MMP-2 regulates Erk1/2 phosphorylation and aortic dilatation in Marfan syndrome

Abstract

Rationale: Aneurysm and dissection of the ascending thoracic aorta are the main cardiovascular complications of Marfan syndrome (MFS) resulting in premature death. Studies using mouse models of MFS have shown that activation of transforming growth factor-beta (TGF-β) and the concomitant upregulation of matrix metalloproteinases (MMPs) contribute to aneurysm development. Our previous study showed that doxycycline delayed aneurysm rupture in a mouse model of MFS, Fbn1(mgR/mgR). Losartan has been shown to prevent aneurysms in another mouse model of MFS, Fbn1(C1039G/+), through inhibition of the Erk1/2 pathway. However, the role of MMP-2 in MFS and effect of losartan on the lifespan of MFS mice remain unknown.

Objective: We investigated the role of MMP-2 in MFS and compared the effects of losartan and doxycycline on aortic dilatation and survival in Fbn1(mgR/mgR) mice.

Methods and results: By life table analysis, we found that losartan and doxycycline improved the survival of Fbn1(mgR/mgR) mice. Gelatin zymography and Western blot data showed that only doxycycline inhibited MMP-2 expression, whereas both drugs decreased Erk1/2 phosphorylation. When combined, only one of nine mice died within the 30-week study; aortic histology and diameter were normalized and the effects on Smad2 phosphorylation was additive. To further explore the role of MMP-2 in MFS, we created MMP-2-deficient Fbn1(mgR/mgR) mice. MMP-2 deletion inhibited activation of TGF-β and phosphorylation of Erk1/2 and Smad2 and prolonged the lifespan of the mice.

Conclusions: These studies demonstrated that inhibition of MMP-2 by doxycycline delayed the manifestations of MFS, in part, through its ability to decrease active TGF-β and the noncanonical signaling cascade downstream of TGF-β. This study further suggested that targeting TGF-β signaling at different points might be a more effective strategy for inhibiting disease progression.

Figure 1

Phosphorylation of Smad2 and Erk1/2 in the aortic tissues and smooth muscle cells from Fbn1^mgR/mgR mice and wild type (WT) littermate controls analyzed by Western blotting (A&C) and MMP-2 and -9 levels analyzed by gelatin zymography (B&C). At the age of 8 weeks, mouse thoracic aortas were harvested from WT control and Fbn1^mgR/mgR mice treated without, or with doxycycline (Doxy)(0.5 g/L), or losartan (Los)(0.6 g/L), or doxycycline (0.5 g/L) and losartan (0.6 g/L) added to the drinking water. Aortic proteins were extracted. A) Representative Western blot showing the immunoreactivity to antibodies for phosphorylated smad2 and Erk1/2 (left panel) and relative expression was quantified (right panel) using 5 or more aortic specimens per group; B) A representative gelatin zymogram of MMP-2 and -9 expression using 10 μg of aortic protein per lane with quantification of relative MMP-2 levels in the aortas (n=4) (right panel). C) Aortic smooth muscle cells from wild type and Fbn1^mgR/mgR mice were isolated and received no treatment (none), doxycycline (100 μg/ml), losartan (100 μg/ml), or combination of doxycycline (100 μg/ml) and losartan (100 μg/ml). Cellular proteins (25 μg) were analyzed for phosphorylation of Smad2 and Erk1/2 using Western blot (upper panel); MMP-2 expression in cell conditioned media were analyzed by zymography (lower panel). D) TGF-β levels in the aortic tissue of Fbn1^mgR/mgR mice with or without treatment were analyzed by Western blotting. Total TGF-β and the ratio active TGF-β to total TGF-β were quantified (n=4) (a&b). Western blots and zymograms are representatives of 3–5 separate experiments. β-actin expression served as internal loading control, * p < 0.05; # p < 0.01; † p < 0.001.

