Heterozygous activin receptor-like kinase 1 (ALK1) mutations are associated with two vascular diseases: hereditary hemorrhagic telangiectasia (HHT) and more rarely pulmonary arterial hypertension (PAH). Here, we aimed to understand the impact of ALK1 mutations on BMP9 and BMP10 transcriptomic responses in endothelial cells. Endothelial colony-forming cells (ECFCs) and microvascular endothelial cells (HMVECs) carrying loss of function ALK1 mutations were isolated from newborn HHT and adult PAH donors, respectively. RNA-sequencing was performed on each type of cells compared to controls following an 18 h stimulation with BMP9 or BMP10. In control ECFCs, BMP9 and BMP10 stimulations induced similar transcriptomic responses with around 800 differentially expressed genes (DEGs). ALK1-mutated ECFCs unexpectedly revealed highly similar transcriptomic profiles to controls, both at the baseline and upon stimulation, and normal activation of Smad1/5 that could not be explained by a compensation in cell-surface ALK1 level. Conversely, PAH HMVECs revealed strong transcriptional dysregulations compared to controls with > 1200 DEGs at the baseline. Consequently, because our study involved two variables, ALK1 genotype and BMP stimulation, we performed two-factor differential expression analysis and identified 44 BMP9-dysregulated genes in mutated HMVECs, but none in ECFCs. Yet, the impaired regulation of at least one hit, namely lunatic fringe (LFNG), was validated by RT-qPCR in three different ALK1-mutated endothelial models. In conclusion, ALK1 heterozygosity only modified the BMP9/BMP10 regulation of few genes, including LFNG involved in NOTCH signaling. Future studies will uncover whether dysregulations in such hits are enough to promote HHT/PAH pathogenesis, making them potential therapeutic targets, or if second hits are necessary.

杂合激活素受体样激酶 1 ( ALK1 ) 突变与两种血管疾病相关：遗传性出血性毛细血管扩张症 (HHT) 和罕见的肺动脉高压 (PAH)。在这里，我们旨在了解ALK1突变对内皮细胞中 BMP9 和 BMP10 转录组反应的影响。分别从新生儿 HHT 和成人 PAH 供体中分离出携带功能缺失ALK1突变的内皮集落形成细胞 (ECFC) 和微血管内皮细胞 (HMVEC)。在用 BMP9 或 BMP10 刺激 18 小时后，对每种类型的细胞进行 RNA 测序，并与对照细胞进行比较。在对照 ECFC 中，BMP9 和 BMP10 刺激诱导了大约 800 个差异表达基因 (DEG) 的类似转录组反应。 ALK1突变的 ECFC 出乎意料地显示出与对照高度相似的转录组谱，无论是在基线还是在刺激后，以及 Smad1/5 的正常激活，这无法通过细胞表面 ALK1 水平的补偿来解释。相反，与基线> 1200 DEG 的对照相比，PAH HMVEC 显示出强烈的转录失调。因此，由于我们的研究涉及两个变量，即 ALK1 基因型和 BMP 刺激，因此我们进行了双因素差异表达分析，并在突变的 HMVEC 中鉴定出 44 个 BMP9 失调基因，但在 ECFC 中没有发现。然而，RT-qPCR 在三种不同的ALK1突变内皮模型中验证了至少一种打击（即疯子边缘 ( LFNG )）的受损调节。总之， ALK1杂合性仅改变了少数基因的 BMP9/BMP10 调控，包括参与 NOTCH 信号传导的LFNG 。 未来的研究将揭示此类打击中的失调是否足以促进 HHT/PAH 发病机制，使其成为潜在的治疗目标，或者是否有必要进行第二次打击。