mg/kg, IV) at baseline and following intradermal injection of αAnalogue (100 pmol, daily) or vehicle (Veh) (15 µL, n=6). A, Blood flow responses expressed as % change from baseline. B, Representative Full-field Laser Perfusion Imager pictures alongside gray/black photo showing blood flow at baseline and 15 minutes after treatment. C, Blood flow assessed by area under the curve (AUC) for 30 minutes following vehicle or αAnalogue administration (n=6). Data showed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 versus vehicle-treated; #P<0.05, ##P<0.01, ###P<0.001 for αAnalogue treated (A, repeated-measures 2-way ANOVA + Bonferroni post hoc test; C, 2-way ANOVA + Bonferroni post hoc test). α-CGRP indicates α-calcitonin gene–related peptide.

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  Figure 2.. Daily systemic treatment with α-CGRP analogue (αAnalogue) protects against angiotensin II (AngII)–induced hypertension and vascular damage. Mice were infused with AngII (A, 1.1 mg·kg–1·d-–1) or control (S, saline) for 14 days and treated daily with vehicle (V) or αAnalogue (50 nmol/kg, SC). A, Systolic blood pressure was measured by radiotelemetry. Results expressed as 6-hour average. Mice experience a 12/12 hour light/dark cycle, with the dark cycle shown in the gray striped area. Arrow represents the start of daily treatment. B, Protein expression of total eNOS in aorta (n=4–5). C, Representative images of Masson trichrome-stained aortic sections. D, Quantification of smooth muscle wall width (n=4–5; scale bars, 100 µm). Protein expression of NADPH oxidase-2 (NOX-2) (E), heme oxygenase-1 (HO-1) (F), nitrotyrosine in aorta (n=4–6) (G). H, Protein expression of nitrotyrosine in mesenteric vessels (n=6–7). Results shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 versus vehicle-treated saline-infused; #P<0.05, ##P<0.01, ###P<0.001 for αAnalogue-treated AngII-infused versus vehicle-treated AngII-infused (A, repeated-measures 2-way ANOVA + Bonferroni post hoc test; B through H, 2-way…

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  Figure 3.. α-CGRP analogue (αAnalogue) protects against angiotensin II (AngII)–induced cardiac hypertrophy and fibrosis. Mice were treated as in Figure 2 (AngII, A; Saline, S). A, Left ventricle weight normalized to tibia length ratio (mg/mm). B, Protein expression of α-smooth muscle actin (α-SMA) in heart (n=5). mRNA expression measured by real-time quantitative polymerase chain reaction for transforming growth factor-β1 (TGF-β1) (C), fibronectin (D), collagen type 1 α1 (COL1A1) (E), collagen type 1 α2 (COL1A2) (F), collagen type 3 α1 (COL3A1) (G), collagen type 4 α1 (COL4A1) (H), atrial natriuretic peptide (ANP) (I), brain natriuretic peptide (BNP) (J), matrix metalloproteinase-2 (MMP-2) (K), and sarcoplasmic reticulum Ca2+ ATPase-2 (SERCA-2) (L) in heart (n=5–11). Results expressed as copy numbers per microliter normalized to hypoxanthine-guanine phosphoribosyltransferase, B2M and β-actin, and showed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 versus vehicle-treated saline-infused; #P<0.05, ##P<0.01, ###P<0.001 versus vehicle-treated AngII-infused (2-way ANOVA + Bonferroni post hoc test). α-CGRP indicates α-calcitonin gene–related peptide.

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  Figure 4.. Daily systemic treatment with α-CGRP analogue (αAnalogue) protects against angiotensin II (AngII)–induced cardiac inflammation and oxidative stress. Mice were treated as in Figure 2 (AngII, A; Saline, S). mRNA expression measured by real-time quantitative polymerase chain reaction (n=5–11) for RANTES (A) and glutathione peroxidase-1 (GPX-1) (n=6–11) (B). C, Protein expression of GPX-1 in heart (n=5). mRNA expression for hypoxia-inducible factor-1 (HIF-1α) (D), heme oxygenase-1 (HO-1) (E), and NADPH dehydrogenase quinone-1 (NQO1) (n=5–11) (F). Results expressed as copy numbers per microliter normalized to hypoxanthine-guanine phosphoribosyltransferase, B2M and β-actin, and showed as mean±SEM. G, Protein expression of nitrotyrosine (n=5) in heart (n=6–7). *P<0.05, **P<0.01, ***P<0.001 versus vehicle-treated saline-infused; #P<0.05, ##P<0.01, ###P<0.001 versus vehicle-treated AngII-infused (2-way ANOVA + Bonferroni post hoc test). α-CGRP indicates α-calcitonin gene–related peptide.

