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. 2019 Feb 14;9(1):2071.
doi: 10.1038/s41598-018-38235-6.

In Vivo Characterization of Doxycycline-Mediated Protection of Aortic Function and Structure in a Mouse Model of Marfan Syndrome-Associated Aortic Aneurysm

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In Vivo Characterization of Doxycycline-Mediated Protection of Aortic Function and Structure in a Mouse Model of Marfan Syndrome-Associated Aortic Aneurysm

Jason Z Cui et al. Sci Rep. .
Free PMC article

Abstract

Aortic aneurysm is the most life-threatening complication in Marfan syndrome (MFS) patients. Doxycycline, a nonselective matrix metalloproteinases inhibitor, was reported to improve the contractile function and elastic fiber structure and organization in a Marfan mouse aorta using ex vivo small chamber myography. In this study, we assessed the hypothesis that a long-term treatment with doxycycline would reduce aortic root growth, improve aortic wall elasticity as measured by pulse wave velocity, and improve the ultrastructure of elastic fiber in the mouse model of MFS. In our study, longitudinal measurements of aortic root diameters using high-resolution ultrasound imaging display significantly decreased aortic root diameters and lower pulse wave velocity in doxycycline-treated Marfan mice starting at 6 months as compared to their non-treated MFS counterparts. In addition, at the ultrastructural level, our data show that long-term doxycycline treatment corrects the irregularities of elastic fibers within the aortic wall of Marfan mice to the levels similar to those observed in control subjects. Our findings underscore the key role of matrix metalloproteinases during the progression of aortic aneurysm, and provide new insights into the potential therapeutic value of doxycycline in blocking MFS-associated aortic aneurysm.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Effects of long-term doxycycline intervention on pulse wave velocity and aortic wall stiffness in control and MFS mice. (A) Control mouse: Aortic arch length from “d0” to “d” was measured on B-mode view as the distance; (B) Tracing recordings on pulse wave Doppler-mode view: time interval T1 in the ascending aorta (upper panel) and T2 descending aorta (lower panel). Pulse Wave Velocity formula: PWV = [d − d0]/[T2 − T1]; (C) PWV is significantly increased in MFS mice compared to controls without treatment at 3-month old (**p < 0.01); the difference in PWV is not significant in doxycycline-treated MFS mice compared to controls. PWV is significantly increased in non-treated MFS mice compared to controls at 6-month old (***p < 0.001), and doxycycline-treated MFS mice have significantly lower PWV values than non-treated MFS mice (**p < 0.01), But they do not display significant difference in PWV from either the treated or non-treated control mice, suggesting that long term doxycycline treatment exerted protective effects of on reduction of aortic stiffness in MFS mice compared to non-treated MFS mice starting from 6-month of age. PWV is significantly increased in MFS mice compared to controls without treatment at 9-month old (***p < 0.001); the doxycycline-treated MFS mice have significantly lower PWV than non-treated MFS mice (*p < 0.05), however, they show a significantly increased PWV compared to the treated control mice (**p < 0.01). The same findings as at 9-month old, but PWV in 12-month treated MFS mice continued to decrease further towards the levels in both treated and non-treated control mice, suggesting that long term doxycycline treatment extended protective effects of on reduction of aortic stiffness in MFS mice.
Figure 2
Figure 2
Effects of long-term doxycycline intervention on regional aortic root diameters in control and MFS mice. (A) Representative image of the B-mode view of a mouse aortic arch showing the diameters of three areas of interest, aortic annulus (L1), sinuses of Valsalva (L2), and sinotubular junctions (L3); (B) Aortic annulus diameters are significantly increased in MFS mice compared to controls with and without doxycycline treatment at 3-month old (**p < 0.01). Aortic diameters of the treated Marfan mice are not different from those in controls at age of 6-, 9-month old, while the aortas are all significantly wider in MFS mice without doxycycline treatment (***p < 0.001), suggesting severe aortic root dilation. Diameters of the treated Marfan mice are significantly decreased compared to non-treated MFS mice at age of 12-month old (*p < 0.05). The diameters also show no difference than treated control mice. (C) Aortic diameters at sinuses of Valsalva of the treated Marfan mice are not different from those in controls at age of 6-, 9-month old, while the aortas are all significantly wider in MFS mice without doxycycline treatment (**p < 0.01, ***p < 0.001, respectively). Furthermore, at 6-month old, treated MFS mice display significantly decreased aortic diameters compared to their age-matched MFS counterparts without treatment (*p < 0.05), suggesting potential protective effects of doxycycline on reduction of aortic dilation. (D) Measurements of diameters in sinotubular junctions that is a relatively distant loci of aorta from the left ventricle show that there are no differences in the aortic diameters between MFS and control mice, regardless of doxycycline treatment or not at all age groups, even though trends of dilated aortas in MFS mice can be observed. Diameters of the treated Marfan mice are significantly decreased compared to non-treated MFS mice at age of 12-month old (*p < 0.05). The diameters also show no difference than treated control mice (n = 12–13; Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001).
Figure 3
Figure 3
Effects of long-term doxycycline intervention on MMP-2 and MMP-9 expression in the aortic wall of control and MFS mice. Representative immunohistochemistry staining of total (A) MMP-2 and (B) MMP-9 expression levels within the cross sections of aortic wall isolated from 12-month old control and MFS mice in the presence and absence of doxycycline treatment. (C) Bar graphs representing quantification of MMP-2 and -9 expression levels within the aortic wall sections isolated from 12-month old control and MFS mice in the presence and absence of doxycycline treatment (n = 5; Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001).
