Thesis presented May 31, 2023

Abstract:
Heterozygous loss-of-function mutations in ACVRL1 (encoding activin receptor-like kinase 1; ALK1) are associated with the development of two vascular diseases: hereditary hemorrhagic telangiectasia (HHT) and, more rarely, pulmonary arterial hypertension (PAH). ALK1 is a type 1 TGF-ß family receptor that is predominantly expressed on endothelial cells (ECs) and binds with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10), inducing the phosphorylation of Smad1/5 transcription factors that subsequently regulate target gene expression. ALK1 signaling generally promotes vascular quiescence, while its haploinsufficiency is believed to underly pathogenesis in a number of HHT and PAH cases. The aim of this work is to characterize the transcriptomic response of primary ALK1-mutated ECs to BMP9 and BMP10, in order to better understand the pathogenic molecular mechanisms underlying these two diseases and identify potential therapeutic targets.
Endothelial colony-forming cells (ECFCs) and microvascular endothelial cells (MVECs) carrying heterozygous loss-of-function ALK1 mutations were isolated from cord blood of newborn HHT donors and explanted lungs of PAH patients, respectively. RNA-sequencing was performed on each type of cells compared to control counterparts following an overnight stimulation with BMP9 or BMP10. In both EC models, BMP9 and BMP10 stimulations induced very similar transcriptomic responses with nearly no differentially expressed genes (DEGs) between BMP9 and BMP10 stimulation. Comparison of the transcriptome between control and ALK1-mutated ECFCs revealed very similar profiles, both at the baseline and upon stimulation with BMP9 or BMP10. Consistently, control and mutated ECFCs displayed a similar activation level of Smad1/5 by BMP9, which could not be explained by a compensation in cell-surface ALK1 levels. On the other hand, mutated PAH MVECs, derived from a sick environment, revealed strong transcriptional dysregulations compared to controls, with >1200 DEGs already at the baseline. Two-factor differential expression analysis, which takes into account the two involved variables (ALK1 genotype and BMP stimulation), identified 44 protein-coding genes with impaired regulation by BMP9 in mutated HMVECs, but none in mutated ECFCs. Most of these 44 genes could be validated by RT-qPCR on mutated HMVECs, and surprisingly very few were also validated in mutated ECFCs and/or human umbilical vein ECs (HUVECs; isolated from HHT newborns), but to a milder extent compared to mutated HMVECs. These included LFNG, encoding lunatic fringe, which is a modulator of Notch signaling that in turn is a critical pathway for angiogenesis and arterial specification.
This work revealed that ALK1 heterozygosity does not impair Smad1/5 activation nor strongly affect the transcriptomic response to BMP9 or BMP10, which were shown to exert equivalent transcriptomic roles in vitro. Nevertheless, more in depth bioinformatic analysis highlighted few genes exhibiting impaired regulation by BMP9/BMP10 even in newborn cells. The paucity of transcriptomic dysregulations in mutated ECFCs indicates that the overt changes observed in mutated HMVECs cannot be solely attributed to ALK1 heterozygosity, but might additionally involve second hits (somatic mutation/ inflammation/ angiogenesis) potentially present in the sick lungs of endstage PAH patients. In conclusion, these findings challenge the haploinsufficiency model for HHT and PAH, and favor the notion that a second hit might be necessary for driving vascular pathogenesis, while highlighting few potential therapeutic targets that could be highly sensitive to reduction in functional ALK1 levels and might act as priming events for early disease onset.


Keywords:
HHT, PAH, RNA-sequencing, Transcriptome, BMP9/BMP10, Endothelial Cells

On-line thesis. ​