Cell and Molecular Biology Department, Tulane University, New Orleans, LA (A.M.C., X.Z., A.R.L., S.M.M.).
Department of Pediatrics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans (J.G., J.Z.).
Circulation. 2019 Apr 23;139(17):2049-2063. doi: 10.1161/CIRCULATIONAHA.118.036952.
Hereditary hemorrhagic telangiectasia is an autosomal dominant vascular disorder caused by heterozygous, loss-of-function mutations in 4 transforming growth factor beta (TGFβ) pathway members, including the central transcriptional mediator of the TGFβ pathway, Smad4. Loss of Smad4 causes the formation of inappropriate, fragile connections between arteries and veins called arteriovenous malformations (AVMs), which can hemorrhage leading to stroke, aneurysm, or death. Unfortunately, the molecular mechanisms underlying AVM pathogenesis remain poorly understood, and the TGFβ downstream effectors responsible for hereditary hemorrhagic telangiectasia-associated AVM formation are currently unknown.
To identify potential biological targets of the TGFβ pathway involved in AVM formation, we performed RNA- and chromatin immunoprecipitation-sequencing experiments on BMP9 (bone morphogenetic protein 9)-stimulated endothelial cells (ECs) and isolated ECs from a Smad4-inducible, EC-specific knockout ( Smad4-iECKO) mouse model that develops retinal AVMs. These sequencing studies identified the angiopoietin-Tek signaling pathway as a downstream target of SMAD4. We used monoclonal blocking antibodies to target a specific component in this pathway and assess its effects on AVM development.
Sequencing studies uncovered 212 potential biological targets involved in AVM formation, including the EC surface receptor, TEK (TEK receptor tyrosine kinase) and its antagonistic ligand, ANGPT2 (angiopoietin-2). In Smad4-iECKO mice, Angpt2 expression is robustly increased, whereas Tek levels are decreased, resulting in an overall reduction in angiopoietin-Tek signaling. We provide evidence that SMAD4 directly represses Angpt2 transcription in ECs. Inhibition of ANGPT2 function in Smad4-deficient mice, either before or after AVMs form, prevents and alleviates AVM formation and normalizes vessel diameters. These rescue effects are attributed to a reversion in EC morphological changes, such as cell size and shape that are altered in the absence of Smad4.
Our studies provide a novel mechanism whereby the loss of Smad4 causes increased Angpt2 transcription in ECs leading to AVM formation, increased blood vessel calibers, and changes in EC morphology in the retina. Blockade of ANGPT2 function in an in vivo Smad4 model of hereditary hemorrhagic telangiectasia alleviated these vascular phenotypes, further implicating ANGPT2 as an important TGFβ downstream mediator of AVM formation. Therefore, alternative approaches that target ANGPT2 function may have therapeutic value for the alleviation of hereditary hemorrhagic telangiectasia symptoms, such as AVMs.
遗传性出血性毛细血管扩张症是一种常染色体显性血管疾病，由 4 种转化生长因子β（TGFβ）途径成员中的杂合性、功能丧失突变引起，包括 TGFβ途径的中央转录中介物 Smad4。Smad4 的缺失导致动脉和静脉之间形成不合适的、脆弱的连接，称为动静脉畸形（AVM），可导致出血、中风、动脉瘤或死亡。不幸的是，AVM 发病机制的分子机制仍知之甚少，目前尚不清楚负责遗传性出血性毛细血管扩张症相关 AVM 形成的 TGFβ 下游效应物。
为了确定 TGFβ 途径中参与 AVM 形成的潜在生物学靶标，我们对 BMP9（骨形态发生蛋白 9）刺激的内皮细胞（EC）和 Smad4 诱导的、内皮细胞特异性敲除（Smad4-iECKO）小鼠模型中分离的 EC 进行了 RNA 和染色质免疫沉淀测序实验，该模型会发展出视网膜 AVM。这些测序研究确定了血管生成素-Tek 信号通路是 SMAD4 的下游靶标。我们使用单克隆阻断抗体靶向该通路中的特定成分，并评估其对 AVM 发育的影响。
测序研究揭示了 212 个潜在的与 AVM 形成相关的生物学靶标，包括 EC 表面受体 TEK（TEK 受体酪氨酸激酶）及其拮抗配体 ANGPT2（血管生成素-2）。在 Smad4-iECKO 小鼠中，Angpt2 的表达强烈增加，而 Tek 水平降低，导致血管生成素-Tek 信号通路总体减少。我们提供的证据表明，SMAD4 可直接抑制 EC 中的 Angpt2 转录。在 AVM 形成之前或之后，抑制 Smad4 缺陷小鼠中的 ANGPT2 功能可预防和缓解 AVM 形成并使血管直径正常化。这些挽救作用归因于 EC 形态变化的逆转，例如在缺乏 Smad4 的情况下改变的细胞大小和形状。
我们的研究提供了一种新的机制，即 Smad4 的缺失导致 EC 中 Angpt2 转录增加，从而导致 AVM 形成、血管口径增加和视网膜中 EC 形态的变化。在遗传性出血性毛细血管扩张症的 Smad4 体内模型中阻断 ANGPT2 功能可缓解这些血管表型，进一步表明 ANGPT2 是 AVM 形成的重要 TGFβ 下游介质。因此，针对 ANGPT2 功能的替代方法可能具有缓解遗传性出血性毛细血管扩张症症状（如 AVM）的治疗价值。
