15 research outputs found
Small extracellular vesicle targeting of hypothalamic AMPKα1 promotes weight loss in leptin receptor deficient mice
Background and aims: Leptin receptor (LEPR) deficiency promotes severe obesity and metabolic disorders. However, the current therapeutic options against this syndrome are scarce.
Methods: db/db mice and their wildtypes were systemically treated with neuronal-targeted small extracellular vesicles (sEVs) harboring a plasmid encoding a dominant negative mutant of AMP-activated protein kinase alpha 1 (AMPKα1-DN) driven by steroidogenic factor 1 (SF1) promoter; this approach allowed to modulate AMPK activity, specifically in SF1 cells of the ventromedial nucleus of the hypothalamus (VMH). Animals were metabolically phenotyped. Results: db/db mice intravenously injected with SF1-AMPKα1-DN loaded sEVs showed a marked feeding-independent weight loss and decreased adiposity, associated with increased sympathetic tone, brown adipose tissue (BAT) thermogenesis and browning of white adipose tissue (WAT).Conclusion: Overall, this evidence indicates that specific modulation of hypothalamic AMPK using a sEV-based technology may be a suitable strategy against genetic forms of obesity, such as LEPR deficiencyMinisterio de Ciencia y Universidades co-funded by the FEDER Program of EU (CD: BFU2017-87721; RN: RTI2018-099413-B-I00 and RED2018-102379-T; ML: RTI2018-101840-B-I00, PID2021-128145NB-I00 and PDC2022-133958-I00). âla Caixaâ Foundation (ID100010434), under the agreement LCF/PR/HR19/52160022 (ML); EuroNanoMed III (RA & ML: EURONANOMED2019-050-ENAMEP); European Research Council (RN: ERC Synergy Grant-2019-WATCH-810331)S
Role of non-muscular myosin light chain kinase in vascular inflammation induced by lipopolysaccharide and intermittent hypoxia
La forme non musculaire de la kinase de la chaine lĂ©gĂšre de la myosine (MLCKnm) est une kinase principalement exprimĂ©e par les cellules endothĂ©liales dont le rĂŽle principal est de phosphoryler la chaine lĂ©gĂšre de myosine. Cette phosphorylation modifie la conformation des tĂȘtes de myosine, augmente lâinteraction actine/myosine, et induit une rĂ©traction des cellules endothĂ©liales. Ce processus augmente la permĂ©abilitĂ© de la barriĂšre endothĂ©liale. Lâactivation de MLCKnm permet lâinfiltration de cellules inflammatoires en rĂ©ponse Ă certains stimuli dont le lipopolysaccharide (LPS) bactĂ©rien. Dans ce modĂšle expĂ©rimental de sepsis, la dĂ©ficience de MLCKnm dans un modĂšle murin protĂšge les souris injectĂ©es avec du LPS, associĂ©e Ă une prĂ©vention des stress oxydant et nitrosant ainsi que de lâactivation de voies de signalisation inflammatoire. Cependant les mĂ©canismes molĂ©culaires mis en jeu ne sont pas totalement connus. Dans le contexte inflammatoire, le syndrome dâapnĂ©es/hypopnĂ©es obstructives du sommeil, caractĂ©risĂ© par une obstruction des voies aĂ©riennes lors du sommeil menant Ă une hypoxie intermittente (HI), partage certaines caractĂ©ristiques dans lâactivation inflammatoire observĂ©e lors du sepsis. LâHI modifie le mĂ©tabolisme des cellules endothĂ©liales en diminuant la biodisponibilitĂ© du monoxyde dâazote, augmentant le stress oxydant ainsi que la production de certains facteurs inflammatoires. A long terme, une rĂ©ponse inflammatoire systĂ©mique est observĂ©e augmentant les risques dâathĂ©rosclĂ©rose. Lâobjectif de ce travail est dâĂ©tudier lâimplication de MLCKnm dans lâinflammation vasculaire dans deux modĂšles physiopathologiques, induits par le LPS et lâHI.Non muscular myosin light chain kinase (nmMLCK) is aprotein mainly expressed by endothelial cells whose roleis to phosphorylate myosin light chain. This phosphorylation modifies the conformation of myosin heads, increasing actin/myosin interaction, and inducing endothelial cells retraction. This process increases endothelial barrier permeability. The activation of nmMLCK increases inflammatory cell infiltration in response to several stimuli such as the bacterial lipopolysaccharide (LPS). In this experimental model of sepsis, nmMLCK deficiency in a murine model protects mice injected with LPS, associated with oxidative and nitrative stresses prevention as well as inflammatory pathway inhibition. However, molecular mechanisms are not fully known. In this inflammatory context, obstructive sleep apnea hypopnea syndrome, characterized by obstruction of upper airway during sleep leading to intermittent hypoxia (IH), share several characteristics in inflammatory activation observed during sepsis. IH modifies the metabolism of endothelial cells decreasing nitric oxide bioavailability, increasing oxidative stress aswell as inflammatory mediators. Long-term, systemic inflammatory response is observed increasing atherosclerosis risk. The objective of this work is to study the implication of nmMLCK in vascular inflammation in two pathophysiological models induced by LPS and IH
nmMLCK is essential to trigger endothelial dysfunction, interleukin-6 secretion and endothelial permeability in an experimental model of intermittent hypoxia
National audienc
Polyphenols impact on vascular dysfunction associated with obstructive sleep apnea: a randomized controlled trial
International audienc
Polyphenols impact on vascular dysfunction associated with obstructive sleep apnea: a randomized controlled trial
International audienc
nmMLCK is essential to trigger endothelial dysfunction, interleukin-6 secretion and endothelial permeability in an experimental model of intermittent hypoxia
