Donatienne Tyteca

Acute pancreatitis is a common inflammatory condition of the pancreas that can lead to severe complications such as chronic pancreatitis and pancreatic ductal adenocarcinoma. While vascular remodeling is a hallmark of many inflammatory conditions, the molecular mechanisms underlying vascular changes during pancreatitis remain largely unexplored. This study aimed to investigate the vascular changes associated with acute pancreatitis and to identify the molecular mechanisms underlying these changes. Acute pancreatitis was induced in wild-type mice through caerulein injections and resulted in progressive and substantial vascular changes, characterized by morphological remodeling, increased vessel density, and elevated vascular permeability. These structural changes were accompanied by molecular alterations, including increased expression of endothelial genes and decreased surface expression of vascular endothelial VE-cadherin. Injured acinar cells exhibited a significant increase in VEGF-A expression during pancreatitis. Acinar-specific VEGF-A inactivation led to marked impairments in vascular remodeling, with reduced vessel density and diminished number of vessels, without affecting immune cell infiltration, fibrosis, or acinar-to-ductal metaplasia. Together, our work identifies a mechanism by which increased expression of VEGF-A by acinar cells during pancreatitis is essential for maintaining vascular integrity and for driving vascular remodeling in the inflamed pancreas.

Pancreatitis, or inflammation of the pancreas, is a common gastrointestinal condition. While often acute and self-resolving, it can become chronic and promote pancreatic ductal adenocarcinoma (PDAC), the third deadliest cancer worldwide. Pancreatitis is accompanied by morphological and molecular changes, notably immune cell infiltration, fibrosis, and acinar-to-ductal metaplasia (ADM). ELP3, the catalytic subunit of the Elongator complex, modifies wobble uridine tRNAs to optimize codon translation rates. It is critical to inflammatory processes and cancer in multiple organ systems, yet its role in the pancreas has not been investigated. This study aimed to investigate the expression and implication of ELP3 during pancreatitis induced in mice via repetitive caerulein injections. Acute pancreatitis was accompanied by increased expression of ELP3, which was mainly detected in pancreatic epithelial cells. To assess its function, we genetically inactivated Elp3 in pancreatic epithelial cells. Elp3 deficiency had no detectable effects on pancreas homeostasis, on the initiation and resolution of acute pancreatitis, on the development of chronic pancreatitis, or on pancreatitis-induced PDAC initiation. Our findings indicate that ELP3 is dispensable in pancreatic formation, inflammation and PDAC initiation. Future studies should explore its role in non-epithelial cells and its potential involvement in other PDAC hallmarks, such as therapy resistance.

Acute pancreatitis is a common inflammatory condition of the pancreas that can lead to severe complications such as chronic pancreatitis and pancreatic ductal adenocarcinoma. While vascular remodeling is a hallmark of many inflammatory conditions, the molecular mechanisms underlying vascular changes during pancreatitis remain largely unexplored. This study aimed to investigate the vascular changes associated with acute pancreatitis and to identify the molecular mechanisms underlying these changes. Acute pancreatitis was induced in wild-type mice through caerulein injections and resulted in progressive and substantial vascular changes, characterized by morphological remodeling, increased vessel density, and elevated vascular permeability. These structural changes were accompanied by molecular alterations, including increased expression of endothelial genes and decreased surface expression of vascular endothelial VE-cadherin. Injured acinar cells exhibited a significant increase in VEGF-A expression during pancreatitis. Acinar-specific VEGF-A inactivation led to marked impairments in vascular remodeling, with reduced vessel density and diminished number of vessels, without affecting immune cell infiltration, fibrosis, or acinar-to-ductal metaplasia. Together, our work identifies a mechanism by which increased expression of VEGF-A by acinar cells during pancreatitis is essential for maintaining vascular integrity and for driving vascular remodeling in the inflamed pancreas.