Figure 2

Doxycycline and losartan effects on aneurysm expansion and elastin degradation. A) Aortic diameters for wild type littermate and Fbn1^mgR/mgR (mgR) mice receiving no treatment (No Tx) or doxycycline (Doxy)(0.5 g/L), losartan (Los) (0.6 g/L) or a combination were measured by high frequency ultrasound at 5, 8, and 12 weeks of ages. The aortic diameter of untreated Fbn1^mgR/mgR mice were significantly enlarged compared with wild type control at all time points, # p < 0.002. Doxycycline- and losartan-treatment significantly inhibited aortic enlargement of Fbn1^mgR/mgR mice at 8 and 12 weeks-time-points as compared with untreated Fbn1^mgR/mgR mice (** p = 0.0054 and p = 0.0060, respectively). At 12 weeks-time-point, combination of doxycycline and losartan further inhibited aortic growth compared to doxycycline and losartan alone, † p<0.05. C) Verhoeff-van Gieson staining of elastic fibers of the ascending aorta of wild type and Fbn1^mgR/mgR mice treated with or without of doxycycline and losartan examined at 8 weeks of age; Fbn1^mgR/mgR mice without treatment (a), doxycycline (0.5 g/L) treatment (b), losartan (0.6 g/L) treatment (c), or combination of doxycycline (0.5 g/L) and losartan (0.6 g/L) (d). Wild type littermate (e). B) Elastin breaks per field under 40x magnification in wild type (WT) and Fbn1^mgR/mgR mice: No Tx, or Doxy, or Los, or Doxy &Los (n= 3–5 aortas/group). * p < 0.05; † p < 0.001.

Figure 3

The effect of doxycycline and losartan on the survival of Fbn1^mgR/mgR mice. Fbn1^mgR/mgR mice were untreated (No Tx) or treated with doxycycline (Doxy) (0.5 g/L), or losartan (Los) (0.6 g/L), or combination of doxycycline (0.5 g/L) and losartan (0.6 g/L), in their drinking water. Life table analysis shows improved survival in mice with doxycycline (p <0.001), losartan (p < 0.01), and doxycycline & losartan (p < 0.001) compared to untreated Fbn1^mgR/mgR mice. Compared with single drug treatment, combination of doxycycline and losartan further extends the life span of Fbn1^mgR/mgR mice, p < 0.001.

Figure 4

The effects of MMP-2-deletion on elastolytic activities and TGF-β signaling. A) SMC from wild type (WT) and MMP-2^−/− mice were co-cultured with [³H]elastin and incubated with 50 μg/ml ConA. The elastolytic activities were quantified. Results are expressed as μg elastin degraded/10⁶ cells for 5 days (mean ± SE; n=3). B) MMP-2 and MMP-9 expression in the aortas of MMP-2^+/+/fbn1^mgR/mgR (WT/mgR) and MMP-2^−/−/fbn1^mgR/mgR (MMP-2^−/−/mgR) mice at 8 weeks of age were analyzed by zymography. WT/mgR and MMP-2^−/−/mgR mouse aortic tissues expressed similar levels of MMP-9. C) TGF-β levels and Smad2 phosphorylation in the aortic tissue of WT/mgR and MMP-2^−/−/mgR mice were analyzed by Western blotting; latent and active TGF-β (n = 4) (a) and pSmad2 (n =4) (b) were quantified. D) Representative Western blot analysis of Erk1/2 phosphorylation in the aorta of WT/mgR and MMP-2^−/−/mgR mice (bar graph represents 6/group). * p < 0.05; # p < 0.01.

Figure 5

The effects of MMP-2-deletion on Fbn1^mgR/mgR mice. A) Verhoeff-van Gieson staining of elastic fibers of thoracic ascending aortas of MMP-2^+/+/fbn1^mgR/mgR (WT/mgR)(a) and MMP-2^−/−/fbn1^mgR/mgR (MMP-2^−/−/mgR)(b) mice at 8 weeks of age, representative images from 3–5 independent experiments. B) Elastin breaks per field under 40x magnification in WT/mgR and MMP-2^−/−/mgR mice (n= 3–5 aortas/group); C) Ascending thoracic aorta diameter measured by high frequency ultrasound in WT/mgR and MMP-2^−/−/mgR mice at 5, 8, and 12 weeks of age (* p = 0.0283); D) comparison of average lifespan of WT/mgR with MMP-2^−/−/mgR, *p < 0.0001.