Endothelium- and Fibroblast-Derived C-Type Natriuretic Peptide Prevents the Development and Progression of Aortic Aneurysm

Abstract

Background: Thoracic (TAA) and abdominal (AAA) aortic aneurysm are life-threatening diseases characterized by dilation, inflammation, and structural weakness; development of pharmacological therapies is desperately needed. CNP (C-type natriuretic peptide) plays a key role in vascular homeostasis, mediating vasodilator, anti-inflammatory, and antiatherogenic actions. Since such processes drive AA, we determined the role of endogenous CNP in offsetting pathogenesis.

Methods: Tissue from patients with AA was analyzed to determine the consequences on CNP signaling. Ascending and suprarenal aortic diameters were assessed at baseline and following Ang II (angiotensin II; 1.44 mg/kg per day) infusion in wild-type, endothelium-restricted (ecCNP^-/-), fibroblast-restricted (fbCNP^-/-), global CNP (gbCNP^-/-), or global NPR-C^-/- mice infected with an adeno-associated virus expressing a proprotein convertase subtilisin/kexin type 9 gain-of-function mutation or backcrossed to an apoE^-/- background. At 28 days, aortas were harvested for RT-qPCR (quantitative reverse transcription polymerase chain reaction) and histological analyses. CNP (0.2 mg/kg per day) was infused to rescue any adverse phenotype.

Results: Aneurysmal tissue from patients with TAA and AAA revealed that CNP and NPR-C (natriuretic peptide receptor-C) expression were overtly perturbed. ecCNP^-/-, fbCNP^-/-, and gbCNP^-/- mice exhibited an aggravated phenotype compared to wild-type animals in both ascending and suprarenal aortas, exemplified by greater dilation, fibrosis, elastin degradation, and macrophage infiltration. CNP and NPR-C expression was also dysregulated in murine thoracic AA and abdominal AA, accompanied by increased accumulation of mRNA encoding markers of inflammation, extracellular matrix remodeling/calcification, fibrosis, and apoptosis. CNP also prevented activation of isolated macrophages and vascular smooth muscle cells. An essentially identical phenotype was observed in NPR-C^-/- mice and while administration of CNP protected against disease severity in wild-type animals, this phenotypic rescue was not apparent in NPR-C^-/- mice.

Conclusions: Endothelium- and fibroblast-derived CNP, via NPR-C activation, plays important roles in attenuating AA formation by preserving aortic structure and function. Therapeutic strategies aimed at mimicking CNP bioactivity hold potential to reduce the need for surgical intervention.

Keywords: aortic aneurysm; fibrosis; macrophages; natriuretic peptide, C-type; vascular remodeling.

Figure 1.

Perturbed CNP (C-type natriuretic peptide) and NPR (natriuretic peptide receptor) expression in human and murine thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA). Individual heatmap showing quantification of RNA-seq data from human aortic media of control and sporadic ascending TAA (A), relative mRNA expression of CNP, NPR-B, and NPR-C in aortic samples from patients with nonaortic aneurysm-related disease (control) and individuals with Marfan syndrome (MFS) or AAA (B–D). Heatmaps (E and G) and quantified data (F and H) indicating the relative mRNA expression of markers of fibrosis, inflammation, and apoptosis, and CNP, NPR-B, and NPR-C in aortic samples from ApoE^−/− mice in the absence (control) and presence of an Ang II (angiotensin II) infusion (1.44 mg/kg per day; 28 days); plasma CNP concentrations (I) and thoracic (J) and abdominal (K) aortic diameter in the same animals. Data are represented as mean±SEM. Statistical analysis by 1-way ANOVA with Sidak post hoc test (B, D, F, and H) or 2-tailed Student t test (I, J, and K). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity). n=5 to 18 patients or animals per group.

Figure 2.