The core neuroacanthocytosis syndromes, i.e., chorea-acanthocytosis/VPS13A disease (ChAc) and McLeod syndrome/XK disease (MLS), are respectively due to mutations in VPS13A and XK genes and share similar manifestations including the formation of acanthocytes. We previously showed by lipidomics of red blood cells (RBCs) from ChAc patients slight lipid changes compared to healthy controls. We here evaluated the consequences for RBC morphology, deformability, cytoskeleton and membrane lipid transversal and lateral distribution in five patients with ChAc and two patients with MLS. Compared to healthy donors, the two patient cohorts showed a strong increase of abnormal RBCs including acanthocytes and spheroechinocytes, a decrease in RBC projected surface area and deformability, and a rise in spectrin density. The abundance of cholesterol-enriched domains and the proportion of RBCs with ceramide-enriched patches were also increased while phosphatidylserine surface exposure was slightly decreased. In contrast, the abundance of sphingomyelin-enriched domains was poorly affected. At the individual level, patients showing the highest cholesterol-enriched domain abundance exhibited the highest number of RBCs with ceramide-enriched patches, compatible with RBC maturation defects, whereas patient RBCs exhibiting the highest spectrin membrane density showed the strongest loss of RBC projected surface area and the lowest abundance of sphingomyelin-enriched domains, consistent with RBC membrane alterations. Our study indicated that abnormal RBCs were associated with lipid distribution and cytoskeleton impairments, which appeared to result from both RBC maturation defects and membrane alterations. Moreover, the extent of lipid distribution alteration is well correlated with laboratory parameters typically altered in neuroacanthocytosis and could present an added value in neuroacanthocytosis syndrome evaluation.

The extracellular vesicle release in red blood cell concentrates reflects progressive accumulation of storage lesions and could represent a new measure to be implemented routinely in blood centres in addition to haemolysis. Nevertheless, there is currently no standardized isolation protocol. In a previous publication, we developed a reproducible ultracentrifugation‐based protocol (20,000 × g protocol) that allows to classify red blood cell concentrates into three cohorts according to their vesiculation level. Since this protocol was not adapted to meet routine requirements, the goal of this study was to develop an easier method based on low‐speed centrifugation (2,000 × g protocol) and limited red blood cell concentrate volumes to match with a non‐destructive sampling from the quality control sampling tubing. Despite the presence of contaminants, mainly in the form of albumin and lipoproteins, the material isolated with the 2,000 × g protocol contained red blood cell‐derived vesicular structures. It was reproducible, could predict the number of extracellular vesicles obtained with the 20,000 × g protocol and better discriminated between the three vesiculation cohorts than haemolysis at the legal expiry date of 6 weeks. However, by decreasing red blood cell concentrate volumes to fit with the volume in the quality control tubing, particle yield was highly reduced. Therefore, centrifugation time and relative centrifugal force were adapted (1,000 × g protocol), allowing for the recovery of a similar particle number and composition between small and large volumes sampled from the main unit, in different vesiculation cohorts over time. A similar observation was made with the 1,000 × g protocol between small volumes sampled from the quality control tubing and the mother‐bag. In conclusion, our study paves the way for the use of the 2,000 × g protocol (adapted to a 1,000 × g protocol with the quality control sampling tubing) for particle measurement in blood centres.

