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. 2024 Jul 2;19(7):e0306515.
doi: 10.1371/journal.pone.0306515. eCollection 2024.

Smooth muscle cell phenotypic switching occurs independent of aortic dilation in bicuspid aortic valve-associated ascending aortas

Brittany Balint  1 , Inés García Lascurain Bernstorff  1 , Tanja Schwab  1 , Hans-Joachim Schäfers  1
Affiliations

Smooth muscle cell phenotypic switching occurs independent of aortic dilation in bicuspid aortic valve-associated ascending aortas

Brittany Balint et al. PLoS One. .

Abstract

Background: Bicuspid aortic valves (BAV) are frequently associated with ascending aortic aneurysms. The etiology is incompletely understood, but genetic factors, in addition to flow perturbations, are likely involved. Since loss of contractility and elaboration of extracellular matrix in the vessel wall are features of BAV-associated aortopathy, phenotypic modulation of smooth muscle cells (SMCs) may play a role.

Methods: Ascending aortic tissue was collected intra-operatively from 25 individuals with normal (i.e., tricuspid) aortic valves (TAV) and from 25 individuals with BAVs. For both TAV and BAV, 10 patients had non-dilated (ND) and 15 patients had dilated (D) aortas. SMCs were isolated and cultured from a subset of patients from each group. Aortic tissue and SMCs were fluorescently immunolabeled for SMC phenotypic markers (i.e., alpha-smooth muscle actin (ASMA, contractile), vimentin (synthetic) and p16INK4a and p21Cip1 (senescence). SMCs were also analyzed for replicative senescence in culture.

Results: In normal-sized and dilated BAV aortas, SMCs switched from the contractile state to either synthetic or senescent phenotypes, as observed by loss of ASMA (ND: P = 0.001, D: P = 0.002) and associated increases in vimentin (ND: P = 0.03, D: P = 0.004) or p16/p21 (ND: P = 0.03, D: P<0.0001) compared to TAV. Dilatation of the aorta exacerbated SMC phenotypic switching in both BAV and TAV aortas (all P<0.05). In SMCs cultured from normal and dilated aortas, those isolated from BAV reached replicative senescence faster than those from TAV aortas (all P = 0.02). Furthermore, there was a stark inverse correlation between ASMA and cell passage number in BAV SMCs (ND: P = 0.0006, D: P = 0.01), but not in TAV SMCs (ND: P = 0.93, D: P = 0.20).

Conclusions: The findings of this study provide direct evidence from cell culture studies implying that SMCs switch from the contractile state to either synthetic or senescent phenotypes in the non-dilated BAV aorta. In cultured SMCs from both non-dilated and dilated aortas, we found that this process may precede dilatation and accompany aneurysm development in BAV. Our findings suggest that therapeutically targeting SMC phenotypic modulation in BAV patients may be a viable option to prevent or delay ascending aortic aneurysm formation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Alpha-smooth muscle actin (ASMA) is decreased while vimentin is increased in non-dilated bicuspid aortic valve (BAV)-associated aortas.
A. Fluorescent micrographs of ASMA (top) and vimentin (bottom) in non-dilated aortic tissue from individuals with normal (i.e., tricuspid) aortic valves (TAV; left) and BAVs (right). Higher magnification images of the boxed region are shown to the right of each image. B. Graphs depicting ASMA (top) and vimentin (bottom) expression in the normal ascending aorta from TAV (N = 10) and BAV (N = 10) patients. Horizontal bars represent median values. C. Graph depicting the relationship between ASMA and vimentin expression in the non-dilated aorta of TAV (N = 10) and BAV (N = 10) patients. A.U. = arbitrary units; * = statistical significance; scale bar = 25 μm.
Fig 2
Fig 2. Alpha-smooth muscle actin (ASMA) is decreased and vimentin is increased in smooth muscle cells (SMCs) isolated from non-dilated bicuspid aortic valve (BAV)-associated aortas.
A. Fluorescent micrographs of ASMA (top) and vimentin (bottom) in SMCs isolated from non-dilated aortas associated with normal (i.e., tricuspid) aortic valves (TAV; left) and BAVs (right). Higher magnification images of the boxed region are shown to the right of each image. B. Graphs depicting ASMA (top) and vimentin (bottom) expression in SMCs from from each group (TAV: N = 8, BAV: N = 7). Horizontal bars represent median values. C. Graph depicting the relationship between ASMA and vimentin in SMCs for each group (TAV: N = 8, BAV: N = 7). A.U. = arbitrary units; * = statistical significance; Scale bar = 20 μm.
Fig 3
Fig 3. Dilatation of the ascending aorta impacts alpha-smooth muscle actin (ASMA) and vimentin expression in bicuspid aortic valve (BAV)-associated aortas and smooth muscle cells (SMCs).
Graphs depicting ASMA (A) and vimentin (B) expression in non-dilated (ND) and dilated (D) ascending aortic tissue from tricuspid aortic valve (TAV; left)- and BAV (right)- associated aortas. Graphs depicting ASMA (C) and vimentin (D) expression in SMCs isolated from ND and D aortas from TAV (left) and BAV (right) patients. Horizontal bars represent median values. * = statistical significance.
Fig 4
Fig 4. Alpha-smooth muscle actin (ASMA) is decreased and vimentin is increased in dilated bicuspid aortic valve (BAV)-associated aortas.
A,C. Graphs depicting ASMA (top) and vimentin (bottom) in dilated aortic tissue (A) and smooth muscle cells (SMCs, C) from individuals tricuspid aortic valves (TAV) and BAVs. B,D. Graph depicting relationships between ASMA and vimentin expression in the dilated aorta (B) or isolated SMCs (D) from TAV and BAV patients. Horizontal bars represent median values. A.U. = arbitrary units; * = statistical significance.
Fig 5
Fig 5
Cell cycle inhibitors p16 and p21 are increased in the dilated aorta and in association with bicuspid aortic valves (BAV). A-B. Graphs depicting p16/p21 positivity in the non-dilated aorta (A) and isolated smooth muscle cells (SMCs, B) in individuals with normal (i.e., tricuspid) aortic valves (TAV) or BAVs. C. Graphs depicting p16/p21 positivity in response to aortic dilatation (D) versus non-dilated (ND) in aortic tissue (left) and SMCs (right) from individuals with TAVs and BAVs. D-E. Graphs depicting p16/p21 positivity in the dilated aorta (D) and isolated SMCs (E) in individuals with TAVs or BAVs. Horizontal bars represent median values. * = statistical significance.
Fig 6
Fig 6. Smooth muscle cells (SMCs) from bicuspid aortic valve (BAV) aortas switch from contractile to senescent states over time in culture.
A. Graphs depicting the maximum cell passage reached by SMCs isolated from non-dilated (left) or dilated aortas, from individuals with tricuspid aortic valves (TAVs) or BAVs. B. Graphs depicting the relationship between cell passage and ASMA levels in SMCs isolated from normal-sized (left) and dilated (right) TAV and BAV aortas. Horizontal bars represent median values. * = statistical significance. A.U. = arbitrary units.
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