Abstract：
BACKGROUND: Loeys-Dietz syndrome (LDS) is an inherited disorder predisposing individuals to thoracic aortic aneurysm and dissection. Currently, there are no medical treatments except surgical resection. Although the genetic basis of LDS is well-understood, molecular mechanisms underlying the disease remain elusive, impeding the development of a therapeutic strategy. In addition, aortic smooth muscle cells (SMCs) have heterogenous embryonic origins, depending on their spatial location, and lineage-specific effects of pathogenic variants on SMC function, likely causing regionally constrained LDS manifestations, have been unexplored.
METHODS: We identified an LDS family with a dominant pathogenic variant in the TGFBR1 gene (TGFBR1A230T) causing aortic root aneurysm and dissection. To accurately model the molecular defects caused by this mutation, we used human induced pluripotent stem cells from a subject with normal aorta to generate human induced pluripotent stem cells carrying TGFBR1A230T, and corrected the mutation in patient-derived human induced pluripotent stem cells using CRISPR-Cas9 gene editing. After their lineage-specific SMC differentiation through cardiovascular progenitor cell (CPC) and neural crest stem cell lineages, we used conventional molecular techniques and single-cell RNA sequencing to characterize the molecular defects. The resulting data led to subsequent molecular and functional rescue experiments using activin A and rapamycin.
RESULTS: Our results indicate the TGFBR1A230T mutation impairs contractile transcript and protein levels, and function in CPC-SMC, but not in neural crest stem cell-SMC. Single-cell RNA sequencing results implicate defective differentiation even in TGFBR1A230T/+ CPC-SMC including disruption of SMC contraction and extracellular matrix formation. Comparison of patient-derived and mutation-corrected cells supported the contractile phenotype observed in the mutant CPC-SMC. TGFBR1A230T selectively disrupted SMAD3 (SMAD family member 3) and AKT (AKT serine/threonine kinase) activation in CPC-SMC, and led to increased cell proliferation. Consistently, single-cell RNA sequencing revealed molecular similarities between a loss-of-function SMAD3 mutation (SMAD3c.652delA/+) and TGFBR1A230T/+. Last, combination treatment with activin A and rapamycin during or after SMC differentiation significantly improved the mutant CPC-SMC contractile gene expression and function, and rescued the mechanical properties of mutant CPC-SMC tissue constructs.
CONCLUSIONS: This study reveals that a pathogenic TGFBR1 variant causes lineage-specific SMC defects informing the etiology of LDS-associated aortic root aneurysm. As a potential pharmacological strategy, our results highlight a combination treatment with activin A and rapamycin that can rescue the SMC defects caused by the variant.
METHODS: We identified an LDS family with a dominant pathogenic variant in the TGFBR1 gene (TGFBR1A230T) causing aortic root aneurysm and dissection. To accurately model the molecular defects caused by this mutation, we used human induced pluripotent stem cells from a subject with normal aorta to generate human induced pluripotent stem cells carrying TGFBR1A230T, and corrected the mutation in patient-derived human induced pluripotent stem cells using CRISPR-Cas9 gene editing. After their lineage-specific SMC differentiation through cardiovascular progenitor cell (CPC) and neural crest stem cell lineages, we used conventional molecular techniques and single-cell RNA sequencing to characterize the molecular defects. The resulting data led to subsequent molecular and functional rescue experiments using activin A and rapamycin.
RESULTS: Our results indicate the TGFBR1A230T mutation impairs contractile transcript and protein levels, and function in CPC-SMC, but not in neural crest stem cell-SMC. Single-cell RNA sequencing results implicate defective differentiation even in TGFBR1A230T/+ CPC-SMC including disruption of SMC contraction and extracellular matrix formation. Comparison of patient-derived and mutation-corrected cells supported the contractile phenotype observed in the mutant CPC-SMC. TGFBR1A230T selectively disrupted SMAD3 (SMAD family member 3) and AKT (AKT serine/threonine kinase) activation in CPC-SMC, and led to increased cell proliferation. Consistently, single-cell RNA sequencing revealed molecular similarities between a loss-of-function SMAD3 mutation (SMAD3c.652delA/+) and TGFBR1A230T/+. Last, combination treatment with activin A and rapamycin during or after SMC differentiation significantly improved the mutant CPC-SMC contractile gene expression and function, and rescued the mechanical properties of mutant CPC-SMC tissue constructs.
CONCLUSIONS: This study reveals that a pathogenic TGFBR1 variant causes lineage-specific SMC defects informing the etiology of LDS-associated aortic root aneurysm. As a potential pharmacological strategy, our results highlight a combination treatment with activin A and rapamycin that can rescue the SMC defects caused by the variant.