Background: Breast cancer clinical outcome relies on its intrinsic molecular subtype and mortality is almost exclusively due to metastasis, whose mechanism remains unclear. We recently revealed the specific contribution of plasma membrane cholesterol to the invasion of malignant MCF10CAIa but not premalignant MCF10AT and normal MCF10A cell lines in 2D, through invadopodia formation and extracellular matrix (ECM) degradation. In the present study, we address the impact of breast cancer subtypes, mutations and aggressiveness on cholesterol implication in breast cancer cell invasion and 3D spheroid invasion and growth. Methods: We used nine breast cancer cell lines grouped in four subtypes matching breast tumor classification. Four of these cell lines were also used to generate 3D spheroids. These cell lines were compared for cell invasion in 2D and 3D, spheroid growth in 3D, gelatin degradation, cortactin expression, activation and subcellular distribution as well as cell surface cholesterol distribution and lipid droplets. The effect of plasma membrane cholesterol depletion on all these parameters was determined in parallel and systematically compared with the impact of global matrix metalloproteinase (MMP) inhibition. Results: The six invasive cell lines in 2D were sensitive to partial cholesterol depletion, independently of their subtype, aggressiveness or mutation. Nevertheless, the effect was stronger in the three cell lines able to degrade gelatin. 3D spheroid invasion was also reduced after cholesterol depletion in all breast cancer subtypes tested. Notably, targeting cholesterol was more powerful than MMP inhibition in reducing invasion in both 2D and 3D culture models. Moreover, cholesterol depletion in the six invasive cell lines impaired cortactin distribution in the perinuclear region where invadopodia localized. Breast cancer cell line aggressiveness relied on cholesterol-enriched domains at the ECM-free side and intracellular lipid droplets. Furthermore, the three gelatin-degrading cell lines were characterized by increased cholesterol-enriched submicrometric domains at their ECM-contact side. Conclusion: Together, our data suggest cell surface cholesterol combined with lipid droplet labeling as a breast cancer cell aggressiveness marker. They also open the way to test other cholesterol-targeting drugs in more complex models to further evaluate whether cholesterol could represent a strategy in breast cancer therapy.

Piezo1 is a mechanosensitive ion channel required for various biological processes, but its regulation remains poorly understood. Here, we used erythrocytes to address this question since they display Piezo1 clusters, a strong and dynamic cytoskeleton and three types of submicrometric lipid domains, respectively enriched in cholesterol, GM1 ganglioside/cholesterol and sphingomyelin/cholesterol. We revealed that Piezo1 clusters were present in both the rim and the dimple erythrocyte regions. Upon Piezo1 chemical activation by Yoda1, the Piezo1 cluster proportion mainly increased in the dimple area. This increase was accompanied by Ca²⁺ influx and a rise in echinocytes, in GM1/cholesterol-enriched domains in the dimple and in cholesterol-enriched domains in the rim. Conversely, the effects of Piezo1 activation were abrogated upon membrane cholesterol depletion. Furthermore, upon Piezo1-independent Ca²⁺ influx, the above changes were not observed. In healthy donors with a high echinocyte proportion, Ca²⁺ influx, lipid domains and Piezo1 fluorescence were high even at resting state, whereas the cytoskeleton membrane occupancy was lower. Accordingly, upon decreases in cytoskeleton membrane occupancy and stiffness in erythrocytes from patients with hereditary spherocytosis, Piezo1 fluorescence was increased. Altogether, we showed that Piezo1 was differentially controlled by lipid domains and the cytoskeleton and was favored by the stomatocyte–discocyte–echinocyte transformation.

A specific plasma membrane distribution of the mechanosensitive ion channel Piezo1 is required for cell migration, but the mechanism remains elusive. Here, we addressed this question using WT and Piezo1-silenced C2C12 mouse myoblasts and WT and Piezo1-KO human kidney HEK293T cells. We showed that cell migration in a cell-free area and through a porous membrane decreased upon Piezo1 silencing or deletion, but increased upon Piezo1 activation by Yoda1, whereas migration towards a chemoattractant gradient was reduced by Yoda1. Piezo1 organized into clusters, which were preferentially enriched at the front. This polarization was stimulated by Yoda1, accompanied by Ca²⁺ polarization, and abrogated by partial cholesterol depletion. Piezo1 clusters partially colocalized with cholesterol- and GM1 ganglioside-enriched domains, the proportion of which was increased by Yoda1. Mechanistically, Piezo1 activation induced a differential mobile fraction of GM1 associated with domains and the bulk membrane. Conversely, cholesterol depletion abrogated the differential mobile fraction of Piezo1 associated with clusters and the bulk membrane. In conclusion, we revealed, for the first time, the differential implication of Piezo1 depending on the migration mode and the interplay between GM1/cholesterol-enriched domains at the front during migration in a cell-free area. These domains could provide the optimal biophysical properties for Piezo1 activity and/or spatial dissociation from the PMCA calcium efflux pump.

Corrigendum: Variability of extracellular vesicle release during storage of red blood cell concentrates is associated with differential membrane alterations, including loss of cholesterol-enriched domains