Olivier Meilhac

During ischemia, endothelial cell integrity is compromised, as a consequence, blood barrier homeostasis is disrupted. Therefore, the structural and functional preservation of endothelial cells is paramount when trying to improve outcomes after ischemic injury. Endoplasmic reticulum (ER) stress is increasingly recognized as a key player in ischemic injury through unfolded protein response (UPR) signalling, and its crosstalk with mitochondrial death pathways. This study provides a cost-effective and straightforward method to delve into the relationship between ER stress and ischemia in human microvascular endothelial cells-1 (HMEC-1). HMEC-1 was exposed to 8 h of oxygen–glucose deprivation (OGD) in glucose-free medium with rapidly induced hypoxia. Hypoxia, oxygen consumption, cell viability, apoptosis, and ER stress markers (BiP/GRP78, PERK, ATF6, IRE1/XBP1s, CHOP) were assessed by RT-qPCR and Western blot. Cell viability decreased by approximately 33% following OGD, while CHOP expression increased ~4-fold, indicating significant ER stress induction. The model enables quantification of metabolic stress (OCR), as well as evaluation of viability loss, membrane integrity, apoptotic commitment, and discrimination between ER stress resolution versus maladaptation. Overall, GasPak EZ Pouch Systems provide a reproducible and practical in vitro platform to study ischemic injury down to the mechanistic details of ER-mitochondria signalling. They give the opportunity to evaluate therapeutic approaches that target ER homeostasis to limit apoptosis and/or recovery of metabolic function after ischemia. This method could allow rapid screening of ER stress-modulating interventions aimed at preserving endothelial barrier function, in various ischemic contexts.

During ischemia, endothelial cells' integrity is compromised, as a consequence, blood–barrier homeostasis is disrupted. Therefore, the structural and functional preserva-tion of endothelial cells is paramount when trying to improve outcomes after ischemic injury. Endoplasmic reticulum (ER) stress is increasingly recognized as a key player in ischemic injury through unfolded protein response (UPR) signalling, and its crosstalk with mitochondrial death pathways. This study provides a cost-effective and straightfor-ward method to delve into the relationship between ER stress and ischemia in human microvascular endothelial cells-1 (HMEC-1). HMEC-1 was exposed to 8 hours of oxygen–glucose deprivation (OGD) in glucose-free medium with rapidly induced hypoxia. Hy-poxia, oxygen consumption, cell viability, apoptosis, and ER stress markers (BiP/GRP78, PERK, ATF6, IRE1/XBP1s, CHOP) were assessed by RT-qPCR and Western blot. The mod-el enables quantification of metabolic stress (OCR), as well as evaluation of viability loss, membrane integrity, apoptotic commitment, and discrimination between ER stress reso-lution versus maladaptation. Overall, GasPak EZ Pouch Systems provide a reproducible and practical in vitro platform to study ischemic injury down to its mechanistic details of ER-mitochondria signalling. They give the opportunity to evaluate therapeutic approach-es that target ER homeostasis to limit apoptosis and/or recovery of metabolic function after ischemia. This method could allow rapid screening of ER stress–modulating interven-tions aiming at preserving endothelial barrier function, in various ischemic contexts.

During periodontitis, Porphyromonas gingivalis and its lipopolysaccharides (LPS) may translocate into the bloodstream and alter adipocyte function, aggravating obesity-related disorders. This study aimed to evaluate the inflammatory and metabolic effects of P. gingivalis in obese db/db mice, and to decipher the molecular mechanisms targeted by P. gingivalis or its LPS in 3T3-L1 adipocytes. Then, we determined the ability of three major dietary polyphenols, namely caffeic acid, quercetin and epicatechin, to protect adipocytes under LPS conditions. Results show that obese mice exposed to P. gingivalis exhibited an altered lipid profile with higher triglyceride accumulation, an enhanced pro-inflammatory response and a reduced antioxidant SOD activity in the adipose tissue. In adipose cells, P. gingivalis and LPS induced the TLR2-4/MyD88/NFκB signaling pathway, and promoted IL-6 and MCP-1 secretion. Bacterial stimuli also increased ROS levels and the expression of NOX2, NOX4 and iNOS genes, while they deregulated mRNA levels of Cu/ZnSOD, MnSOD, catalase, GPx and Nrf2. Interestingly, caffeic acid, quercetin and epicatechin protected adipose cells via antioxidant and anti-inflammatory effects. Overall, these findings show the deleterious impact of P. gingivalis on inflammation, oxidative stress and lipid metabolism in obese mice and adipose cells, and highlight the therapeutic potential of polyphenols in mitigating periodontal bacteria-mediated complications during obesity.

