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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.
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.
摘要：
背景：二叶主动脉瓣（BAV）通常与升主动脉瘤相关。病因尚未完全了解，但是遗传因素，除了流动扰动，很可能参与其中。由于血管壁中收缩性的丧失和细胞外基质的形成是BAV相关主动脉病的特征，平滑肌细胞（SMC）的表型调节可能起作用。
方法：术中收集25名正常人的升主动脉组织（即三尖瓣）主动脉瓣（TAV）和25例BAV患者。对于TAV和BAV，10例患者未扩张（ND），15例患者扩张（D）主动脉。从每组的患者亚组中分离并培养SMC。对主动脉组织和SMC进行SMC表型标记的荧光免疫标记(即，α-平滑肌肌动蛋白(ASMA，收缩)，波形蛋白（合成）和p16INK4a和p21Cip1（衰老）。还分析了SMC在培养物中的复制衰老。
结果：在正常大小和扩张的BAV主动脉中，SMC从收缩状态转变为合成或衰老表型，如通过ASMA的损失(ND:P=0.001,D:P=0.002)和波形蛋白(ND:P=0.03,D:P=0.004)或p16/p21(ND:P=0.03,D:P<0.0001)与TAV相比所观察到的。主动脉扩张加剧了BAV和TAV主动脉的SMC表型转换（均P<0.05）。在正常和扩张主动脉培养的SMC中，从BAV中分离的那些比从TAV主动脉中分离的那些更快地达到复制衰老（所有P=0.02）。此外，BAVSMC中ASMA与细胞传代数之间存在明显的负相关（ND：P=0.0006，D：P=0.01），但在TAVSMC中没有（ND：P=0.93，D：P=0.20）。
结论：这项研究的结果提供了细胞培养研究的直接证据，暗示SMC在非扩张的BAV主动脉中从收缩状态转变为合成或衰老表型。在来自非扩张和扩张主动脉的培养SMC中，我们发现,在BAV中,这一过程可能先于扩张,并伴随动脉瘤的发展.我们的发现表明，在BAV患者中治疗靶向SMC表型调节可能是预防或延迟升主动脉瘤形成的可行选择。
方法：术中收集25名正常人的升主动脉组织（即三尖瓣）主动脉瓣（TAV）和25例BAV患者。对于TAV和BAV，10例患者未扩张（ND），15例患者扩张（D）主动脉。从每组的患者亚组中分离并培养SMC。对主动脉组织和SMC进行SMC表型标记的荧光免疫标记(即，α-平滑肌肌动蛋白(ASMA，收缩)，波形蛋白（合成）和p16INK4a和p21Cip1（衰老）。还分析了SMC在培养物中的复制衰老。
结果：在正常大小和扩张的BAV主动脉中，SMC从收缩状态转变为合成或衰老表型，如通过ASMA的损失(ND:P=0.001,D:P=0.002)和波形蛋白(ND:P=0.03,D:P=0.004)或p16/p21(ND:P=0.03,D:P<0.0001)与TAV相比所观察到的。主动脉扩张加剧了BAV和TAV主动脉的SMC表型转换（均P<0.05）。在正常和扩张主动脉培养的SMC中，从BAV中分离的那些比从TAV主动脉中分离的那些更快地达到复制衰老（所有P=0.02）。此外，BAVSMC中ASMA与细胞传代数之间存在明显的负相关（ND：P=0.0006，D：P=0.01），但在TAVSMC中没有（ND：P=0.93，D：P=0.20）。
结论：这项研究的结果提供了细胞培养研究的直接证据，暗示SMC在非扩张的BAV主动脉中从收缩状态转变为合成或衰老表型。在来自非扩张和扩张主动脉的培养SMC中，我们发现,在BAV中,这一过程可能先于扩张,并伴随动脉瘤的发展.我们的发现表明，在BAV患者中治疗靶向SMC表型调节可能是预防或延迟升主动脉瘤形成的可行选择。
