Publications

Abstract 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.

Abstract Pulmonary hypertension (PH) increases the mortality of preterm infants with bronchopulmonary dysplasia (BPD). There are no curative therapies for this disease. Lung endothelial carnitine palmitoyltransferase 1a (Cpt1a), the rate‐limiting enzyme of the carnitine shuttle system, is reduced in a rodent model of BPD. It is unknown whether endothelial Cpt1a reduction causes pulmonary vascular (PV) remodeling. The latter can be the result of endothelial‐mesenchymal transition (EndoMT). Here, endothelial cell (EC)‐specific Cpt1a KO and WT mice (<12 h old) are exposed to hyperoxia (70% O 2 ) for 14 days and allow them to recover in normoxia until postnatal day 28. Hyperoxia causes PH, which is aggravated in EC‐specific Cpt1a KO mice. Upregulating endothelial Cpt1a expression inhibits hyperoxia‐induced PV remodeling. Hyperoxia causes lung EndoMT, detected by immunofluorescence, scRNA‐sequencing, and EC lineage tracing, which is further increased in EC‐specific Cpt1a KO mice. Blocking EndoMT inhibits hyperoxia‐induced PV remodeling. Male mice under the same high oxygen conditions develop a higher degree of PH than females, which is associated with reduced endothelial Cpt1a expression. Conclusively, neonatal hyperoxia causes PH by decreasing endothelial Cpt1a expression and upregulating EndoMT. This provides a valuable strategy for developing targeted therapies by upregulating endothelial Cpt1a levels or inhibiting EndoMT to treat BPD‐associated PH.

High levels of the plasminogen activators, but also their inhibitor, plasminogen activator inhibitor 1 (PAI-1), have been documented in neovascularization of severe ocular pathologies such as diabetic retinopathy or age-related macular degeneration (AMD). AMD is the primary cause of irreversible photoreceptors loss, and current therapies are limited. PAI-1 has recently been shown to be essential for tumoral angiogenesis. We report here that deficient PAI-1 expression in mice prevented the development of subretinal choroidal angiogenesis induced by laser photocoagulation. When systemic and local PAI-1 expression was achieved by intravenous injection of a replication-defective adenoviral vector expressing human PAI-1 cDNA, the wild-type pattern of choroidal angiogenesis was restored. These observations demonstrate the proangiogenic activity of PAI-1 not only in tumoral models, but also in choroidal experimental neovascularization sharing similarities with human AMD. They identify therefore PAI-1 as a potential target for therapeutic ocular anti-angiogenic strategies.

Unlike vascular endothelial growth factor (VEGF)-A, the effect of VEGF-C on tumor angiogenesis, vascular permeability, and leukocyte recruitment is not known. To this end, we quantified in vivo growth and vascular function in tumors derived from two VEGF-C-overexpressing (VC+) and mock-transfected cell lines (T241 fibrosarcoma and VEGF-A-/- embryonic stem cells) grown in murine dorsal skinfold chambers. VC+ tumors grew more rapidly than mock-transfected tumors and exhibited parallel increases in tumor angiogenesis. Furthermore, VEGF-C overexpression elevated vascular permeability in T241 tumors, but not in VEGF-A-/- tumors. Surprisingly, unlike VEGF-A, VEGF-C did not increase leukocyte rolling or adhesion in tumor vessels. Administration of VEGF receptor (VEGFR)-2 neutralizing antibody DC101 reduced vascular density and permeability of both VC+ and mock-transduced T241 tumors. These data suggest that VEGFR-2 signaling is critical for tumor angiogenesis and vascular permeability and that VEGFR-3 signaling does not compensate for VEGFR-2 blockade. An alternate VEGFR, VEGFR-1 or neuropilin-1, may modulate adhesion of leukocytes to tumor vessels.

<b>Background</b>Neutrophils are essential in the early innate immune response to pathogens. Harnessing their antimicrobial powers, without driving excessive and damaging inflammatory responses, represents an attractive therapeutic possibility. The neutrophil population is increasingly recognised to be more diverse and malleable than was previously appreciated. Hypoxic signalling pathways are known to regulate important neutrophil behaviours and, as such, are potential therapeutic targets for regulating neutrophil antimicrobial and inflammatory responses.<b>Methods</b>We used a combination of in vivo and ex vivo models, utilising neutrophil and myeloid specific PHD1 or PHD3 deficient mouse lines to investigate the roles of oxygen sensing prolyl hydroxylase enzymes in the regulation of neutrophilic inflammation and immunity. Mass spectrometry and Seahorse metabolic flux assays were used to analyse the role of metabolic shifts in driving the downstream phenotypes.<b>Results</b>We found that PHD1 deficiency drives alterations in neutrophil metabolism and recruitment, in an oxygen dependent fashion. Despite this, PHD1 deficiency did not significantly alter ex vivo neutrophil phenotypes or in vivo outcomes in mouse models of inflammation. Conversely, PHD3 deficiency was found to enhance neutrophil antibacterial properties without excessive inflammatory responses. This was not linked to changes in the abundance of core metabolites but was associated with increased oxygen consumption and increased mitochondrial reactive oxygen species (mROS) production.<b>Conclusions</b>PHD3 deficiency drives a favourable neutrophil phenotype in infection and, as such, is an important potential therapeutic target.