摘要：
背景：Loeys-Dietz综合征（LDS）是一种遗传性疾病，易患胸主动脉瘤和夹层。目前,除了手术切除，没有药物治疗。虽然LDS的遗传基础是很好理解的，这种疾病的分子机制仍然难以捉摸，阻碍治疗策略的发展。此外,主动脉平滑肌细胞（SMC）具有异质性胚胎起源，根据它们的空间位置，以及致病变体对SMC功能的谱系特异性影响，可能导致区域受限的LDS表现，没有被探索过。
方法：我们确定了一个LDS家族，该家族在TGFBR1基因（TGFBR1A230T）中具有显性致病变异，导致主动脉根部动脉瘤和夹层。为了准确模拟这种突变引起的分子缺陷，我们使用来自正常主动脉的人诱导多能干细胞来产生携带TGFBR1A230T的人诱导多能干细胞，并使用CRISPR-Cas9基因编辑纠正了患者来源的人诱导多能干细胞中的突变。在通过心血管祖细胞（CPC）和神经c干细胞谱系进行谱系特异性SMC分化后，我们使用常规的分子技术和单细胞RNA测序来表征分子缺陷。所得数据导致随后使用活化素A和雷帕霉素的分子和功能拯救实验。
结果：我们的结果表明TGFBR1A230T突变损害收缩转录和蛋白质水平，并在CPC-SMC中发挥作用，但不是在神经c干细胞-SMC中。单细胞RNA测序结果暗示即使在TGFBR1A230T/+CPC-SMC中也存在分化缺陷,包括SMC收缩和细胞外基质形成的破坏。患者来源的和突变校正的细胞的比较支持在突变体CPC-SMC中观察到的收缩表型。TGFBR1A230T选择性破坏CPC-SMC中的SMAD3（SMAD家族成员3）和AKT（AKT丝氨酸/苏氨酸激酶）激活，并导致细胞增殖增加。始终如一，单细胞RNA测序揭示了功能缺失的SMAD3突变(SMAD3c.652delA/+)和TGFBR1A230T/+之间的分子相似性.最后,在SMC分化过程中或之后，用激活素A和雷帕霉素联合治疗显着改善了突变体CPC-SMC收缩基因的表达和功能，并挽救了突变CPC-SMC组织构建体的机械性能。
结论：本研究揭示了一种致病性TGFBR1变异体可导致谱系特异性SMC缺陷，提示LDS相关主动脉根瘤的病因。作为一种潜在的药理学策略，我们的结果强调了活化素A和雷帕霉素的联合治疗可以挽救由变体引起的SMC缺陷.
方法：我们确定了一个LDS家族，该家族在TGFBR1基因（TGFBR1A230T）中具有显性致病变异，导致主动脉根部动脉瘤和夹层。为了准确模拟这种突变引起的分子缺陷，我们使用来自正常主动脉的人诱导多能干细胞来产生携带TGFBR1A230T的人诱导多能干细胞，并使用CRISPR-Cas9基因编辑纠正了患者来源的人诱导多能干细胞中的突变。在通过心血管祖细胞（CPC）和神经c干细胞谱系进行谱系特异性SMC分化后，我们使用常规的分子技术和单细胞RNA测序来表征分子缺陷。所得数据导致随后使用活化素A和雷帕霉素的分子和功能拯救实验。
结果：我们的结果表明TGFBR1A230T突变损害收缩转录和蛋白质水平，并在CPC-SMC中发挥作用，但不是在神经c干细胞-SMC中。单细胞RNA测序结果暗示即使在TGFBR1A230T/+CPC-SMC中也存在分化缺陷,包括SMC收缩和细胞外基质形成的破坏。患者来源的和突变校正的细胞的比较支持在突变体CPC-SMC中观察到的收缩表型。TGFBR1A230T选择性破坏CPC-SMC中的SMAD3（SMAD家族成员3）和AKT（AKT丝氨酸/苏氨酸激酶）激活，并导致细胞增殖增加。始终如一，单细胞RNA测序揭示了功能缺失的SMAD3突变(SMAD3c.652delA/+)和TGFBR1A230T/+之间的分子相似性.最后,在SMC分化过程中或之后，用激活素A和雷帕霉素联合治疗显着改善了突变体CPC-SMC收缩基因的表达和功能，并挽救了突变CPC-SMC组织构建体的机械性能。
结论：本研究揭示了一种致病性TGFBR1变异体可导致谱系特异性SMC缺陷，提示LDS相关主动脉根瘤的病因。作为一种潜在的药理学策略，我们的结果强调了活化素A和雷帕霉素的联合治疗可以挽救由变体引起的SMC缺陷.
