Impaired vascular contractility and aortic wall degeneration in fibulin-4 deficient mice: effect of angiotensin II type 1 (AT1) receptor blockade

Abstract

Medial degeneration is a key feature of aneurysm disease and aortic dissection. In a murine aneurysm model we investigated the structural and functional characteristics of aortic wall degeneration in adult fibulin-4 deficient mice and the potential therapeutic role of the angiotensin (Ang) II type 1 (AT(1)) receptor antagonist losartan in preventing aortic media degeneration. Adult mice with 2-fold (heterozygous Fibulin-4(+/R)) and 4-fold (homozygous Fibulin-4(R/R)) reduced expression of fibulin-4 displayed the histological features of cystic media degeneration as found in patients with aneurysm or dissection, including elastin fiber fragmentation, loss of smooth muscle cells, and deposition of ground substance in the extracellular matrix of the aortic media. The aortic contractile capacity, determined by isometric force measurements, was diminished, and was associated with dysregulation of contractile genes as shown by aortic transcriptome analysis. These structural and functional alterations were accompanied by upregulation of TGF-β signaling in aortas from fibulin-4 deficient mice, as identified by genome-scaled network analysis as well as by immunohistochemical staining for phosphorylated Smad2, an intracellular mediator of TGF-β. Tissue levels of Ang II, a regulator of TGF-β signaling, were increased. Prenatal treatment with the AT(1) receptor antagonist losartan, which blunts TGF-β signaling, prevented elastic fiber fragmentation in the aortic media of newborn Fibulin-4(R/R) mice. Postnatal losartan treatment reduced haemodynamic stress and improved lifespan of homozygous knockdown fibulin-4 animals, but did not affect aortic vessel wall structure. In conclusion, the AT(1) receptor blocker losartan can prevent aortic media degeneration in a non-Marfan syndrome aneurysm mouse model. In established aortic aneurysms, losartan does not affect aortic architecture, but does improve survival. These findings may extend the potential therapeutic application of inhibitors of the renin-angiotensin system to the preventive treatment of aneurysm disease.

Figure 1. Survival of fibulin-4 mice.

(A) Distribution of the three genotypes at 0 and 3 weeks of age. The grey bars show the expected Mendelian distribution and the overlying bars the observed distribution of the different genotypes. Fibulin-4 mice are born in a Mendelian distribution (n = 10–20). Already after three weeks, this distribution is lost (n = 50–180, p<0.0001). (B) Kaplan-Meier survival curves of Fibulin-4^+/+, Fibulin-4^+/R and Fibulin-4^R/R mice alive at the age of three weeks (n = 50–180). After 21 weeks, 96% of wild type Fibulin-4^+/+ and 92% of Fibulin-4^+/R mice survived. Survival of Fibulin-4^R/R mice dramatically decreased to 33% (p<0.0001 vs. wild type). Note that the survival curve starts with all mice alive at the age of three weeks. Symbols indicate censored data.

Figure 2. Architecture of ascending thoracic aortas.

In adult Fibulin-4^+/R and Fibulin-4^R/R aortas there is an increase in aortic wall thickness (A–C), glycosaminoglycan depositions (blue areas) (D–F), elastic fiber fragmentation (G–I) and loss of smooth muscle cells in the media (J–L), also quantified (M).

Figure 3. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) measured using an intra-aortic microtip pressure transducer catheter.

With decreasing expression of Fibulin-4, DBP decreased and pulse pressure (PP) increased (p for trend 0.009 and <0.001 resp.). Data are mean±SEM of 4–17 mice. *p<0.05 vs. Fibulin-4^+/+ and Fibulin-4^+/R (two-way ANOVA).

Figure 4. Contractility mediated by KCl, phenylephrine and angiotensin II.

(A) In ascending aortas, KCl-induced contractility decreased in a gene dose-dependently in Fibulin-4^+/R and Fibulin-4^R/R mice (p for trend <0.001). (B) In descending aortas, phenylephrine-induced contractility decreased gene dose-dependently in Fibulin-4^+/R and Fibulin-4^R/R mice (p for trend 0.004). Data are mean±SEM of 6–18 experiments, *p<0.05 vs. Fibulin-4^+/+ mice. (C–F) Effect of angiotensin II on (C) ascending thoracic aortas, (D) descending thoracic aortas, (E) abdominal aortas and (F) iliac arteries. Data (mean±SEM of 3–6 experiments) are shown as a percentage of the response to 100 mmol/L KCl.

Figure 5. Calcium signaling pathway in a resting muscle cell (Fibulin-4^+/R vs. Fibulin-4^+/+ aortas).

Colors show up- (red) and downregulation (green) of molecules involved in muscle cell contraction.

Figure 6. Increased levels of pSmad2 and angiotensin II in fibulin-4 mutant aortas.

(A) Immunhistochemistry reveals a graded increase in expression and nuclear translocation of pSmad2 in the aortic media of adult fibulin-4 deficient mice. (B) With reduced fibulin-4 expression, tissue (but not blood) Ang II levels increase (p for trend 0.004). Data are shown as mean±SEM of 4–18 experiments. *p<0.05 vs. Fibulin-4^+/+. (C) Relative mRNA expression of Fibulin-4 and Ang II receptors. As published previously, a substantial decrease of fibulin-4 was observed in Fibulin-4^+/R and Fibulin-4^R/R mice when compared to wild type littermates. Both the renal and aortic arch AT₁b receptor content was larger in Fibulin-4^R/R mice when compared to Fibulin-4^+/+ and Fibulin-4^+/R mice. No differences in AT₁a or AT₂ receptor expression were observed between the different genotypes (n = 3–10). AT1aR, angiotensin II type 1a receptor; AT1bR, angiotensin II type 1b receptor; AT2R, angiotensin II type 2 receptor.

Figure 7. Aortic aneurysm treatment with losartan.

(A) Elastic fiber fragmentation in newborn Fibulin-4^R/R mice could be prevented with losartan, but not with propranolol or placebo. (B) Vessel wall thickness of thoracic aortas from newborn Fibulin-4^+/+ and treated Fibulin-4^R/R mice. Losartan treatment of Fibulin-4^R/R mice recovered vessel wall thickness. (C) Losartan treatment of adult Fibulin-4^R/R mice did not improve elastic fiber fragmentation. (D) Vessel wall thickness increased after postnatal losartan treatment. (E) Postnatal treatment with losartan did not reduce the amount of pSmad2 positive cells, nor did it affect lumen diameter (F). *p<0.05 vs. wild type, #p<0.05 vs. placebo-treated Fibulin-4^R/R mice, n = 4–5.