Global CNP (C-type natriuretic peptide) deletion exacerbates thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA). Thoracic (A–C) and abdominal (D–F) aortic diameter (A and D), representative ultrasound and full-length aortic images (B and E), and relative mRNA expression of NPRs (natriuretic peptide receptors; NPR-B and NPR-C) in aortic samples from wild-type (WT) mice and global CNP-null animals (gbCNP^−/−) pretreated with AAV.mPCSK9^D377Y and fed a Western diet, in the presence of an Ang II (angiotensin II) infusion (1.44 mg/kg per day; 28 days); representative immunohistochemical images of the aortic wall in WT and gbCNP^−/− mice following Ang II infusion (G) with quantification of elastin degradation (H), fibrosis (I), and α-SMA (α-smooth muscle actin; J; scale bar, 500 or 100 µm). Data are represented as mean±SEM. Statistical analysis by 2-way ANOVA with repeated measures (A and D), 1-way ANOVA with Sidak post hoc test (C and F), or 2-tailed Student t test (H–J). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity). n=4 to 10 animals per group. EVG indicates elastic van Gieson; H&E, hematoxylin and eosin; and PSR, picrosirius red.

Figure 3.

Endothelium-restricted CNP (C-type natriuretic peptide) deletion exacerbates thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA). Thoracic (A–C) and abdominal (D–F) aortic diameters (A and D), representative ultrasound and full-length aortic images (B and E), and relative mRNA expression of NPRs (natriuretic peptide receptors; NPR-B and NPR-C) in aortic samples from wild-type (WT) mice and endothelium-specific CNP-null animals (ecCNP^−/−) on an ApoE^−/− background in the presence of an Ang II (angiotensin II) infusion (1.44 mg/kg per day; 28 days); representative immunohistochemical images of the aortic wall in WT and ecCNP^−/− mice following Ang II infusion (G) with quantification of elastin degradation (H), fibrosis (I), and α-SMA (α-smooth muscle actin; J; scale bar, 500 or 100 µm). Data are represented as mean±SEM. Statistical analysis by 2-way ANOVA with repeated measures (A and D), 1-way ANOVA with Sidak post hoc test (C and F), or 2-tailed Student t test (H–J). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity). n=5 to 9 animals per group. EVG indicates elastic van Gieson; H&E, hematoxylin and eosin; and PSR, picrosirius red.

Figure 4.

Fibroblast-restricted CNP (C-type natriuretic peptide) deletion exacerbates thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA). Thoracic (A–C) and abdominal (D–F) aortic diameters (A and D), representative ultrasound and full-length aortic images (B and E), and relative mRNA expression of NPRs (natriuretic peptide receptors; NPR-B and NPR-C) in aortic samples from wild-type (WT) mice and fibroblast-specific CNP-null animals (fbCNP^−/−) pretreated with AAV.mPCSK9^D377Y and fed a Western diet in the presence of an Ang II (angiotensin II) infusion (1.44 mg/kg per day; 28 days); representative immunohistochemical images of the aortic wall in WT and fbCNP^−/− mice following Ang II infusion (G) with quantification of elastin degradation (H), fibrosis (I), and α-SMA (α-smooth muscle actin; J; scale bar, 500 or 100 µm). Data are represented as mean±SEM. Statistical analysis by 2-way ANOVA with repeated measures (A and D), 1-way ANOVA with Sidak post hoc test (C and F), or 2-tailed Student t test (H–J). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity). n=5 to 10 animals per group. EVG indicates elastic van Gieson; H&E, hematoxylin and eosin; and PSR, picrosirius red.

Figure 5.

Global natriuretic peptide receptor (NPR)-C deletion exacerbates thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA). Thoracic (A–C) and abdominal (D–F) aortic diameters (A and D), representative ultrasound and full-length aortic images (B and E), and relative mRNA expression of NPR-B and NPR-C in aortic samples from wild-type (WT) mice and global NPR-C–null animals (NPR-C^−/−) on an ApoE^−/− background in the presence of an Ang II (angiotensin II) infusion (1.44 mg/kg per day; 28 days); representative immunohistochemical images of the aortic wall in WT and NPR-C^−/− mice following Ang II infusion (G) with quantification of elastin degradation (H), fibrosis (I), and α-SMA (α-smooth muscle actin; J; scale bar, 500 or 100 µm). Data are represented as mean±SEM. Statistical analysis by 2-way ANOVA with repeated measures (A and D), 1-way ANOVA with Sidak post hoc test (C and F), or 2-tailed Student t test (H–J). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity). n=5 to 12 animals per group. EVG indicates elastic van Gieson; H&E, hematoxylin and eosin; and PSR, picrosirius red.

