Single-cell RNA sequencing of cystic fibrosis liver disease explants reveals endothelial complement activation.

BACKGROUND & AIMS: Cystic fibrosis (CF) is considered a multisystemic disorder in which CF-associated liver disease (CFLD) is the third most common cause of mortality. Currently, no effective treatment is available for CFLD because its pathophysiology is still unclear. Interestingly, CFLD exhibits identical vascular characteristics as non-cirrhotic portal hypertension, recently classified as porto-sinusoidal vascular disorders (PSVD). METHODS: Since endothelial cells (ECs) are an important component in PSVD, we performed single-cell RNA sequencing (scRNA-seq) on four explant livers from CFLD patients to identify differential endothelial characteristics which could contribute to the disease. We comprehensively characterized the endothelial compartment and compared it with publicly available scRNA-seq datasets from cirrhotic and healthy livers. Key gene signatures were validated ex vivo on patient tissues. RESULTS: We found that ECs from CF liver explants are more closely related to healthy than cirrhotic patients. In CF patients we also discovered a distinct population of liver sinusoidal ECs-coined CF LSECs-upregulating genes involved in the complement cascade and coagulation. Finally, our immunostainings further validated the predominant periportal location of CF LSECs. CONCLUSIONS: Our work showed novel aspects of human liver ECs at the single-cell level thereby supporting endothelial involvement in CFLD, and reinforcing the hypothesis that ECs could be a driver of PSVD. Therefore, considering the vascular compartment in CF and CFLD may help developing new therapeutic approaches for these diseases.

Transcriptomic analysis of CFTR-impaired endothelial cells reveals a pro-inflammatory phenotype.

Cystic fibrosis (CF) is a life-threatening disorder characterised by decreased pulmonary mucociliary and pathogen clearance, and an exaggerated inflammatory response leading to progressive lung damage. CF is caused by bi-allelic pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel. CFTR is expressed in endothelial cells (ECs) and EC dysfunction has been reported in CF patients, but a role for this ion channel in ECs regarding CF disease progression is poorly described.We used an unbiased RNA sequencing approach in complementary models of CFTR silencing and blockade (by the CFTR inhibitor CFTRinh-172) in human ECs to characterise the changes upon CFTR impairment. Key findings were further validated in vitro and in vivo in CFTR-knockout mice and ex vivo in CF patient-derived ECs.Both models of CFTR impairment revealed that EC proliferation, migration and autophagy were downregulated. Remarkably though, defective CFTR function led to EC activation and a persisting pro-inflammatory state of the endothelium with increased leukocyte adhesion. Further validation in CFTR-knockout mice revealed enhanced leukocyte extravasation in lung and liver parenchyma associated with increased levels of EC activation markers. In addition, CF patient-derived ECs displayed increased EC activation markers and leukocyte adhesion, which was partially rescued by the CFTR modulators VX-770 and VX-809.Our integrated analysis thus suggests that ECs are no innocent bystanders in CF pathology, but rather may contribute to the exaggerated inflammatory phenotype, raising the question of whether normalisation of vascular inflammation might be a novel therapeutic strategy to ameliorate the disease severity of CF.

Comparative meta-analysis of cystic fibrosis cell models suggests partial endothelial-to-mesenchymal transition.

The mesenchymal conversion of epithelial cells (EMT) has been suggested as a potential contributor in cystic fibrosis (CF) disease progression. Endothelial cells (EndCs), the cells lining blood vessels, express functional CFTR and CFTR impairment promotes endothelial activation and dysfunction. However, if the mesenchymal switch also exists in CF EndCs remains uncharacterized. To understand whether the endothelial-to-mesenchymal transition (EndMT) could occur in CF, we have conducted a transcriptomic meta-analysis of primary CFTR-impaired and patient-derived EndCs, and further compared our results to data from CF epithelial cells (EpCs) where EMT has been demonstrated. As compared to EpCs, we show that CFTR-impaired EndCs display a limited signature of EndMT, and that expression of the mesenchymal inducer Twist1 remained unchanged. Nonetheless, the use of CFTR modulators reduced the expression of mesenchymal markers from CF patient-derived EndCs, suggesting an additional therapeutic added-value next to the known effect on CFTR ion transport.

BRAF, A gatekeeper controlling endothelial permeability.

The RAF/MEK/ERK signal transduction pathway is commonly deregulated in cancer and is activated by various stimuli regulating a variety of cell responses. In wild-type endothelial cells, upon permeability stimuli, ROKα, RAF1, BRAF, and RAP1 become activated, inducing a cascade of reactions resulting in F-actin remodeling and increased cell permeability. Here, Dorard et al. showed that BRAF ablated cells had more RAF1/ROKα dimerization and relocalization to VE-cadherin occurred, ultimately leading to less F-actin content and reduced vessel permeability.