National audienc
Non-muscular myosin light chain kinase triggers intermittent hypoxia-induced interleukin-6 release, endothelial dysfunction and permeability
Abstract Obstructive sleep apnea is characterized by intermittent hypoxia (IH) which alters endothelial function, induces inflammation and accelerates atherosclerosis-induced cardiovascular diseases. The non-muscular myosin light chain kinase (nmMLCK) isoform contributes to endothelial cell-cell junction opening. Deletion of nmMLCK protects mice from death in septic shock models and prevents atherosclerosis in high-fat diet-fed mice. The aim of the study was to analyze the implication of nmMLCK in IH-induced vascular inflammation. Human aortic endothelial cells were exposed to 6âhours of IH in absence or presence of nmMLCK inhibitors, ML-7 (5â”M) or PIK (150â”M). IH increased reactive oxygen species (ROS) and nitric oxide (NO) production, p65-NFÎșB activation and IL-6 secretion. While nmMLCK inhibition did not prevent IH-induced ROS production and p65-NFÎșB activation, it decreased NO production and partially prevented IL-6 secretion. IH-induced IL-6 secretion and vesicle-associated membrane protein-associated vesicles re-organization were inhibited in presence of the inhibitor of protein secretion, brefeldin A, or ML-7. IH increased monocytes transendothelial migration that was partially prevented by ML-7. Finally, IH reduced endothelium-dependent relaxation to acetylcholine of aortas from wild-type but not those taken from nmMLCK-deficient mice. These results suggest that nmMLCK participates to IH-induced endothelial dysfunction resulting from cytokines secretion and endothelial permeability
Interaction in endothelium of non-muscular myosin light-chain kinase and the NF-ÎșB pathway is critical to lipopolysaccharide-induced vascular hyporeactivity
During sepsis, endothelial barrier dysfunction contributes to cardiovascular failure, mainly through the release of oxidative metabolites by penetrant leukocytes. We reported the non-muscular isoform of myosin light chain kinase (nmMLCK) playing a pivotal role in endotoxin shock injury associated with oxidative and nitrative stresses, and vascular hyporeactivity. The present study was aimed at understanding the molecular mechanism of lipopolysaccharide (LPS)-induced vascular alterations as well as studying a probable functional association of nmMLCK with nuclear factor Îș-light-chain enhancer of activated B cells (NF-ÎșB). Aortic rings from mice were exposed in vitro to LPS and, then, vascular reactivity was measured. Human aortic endothelial cells (HAoECs) were incubated with LPS, and interaction of nmMLCK with NF-ÎșB was analysed. We provide evidence that nmMLCK deletion prevents vascular hyporeactivity induced by in vitro LPS treatment but not endothelial dysfunction in the aorta. Deletion of nmMLCK inhibits LPS-induced NF-ÎșB activation and increases nitric oxide (NO) release via induction of inducible NO synthase (iNOS) within the vascular wall. Also, removal of endothelium prevented both NF-ÎșB and iNOS expression in aortic rings. Among the proinflammatory factors released by LPS-treated endothelial cells, interleukin-6 accounts for the induction of iNOS on smooth muscle cells in response to LPS. Of particular interest is the demonstration that, in HAoECs, LPS-induced NF-ÎșB activation occurs via increased MLCK activity sensitive to the MLCK inhibitor, ML-7, and physical interactions between nmMLCK and NF-ÎșB. We report for the first time on NF-ÎșB as a novel partner of nmMLCK within endothelial cells. The present study demonstrates a pivotal role of nmMLCK in vascular inflammatory pathologies
Nonmuscle Myosin Light Chain Kinase: A Key Player in Intermittent HypoxiaâInduced Vascular Alterations
International audienc
Faecesâderived extracellular vesicles participate in the onset of barrier dysfunction leading to liver diseases
International audienceThe role of extracellular vesicles (EVs) from faeces (fEVs) and small circulating EVs (cEVs) in liver diseases such as non-alcoholic fatty diseases (NAFLD) and nonalcoholic steatohepatitis (NASH) has not been demonstrated. fEVs and cEVs of healthy donors, NAFLD and NASH patients were isolated and characterized. The effects of EVs were evaluated in intestinal, endothelial, Kupffer and stellate cells. Nonmuscular myosin light chain kinase (nmMLCK) deficient mice were used in vivo. Bacterial origins of fEVs were analysed by 16s rDNA gene sequencing. fEVs and small cEVs were composed of prokaryotic and eukaryotic origins. Only NASH-fEVs exerted deleterious effects. NASH-fEVs increased intestinal permeability and reduced expression of tight junction proteins that were prevented by nmMLCK inhibition, increased endothelial cell permeability and inflammatory cytokines and chemokines requiring TLR4/lipopolysaccharide pathway. NASH-fEVs and NASH-cEVs activated profibrotic and proinflammatory proteins of hepatic stellate cells. Treatment with NASH-fEVs evoked an increase in intestinal permeability in wild type but not in nmMLCK deficient mice. Bacterial origins of fEVs were different between NAFLD and NASH patients and 16 amplicon sequence variants were differentially abundant. We demonstrate that fEVs actively participate in barrier dysfunctions leading to liver injuries underscoring the role of nmMLCK and lipopolysaccharide carried by fEVs