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

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. 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. 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). 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.

Balint B ，Bernstorff IGL ，Schwab T ，Schäfers HJ ... -  《PLoS One》

Seno-destructive smooth muscle cells in the ascending aorta of patients with bicuspid aortic valve disease.

Ascending aortic aneurysms constitute an important hazard for individuals with a bicuspid aortic valve (BAV). However, the processes that degrade the aortic wall in BAV disease remain poorly understood. We undertook in situ analysis of ascending aortas from 68 patients, seeking potentially damaging cellular senescence cascades. Aortas were assessed for senescence-associated-ß-galactosidase activity, p16Ink4a and p21 expression, and double-strand DNA breaks. The senescence-associated secretory phenotype (SASP) of cultured-aged BAV aortic smooth muscle cells (SMCs) was evaluated by transcript profiling and consequences probed by combined immunofluorescence and circular polarization microscopy. The contribution of p38 MAPK signaling was assessed by immunostaining and blocking strategies. We uncovered SMCs at varying depths of cellular senescence within BAV- and tricuspid aortic valve (TAV)-associated aortic aneurysms. Senescent SMCs were also abundant in non-aneurysmal BAV aortas but not in non-aneurysmal TAV aortas. Multivariable analysis revealed that BAV disease independently associated with SMC senescence. Furthermore, SMC senescence was heightened at the convexity of aortas associated with right-left coronary cusp fusion. Aged BAV SMCs had a pronounced collagenolytic SASP. Moreover, senescent SMCs in the aortic wall were enriched with surface-localized MMP1 and surrounded by weakly birefringent collagen fibrils. The senescent-collagenolytic SMC phenotype depended on p38 MAPK signaling, which was chronically activated in BAV aortas. We have identified a cellular senescence-collagen destruction axis in at-risk ascending aortas. This novel "seno-destructive" SMC phenotype could open new opportunities for managing BAV aortopathy. FUND: Canadian Institutes of Health Research, Lawson Health Research Institute, Heart and Stroke Foundation of Ontario/Barnett-Ivey Chair.

Balint B ，Yin H ，Nong Z ，Arpino JM ，O'Neil C ，Rogers SR ，Randhawa VK ，Fox SA ，Chevalier J ，Lee JJ ，Chu MWA ，Pickering JG ... -  《EBioMedicine》

Aortic regurgitation provokes phenotypic modulation of smooth muscle cells in the normal ascending aorta.

Aortic complications are more likely to occur in patients with ascending aortic aneurysms and concomitant aortic regurgitation (AR). AR may have a negative influence on the aortic wall structure even in patients with tricuspid aortic valves and absence of aortic dilatation. It is unknown whether smooth muscle cell (SMC) changes are a feature of AR-associated aortic remodeling. Nondilated aortic samples were harvested intraoperatively from individuals with normal aortic valves (n = 10) or those with either predominant aortic stenosis (AS) (n = 20) or AR (n = 35). Tissue from each patient was processed for immunohistochemistry or used for the extraction of medial SMCs. Tissue and cells were stained for markers of SMC contraction (alpha-smooth muscle actin), synthesis (vimentin) and senescence (p16INK4A and p21Cip1 [p16/p21]). Replicative capacity was analyzed in cultured SMCs from AS- and AR-associated aortas. A subanalysis compared SMCs from individuals with either tricuspid aortic valves or bicuspid aortic valves to evaluate the effect of aortic valve morphology. In aortic tissue samples, AR was associated with decreased alpha-smooth muscle actin and increased vimentin, p16 and p21 compared with normal aortic valves and AS. In cell culture, SMCs from AR-aortas had decreased alpha-smooth muscle actin and increased vimentin compared with SMCs from AS-aortas. AR-associated SMCs had increased p16 and p21 expression, and they reached senescence earlier than SMCs from AS-aortas. In AR, SMC changes were more pronounced with the presence of a bicuspid aortic valve. AR itself negatively influences SMC phenotype in the ascending aortic wall. This AR-specific effect is independent of aortic diameter and aortic valve morphology, although it is more pronounced with bicuspid aortic valves. These findings provide insight into the mechanisms of AR-related aortic remodeling, and they provide a model for studying SMC-specific therapies in culture.