High-density lipoproteins exert vasculoprotective effects, mainly through apolipoprotein A1, which has led to the development of treatments based on apolipoprotein A1 nanoparticles (A1NPs) administered intravenously, mainly for the treatment of cardiovascular diseases. However, their potential as therapy for lung pathologies has not yet been explored. In this work, we produced A1NPs using microfluidics and characterized their therapeutic potential for lung delivery. Their morphology was characterized by dynamic light scattering and transmission electron microscopy. A1NPs toxicity and cellular uptake were performed on both endothelial (HMEC-1) and epithelial (A549) cells and their anti-inflammatory activity was evaluated on TNF-α-stimulated HMEC-1. A1NPs biodistribution was explored in lung mice after aerosolization and their transcytosis was further investigated using A549 air-liquid interface model. Our results demonstrate that the microfluidic synthesis of A1NPs was reproducible and yielded discoidal particles with sizes ranging from 7-12 nm. A1NPs were internalized by both cells without being cytotoxic and significantly reduced IL-6 expression. Aerosolization resulted in homogeneous distribution in lungs, without causing an immunogenic response. A fraction of A1NPs crossed alveolar epithelial cells both in vitro and in vivo , paving the way for future therapeutic strategies targeting not only the lungs, but also other peripheral organs. These results are promising for the use of A1NPs as vectors for therapeutic molecules, which could exert synergistic protective effects with Apolipoprotein A1. This is the first study to show the non-invasive administration of A1NPs by aerosolization, which may improve their bioavailability in lungs and appears to be a promising approach for treating lung diseases. GRAPHICAL

SARS-CoV-2 infection affects both lipid metabolism and lung function. The severity of the disease has been associated with reduced levels of both high-density lipoprotein (HDL) and low-density lipoprotein cholesterol. Despite the crucial role that these nanoparticles play in SARS-CoV-2 infection, few studies have examined their structure during COVID-19 beyond HDL quantity. The study aimed to assess apolipoprotein levels in COVID-19 patients who either survived or died following ICU admission. In addition, ICU survivors and non-survivors were compared for HDL particle size and proteome. Between February and April 2020, our study enrolled 37 COVID-19 patients upon their intensive care unit admission. Among them, 18 survived the disease, while 19 succumbed to it. We used mass spectrometry to assess plasma levels of 14 apolipoproteins and LCAT. Additionally, we analyzed HDL subpopulation distribution by utilizing native polyacrylamide gel electrophoresis. HDL particles were isolated from both surviving and non-surviving patients using ultracentrifugation, followed by characterization of their proteomes with NanoLC-MS/MS. Plasma apolipoproteins, including Apo A-II, Apo Cs (I, II, III), Apo H, Apo J, Apo M, and LCAT, were decreased in patients who did not survive COVID-19. However, no alterations were noted in the distribution of HDL subpopulations in relation to mortality. HDL composition was further altered based on mortality, displaying a decline in Apo H and paraoxonase 3. In conclusion, we have shown an alteration in plasma apolipoproteins and HDL composition between surviving COVID-19 patients and non-survivors. Some markers, such as Apo H, are more predictive than baseline lipid concentrations such as HDL-C. These markers appear to provide a more accurate indication of mortality during COVID-19 compared with baseline lipid concentrations such as HDL-C.

The establishment of effective antiviral responses within host cells is intricately related to their metabolic status, shedding light on immunometabolism. In this study, we investigated the hypothesis that cellular reliance on glutamine metabolism contributes to the development of a potent antiviral response. We evaluated the antiviral response in the presence or absence of L-glutamine in the culture medium, revealing a bivalent response hinging on cellular metabolism. While certain interferon-stimulated genes (ISGs) exhibited higher expression in an oxidative phosphorylation (OXPHOS)-dependent manner, others were surprisingly upregulated in a glycolytic-dependent manner. This metabolic dichotomy was influenced in part by variations in interferon-β (IFN-β) expression. We initially demonstrated that the presence of L-glutamine induced an enhancement of OXPHOS in A549 cells. Furthermore, in cells either stimulated by poly:IC or infected with dengue virus and Zika virus, a marked increase in ISGs expression was observed in a dose-dependent manner with L-glutamine supplementation. Interestingly, our findings unveiled a metabolic dependency in the expression of specific ISGs. In particular, genes such as ISG54, ISG12 and ISG15 exhibited heightened expression in cells cultured with L-glutamine, corresponding to higher OXPHOS rates and IFN-β signaling. Conversely, the expression of viperin and 2′-5′-oligoadenylate synthetase 1 was inversely related to L-glutamine concentration, suggesting a glycolysis-dependent regulation, confirmed by inhibition experiments. This study highlights the intricate interplay between cellular metabolism, especially glutaminergic and glycolytic, and the establishment of the canonical antiviral response characterized by the expression of antiviral effectors, potentially paving the way for novel strategies to modulate antiviral responses through metabolic interventions.

PCSK9 best-known and studied function is to induce the hepatic degradation of the low-density lipoprotein receptor (LDLR), thereby increasing the concentration of LDL-cholesterol (LDL-C) in the blood. Beyond its effects on LDL, recent studies have reported pleiotropic effects of PCSK9 notably in septic shock, vascular inflammation, viral infection, and cancer. While the functional and structural integrity of peripheral nerves are critically influenced by circulating lipids, the impact of PCSK9 in the peripheral nervous system is unknown. In this study, we investigated the consequences of PCSK9 deficiency on peripheral nerves. We found that PCSK9 deletion in mice leads to peripheral neuropathy characterized by a reduction of thermal and mechanical pain sensations. PCSK9 deficient mice also presented skin structural changes with a reduction of number of terminal nociceptive Schwann cells, Remak fiber axonal swelling, as well as hypomyelination of small nerve fibers. Interestingly, peripheral nerves of PCSK9 deficient mice presented an upregulation of the fatty acid transporter CD36 expression which correlated with an increase in nerve lipid contents and structural mitochondrial abnormalities. Our findings demonstrate that PCSK9 plays a critical role in the peripheral nerves by regulating lipid homeostasis, and its deficiency could lead to the development of symptoms related to peripheral neuropathy. Graphical PCSK9 modulates nerve energy metabolism and health. Created with BioRender.com.