Figure 4
Figure 4
Correlations between aortic structure and function over time in treated and non-treated control and MFS mice. Linear regressions presenting the correlations between aortic diameters measurements and PWV in (A) non-treated control mice, (B) non-treated MFS mice, (C) doxycycline-treated control mice, and (D) doxycycline-treated MFS mice. The gap of the linear regressions between sinotubular junction and aortic root is narrowed, while the gap between these two regions and sinus of Valsalva is wider, in MFS mice compared to controls, regardless on doxycycline treatment or not. Out of three aortic regions, correlation in the region of sinus of Valsalva stands out as the highest level and warrants further investigation.
Figure 5
Figure 5
Correlations between PWV and aortic diameters at the region of sinus of Valsalva in different age groups. (A) At 3 months of age, there is no positive correlation with PWV in control and MFS mice in the presence or absence of doxycycline treatment. (B) At 6 months of age, there is a positive correlation with PWV in non-treated control mice (R2 = 0.96), as well as in non-treated and treated MFS mice. Linear regression level in treated MFS mice falls under the non-treated MFS mice starting at 6-month of age. (C) At 9-month old, correlation between PWV and aortic diameters continues to fall in treated MFS mice, while non-treated MFS remain dominant at the higher range. (D) At 12-month old, linear regression of the correlation in treated MFS group falls down to the level that is similar to the non-treated and treated control mice, while in non-MFS group it continues to stay at the higher level.
Figure 6
Figure 6
Comparison of cardiovascular gross structure and elastic morphology in treated and non-treated control and MFS mice. Representative images of dissected heart and aorta samples from (A,B) non-treated control mouse, (C,D) non-treated Marfan mouse, and (E,F) doxycycline-treated Marfan mouse at 12 months of age. (A,B) Intact mouse aorta with the typical three major branches in the aortic arch, which are, from proximal to distal, the brachiocephalic trunk, the left common carotid artery, and the left subclavian artery, attached to the normal shape of a control mouse heart. (C,D) Severe aortic dilation in MFS mouse without doxycycline treatment, a “balloon-like” dilation is observed prior to the aortic arch three branches. The left ventricle of MFS mouse heart becomes dilated. (E,F) Relatively normal shape and size aorta and heart can be observed in MFS mouse with doxycycline treatment.
Figure 7
Figure 7
Measurements of normalized LV mass in treated and non-treated control and MFS mice. Without doxycycline treatment, Marfan mice showed significant increase in normalized LV mass compared to controls at 3, 6, 9, and 12 months of age (**p < 0.01, *p < 0.05, respectively). However, with doxycycline treatment, except for 3-month groups that LV mass was significantly increased in Marfan mice (**p < 0.01), no difference between treated Marfan and control mice from 6 months on, suggesting potential protective effects of doxycycline on reduction of LV mass (n = 12–13; Mean ± SEM).
Figure 8
Figure 8
Representative images of van Gieson’s staining of the aortic root in treated and non-treated control and MFS mice. (A) Non-treated control, (B) non-treated Marfan, (C) doxycycline-treated control, and (D) doxycycline-treated Marfan mice at 12-month of age illustrate elastin (dark blue/purple) and collagen (light pink). Long-term doxycycline treatment shows protective effects on maintaining elastin fibers organization and aortic wall integrity on Marfan mice; however, it seems that this treatment cannot completely reverse the morphology as in Marfan mice.
Figure 9
Figure 9
Ultrastructural changes at elastin break points in aortic section isolated from treated and non-treated control and MFS mice. Representative TEM images of ultrastructure of aortic elastin from (A) non-treated control mice, (B) non-treated Marfan mice, and (C) doxycycline-treated Marfan mice at 12 months of age. The boarder of the broken elastic fibers is relatively clean and sharp in control aorta; while irregular and fuzzy boarder can be observed in MFS mice. However, with long-term doxycycline treatment, it seems that the elastic fibers regain a normal shape on boarder lines, reversing the irregularities to the levels as those in their control counterparts. (D) Bar graphs showing the significance of observed differences in elastin irregularities index (circumference/width) in control, and non-treated and treated MFS mouse aorta. The irregularities index of elastin is significantly increased in MFS mice compared to controls (***p < 0.001), while elastic fibers in doxycycline-treated MFS mice aorta have significantly lower irregularities index than non-treated MFS mice (***p < 0.001), normalizing the irregularities to the levels observed in control mice (n = 4; Mean ± SEM).

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References

    1. Dietz HC, et al. Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene. Nature. 1991;352:337–339. doi: 10.1038/352337a0. - DOI - PubMed
    1. Pyeritz RE. The Marfan syndrome. Annu Rev Med. 2000;51:481–510. doi: 10.1146/annurev.med.51.1.481. - DOI - PubMed
    1. Judge DP, Dietz HC. Marfan’s syndrome. Lancet. 2005;366:1965–1976. doi: 10.1016/S0140-6736(05)67789-6. - DOI - PMC - PubMed
    1. Kielty CM, Sherratt MJ, Marson A, Baldock C. Fibrillin microfibrils. Adv Protein Chem. 2005;70:405–436. doi: 10.1016/S0065-3233(05)70012-7. - DOI - PubMed
    1. Chung AW, et al. Loss of elastic fiber integrity and reduction of vascular smooth muscle contraction resulting from the upregulated activities of matrix metalloproteinase-2 and -9 in the thoracic aortic aneurysm in Marfan syndrome. Circ Res. 2007;101:512–522. doi: 10.1161/CIRCRESAHA.107.157776. - DOI - PubMed

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