Balint B ，Bernstorff IGL ，Schwab T ，Schäfers HJ ... -  《-》

Targeted gene expression analyses and immunohistology suggest a pro-proliferative state in tricuspid aortic valve-, and senescence and viral infections in bicuspid aortic valve-associated thoracic aortic aneurysms.

Despite the potential life-threatening consequences of thoracic aortic aneurysms (TAAs), the pathogenesis of these diseases is still poorly understood. While some aspects of TAA formation have been elucidated, the role of vascular smooth muscle cells (SMCs) in both bicuspid aortic valve (BAV)-associated and degenerative tricuspid aortic valve (TAV)-associated TAAs has not yet been fully unravelled. Thus, this work was aimed at uncovering processes in SMC biology that may contribute to TAA formation. Using isolated SMCs and tissue samples from TAAs linked to BAV syndrome, TAV-associated degenerative TAAs and control aortas, we performed targeted mRNA expression profile analyses and conducted immunohistological analyses on aortic wall tissue sections. While SMC expression profiles and tissue analyses in TAV-TAAs clearly point toward a pro-proliferative state of the aortic media SMCs, BAV-TAA SMCs and tissue provide evidence for DNA damage, DNA damage response signalling as well as profound TLR-3 signalling. The data presented in this study emphasizes the importance of SMCs in TAA development. Furthermore, our results provide evidence that the state of SMCs in the BAV-TAA (senescent) and TAV-TAA (pro-proliferative) differs significantly. For the first time, we also present findings that may argue for the occurrence of a viral infection in BAV-TAA SMCs.

Blunder S ，Messner B ，Scharinger B ，Doppler C ，Zeller I ，Zierer A ，Laufer G ，Bernhard D ... -  《-》

Age-dependent phenotypic modulation of smooth muscle cells in the normal ascending aorta.

Ascending aortic aneurysms are associated with pre-existing conditions, including connective tissue disorders (i.e., Marfan syndrome) and bicuspid aortic valves. The underlying mechanisms remain uncertain. Even less is known regarding ascending aortic aneurysms in individuals with normal (i.e., tricuspid) aortic valves (TAV), and without known aneurysm-associated disorders. Regardless of etiology, the risk of aortic complications increases with biological age. Phenotypic modulation of smooth muscle cells (SMCs) is a feature of ascending aortic aneurysms, whereby contractile SMCs are replaced with synthetic SMCs that are capable of degrading the aortic wall. We asked whether age itself causes dysfunctional SMC phenotype modulation, independent of aortic dilatation or pre-existing aneurysm-associated diseases. Non-dilated ascending aortic samples were obtained intra-operatively from 40 patients undergoing aortic valve surgery (range: 20-82 years old, mean: 59.1 ± 15.2). Patients with known genetic diseases or aortic valve malformations were excluded. Tissue was divided, and a portion was formalin-fixed and immunolabeled for alpha-smooth muscle actin (ASMA), a contractile SMC protein, and markers of synthetic (vimentin) or senescent (p16/p21) SMCs. Another fragment was used for SMC isolation (n = 10). Cultured SMCs were fixed at cell passage 2 and stained for phenotype markers, or were cultured indefinitely to determine replicative capacity. In whole tissue, ASMA decreased (R2 = 0.47, P < 0.0001), while vimentin increased (R2 = 0.33, P = 0.02) with age. In cultured SMCs, ASMA decreased (R2 = 0.35, P = 0.03) and vimentin increased (R2 = 0.25, P = 0.04) with age. p16 (R2 = 0.34, P = 0.02) and p21 (R2 = 0.29, P = 0.007) also increased with age in SMCs. Furthermore, the replicative capacity of SMCs from older patients was decreased compared to that of younger patients (P = 0.03). By investigating non-dilated aortic samples from individuals with normal TAVs, we found that age itself has a negative impact on SMCs in the ascending aortic wall, whereby SMCs switched from the contractile phenotype to maladaptive synthetic or senescent states with increased age. Therefore, based on our findings, modification of SMC phenotype should be studied as a therapeutic consideration against aneurysms in the future, regardless of etiology.

Balint B ，Bernstorff IGL ，Schwab T ，Schäfers HJ ... -  《-》