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  Figure 5.. Daily systemic treatment with α-CGRP analogue (αAnalogue) protects against angiotensin II (AngII)–induced renal fibrosis, dysfunction, and inflammation. Mice were treated as in Figure 2 (AngII, A; Saline, S). mRNA (A) and protein expression of α-SMA (B) in kidney. mRNA expression of TGF-β1 (C), collagen type 1a1 (COL1A1) (D), cystatin C (E), and neutrophil gelatinase-associated lipocalin (NGAL) (F) in kidney. Creatinine levels in kidney (G) and plasma (H) (n=6–8). IL-6 (I), TNF-α (J), and noradrenaline (NA) (K) levels in kidney (n=6–9). L, Protein expression of klotho in kidney. mRNA expression measured by real-time quantitative polymerase chain reaction (n=6–8), expressed as copy numbers per microliter normalized to hypoxanthine-guanine phosphoribosyltransferase, B2M, and β-actin. Results shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 versus vehicle-treated saline-infused; #P<0.05, ##P<0.01 versus vehicle-treated AngII-infused (2-way ANOVA + Bonferroni post hoc test). α-CGRP indicates α-calcitonin gene–related peptide; IL-6, interleukin 6; α-SMA; α-smooth muscle actin; TGF-β1; transforming growth factor-β1; and TNF-α, tumor necrosis factor-α.

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  Figure 6.. Effects of daily systemic treatment with α-CGRP analogue (αAnalogue) on CGRP receptor expression in angiotensin II (AngII)–induced hypertension. Mice were treated as in Figure 2 (AngII, A; saline, S, V, vehicle; α-CGRP analogue; C). Protein expression of calcitonin receptor-like receptor (CLR) (A) and receptor-associated membrane protein-1 (RAMP1) (B) in aorta (n=6). Protein expression of CLR (C) and RAMP1 (D) in mesenteric vessels (n=6). Protein expression of CLR (E) and RAMP1 (F) in heart (n=6–7). Protein expression of CLR (G) and RAMP1 (H) in kidney (n=5–6). Results showed as mean±SEM.*P<0.05, ***P<0.001 versus vehicle-treated saline-infused; #P<0.05, ###P<0.001 versus vehicle-treated AngII-infused (2-way ANOVA + Bonferroni post hoc test). α-CGRP indicates α-calcitonin gene–related peptide.

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  Figure 7.. α-CGRP analogue (αAnalogue) limits angiotensin II (AngII)–induced hypertension. Mice were infused with AngII (1.1 mg·kg–1·d–1 for 14 days) and treated daily with vehicle or αAnalogue (50 nmol/kg, SC) at day 7 to 14 of infusion (n=4). A, Systolic blood pressure was measured by radiotelemetry. Results expressed as 6-hour average. Mice experience a 12/12 hour light/dark cycle, with the dark cycle shown in the gray striped area. Arrow represents the start of daily treatment. B, Left ventricle weight normalized to tibia length ratio (mg/mm). Representative heart sections (Top) and analysis (Bottom) showing cardiac hypertrophy by cardiomyocyte borders outlined using wheat germ agglutinin (scale bars, 20 µm) (C) and cardiac fibrosis by Picrosirius Red staining (scale bars, 200 µm) (D). E, Protein expression of nitrotyrosine in heart. Results showed as mean±SEM.*P<0.05, **P<0.01, ***P<0.001 versus vehicle-treated (A, repeated-measures 2-way ANOVA + Bonferroni post hoc test; B through E, 2-way ANOVA + Bonferroni post hoc test). α-CGRP indicates α-calcitonin gene–related peptide.

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  Figure 8.. α-CGRP analogue (αAnalogue) preserves heart function post–AAC-induced cardiac hypertrophy and heart failure. Mice were treated daily with vehicle and αAnalogue (50 nmol/kg, SC) postsurgery for 5 weeks (n=6–8). Ejection fraction (%) (A), septum wall thickness at diastole (mm) (B), left ventricle mass normalized to tibia length (mg/mm) (C), and wet lung mass (mg) normalized to tibia length (D). Representative images (E) and quantification of cardiomyocyte cross-sectional area (F) in heart using wheat germ agglutinin staining (scale bars, 20 µm). Quantification (G) and representative images of fibrosis in heart (H) using picrosirius red staining (scale bars, 200 µm). Representative images (I) and quantification of capillary density in heart (J) using isolectin-B4 staining (scale bars, 20 µm). K and L, Protein expression of nitrotyrosine in heart. Quantification (M) and representative images of apoptosis (N) using TUNEL staining (scale bars, 50 µm). Results showed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 versus vehicle-treated sham mice; #P<0.05, ##P<0.01, ###P<0.001 versus vehicle-treated AAC mice (2-way ANOVA + Bonferroni post hoc test). AAC indicates abdominal aorta…