Figure 6.

Therapeutic administration of CNP (C-type natriuretic peptide) or NPR-C (natriuretic peptide receptor-C) agonist prevents disease progression of thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) in wild-type (WT) but not in NPR-C^−/− mice. Thoracic (A–C) and abdominal (C–E) aortic diameters (A and D), representative ultrasound (B and E), and full-length aortic images (C) in wild-type (WT) and global NPR-C–null animals (NPR-C^−/−) pretreated with AAV.mPCSK9^D377Y and fed a Western diet in the absence and presence of CNP (0.2 mg/kg per day) or the NPR-C agonist cANF^– (0.4 mg/kg per day) during concomitant Ang II (angiotensin II) infusion (1.44 mg/kg per day; 28 days). Data are represented as mean±SEM. Statistical analysis by 2-way ANOVA with repeated measures (A and D) or 2-tailed Student t test (F and G). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity). n=5 to 8 animals per group. cANF indicates atrial natriuretic factor-des[Gln18, Ser19, Gly20, Leu21, Gly22] ANF4-23-NH2.

Figure 7.

Markers of inflammation, remodeling, differentiation, and apoptosis are exacerbated by Ang II (angiotensin II) in human aortic smooth muscle cells but rescued by CNP (C-type natriuretic peptide). Expression of NPRs (natriuretic peptide receptors; NPR-B and NPR-C; A–D) and markers of inflammation (CCL2 [chemokine C-C motif ligand 2]; E), remodeling (fibronectin, F; BMPR2 [bone morphogenetic receptor type 2], G), phenotypic switching (smoothelin, H; Klf4 [Kruppel like factor-4], I), and apoptosis (Bax [bcl-2-like protein 4], J) in primary human aortic smooth muscle cells in the absence (baseline) and presence of Ang II (200 nmol/L; 3–12 hours, A and B or at 12 hours, C–J) with concomitant administration of CNP (100 nmol/L), the NPR-C agonist cANF^– (200 nmol/L), or CNP plus the NPR-C antagonist M372049 (10 μmol/L). Data are represented as mean±SEM. Statistical analysis by 1-way ANOVA with Sidak post hoc test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity). n=4 independent experiments per group. cANF indicates atrial natriuretic factor-des[Gln18, Ser19, Gly20, Leu21, Gly22] ANF4-23-NH2.

Figure 8.

Genetic deletion of CNP (C-type natriuretic peptide)/NPR-C (natriuretic peptide receptor-C) signaling exacerbates the proinflammatory phenotype in experimental aortic aneurysm (AA) and in isolated macrophages. Heatmap of markers of inflammation (A) and representative immunofluorescence images of macrophage infiltration using anti-Mac-2 (galectin-3) with quantified data (B) in wild-type (WT), global (gbCNP^−/−), endothelium-restricted (ecCNP^−/−), and fibroblast-restricted (fbCNP^−/−) CNP-null mice and NPR-C^−/− animals following Ang II (angiotensin II) infusion (1.44 mg/kg per day; 28 days; scale bar, 500 or 100 µm). Expression of markers of inflammation (NOS2, IL-6, and IL-1) in isolated murine macrophages in culture in the absence (control) and presence of lipopolysaccharide (LPS; 100 ng/mL) with concomitant administration of CNP (100 nmol/L) or the NPR-C agonist cANF^– (200 nmol/L; C and D). Data are represented as mean±SEM. Statistical analysis by 2-tailed Student t test (B) or 1-way ANOVA with Sidak post hoc test (C and D). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity). n=5 independent experiments per group. cANF indicates atrial natriuretic factor-des[Gln18, Ser19, Gly20, Leu21, Gly22] ANF4-23-NH2; and DAPI, 4′,6-diamidino-2-phenylindole.