Division of Molecular Cardiovascular Biology (A.O., B.A.G., R.W., C.M.A., C.O.S., D.P.M., K.E.Y.), Cincinnati Children's Hospital Medical Center, OH.
Molecular and Developmental Biology Graduate Program (A.O., S.M.), University of Cincinnati, OH.
Arterioscler Thromb Vasc Biol. 2023 Aug;43(8):1478-1493. doi: 10.1161/ATVBAHA.123.319424. Epub 2023 Jun 29.
Specialized valve endothelial cell (VEC) populations are localized oriented to blood flow in developing aortic and mitral valves, but their roles in valve development and disease are unknown. In the aortic valve (AoV), a population of VECs on the fibrosa side expresses the transcription factor Prox1 together with genes found in lymphatic ECs. In this study, we examine Prox1's role in regulating a lymphatic-like gene network and promoting VEC diversity required for the development of the stratified trilaminar extracellular matrix (ECM) of murine AoV leaflets.
To determine whether disruption of Prox1 localization affects heart valve development, we generated mice ( gain-of-function) in which Prox1 is overexpressed on the ventricularis side of the AoV beginning in embryonic development. To identify potential targets of Prox1, we performed cleavage under targets and release using nuclease on wild-type and gain-of-function AoVs with validation by colocalization in vivo using RNA in situ hybridization in gain-of-function AoVs. Natural induction of Prox1 and target gene expression was evaluated in myxomatous AoVs in a mouse model of Marfan syndrome ().
The overexpression of Prox1 is sufficient to cause enlargement of AoVs by postnatal day (P)0, as well as a decrease in ventricularis-specific gene expression and disorganized interstitial ECM layers at P7. We identified potential targets of Prox1 known to play roles in lymphatic ECs including , , , and . Ectopic Prox1 colocalized with induced , , and expression in gain-of-function AoVs. Moreover, in Marfan syndrome myxomatous AoVs, endogenous Prox1, and its identified targets, were ectopically induced in ventricularis side VECs.
Our results support a role for Prox1 in localized lymphatic-like gene expression on the fibrosa side of the AoV. Furthermore, localized VEC specialization is required for development of the stratified trilaminar ECM critical for AoV function and is dysregulated in congenitally malformed valves.
在发育中的主动脉瓣和二尖瓣中，特化的瓣内皮细胞（VEC）群体定位于血流方向，但它们在瓣膜发育和疾病中的作用尚不清楚。在主动脉瓣（AoV）中，纤维侧的 VEC 群体表达转录因子 Prox1 以及在淋巴管内皮细胞（EC）中发现的基因。在这项研究中，我们研究了 Prox1 在调节淋巴管样基因网络中的作用以及促进 VEC 多样性的作用，这对于形成具有分层三层细胞外基质（ECM）的小鼠 AoV 瓣叶是必需的。
为了确定 Prox1 定位的破坏是否会影响心脏瓣膜的发育，我们生成了在胚胎发育开始时在 AoV 的心室面过表达 Prox1 的小鼠（功能获得）。为了确定 Prox1 的潜在靶标，我们使用切口酶靶向和释放技术对野生型和功能获得型 AoV 进行了处理，并通过在功能获得型 AoV 中使用 RNA 原位杂交在体内进行共定位进行了验证。在马凡综合征（）的小鼠模型中评估了粘液样 AoV 中 Prox1 和靶基因表达的自然诱导。
Prox1 的过表达足以导致出生后第 0 天（P）0 时 AoV 的增大，以及心室特异性基因表达减少和 P7 时间质 ECM 层紊乱。我们鉴定了 Prox1 的潜在靶标，这些靶标已知在淋巴管 ECs 中发挥作用，包括、、、和。异位 Prox1 与诱导的、和在功能获得型 AoV 中的表达共定位。此外，在马凡综合征的粘液样 AoV 中，内源性 Prox1 及其鉴定的靶标在心室面 VEC 中异位诱导。
我们的结果支持 Prox1 在 AoV 纤维侧局部淋巴管样基因表达中的作用。此外，局部 VEC 特化对于形成对 AoV 功能至关重要的分层三层 ECM 是必需的，并且在先天性畸形瓣膜中失调。
