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ERK1/2 Has Divergent Roles in LPS-Induced Microvascular Endothelial Cell Cytokine Production and Permeability.

ABSTRACT:

Abstract

Endothelial cells play a major role in inflammatory responses to infection and sterile injury. Endothelial cells express Toll-like receptor 4 (TLR4) and are activated by LPS to express inflammatory cytokines/chemokines, and to undergo functional changes, including increased permeability. The extracellular signal-regulated kinase 1/2 (ERK1/2) mediates pro-inflammatory signaling in monocytes and macrophages, but the role of ERK1/2 in LPS-induced activation of microvascular endothelial cells has not been defined. We therefore studied the role of ERK1/2 in LPS-induced inflammatory activation and permeability of primary human lung microvascular endothelial cells (HMVEC). Inhibition of ERK1/2 augmented LPS-induced IL-6 and vascular cell adhesion protein (VCAM-1) production by HMVEC. ERK1/2 siRNA knockdown also augmented IL-6 production by LPS-treated HMVEC. Conversely, ERK1/2 inhibition abrogated permeability and restored cell-cell junctions of LPS-treated HMVEC. Consistent with the previously described pro-inflammatory role for ERK1/2 in leukocytes, inhibition of ERK1/2 reduced LPS-induced cytokine/chemokine production by primary human monocytes. Our study identifies a complex role for ERK1/2 in TLR4-activation of HMVEC, independent of myeloid differentiation primary response gene (MyD88) and TIR domain-containing adaptor inducing IFN-β (TRIF) signaling pathways. The activation of ERK1/2 limits LPS-induced IL-6 production by HMVEC, while at the same time promoting HMVEC permeability. Conversely, ERK1/2 activation promotes IL-6 production by human monocytes. Our results suggest that ERK1/2 may play an important role in the nuanced regulation of endothelial cell inflammation and vascular permeability in sepsis and injury.

SUBMITTER: Wong E

PROVIDER: S-EPMC8139579 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectivesCatecholaminergic vasopressors are the cornerstone of circulatory shock management. Nevertheless, catecholamines have problematic side effects, arousing a growing interest in noncatecholaminergic agents such as vasopressin or angiotensin-II. However, their respective effects on sepsis-associated microvascular endothelial dysfunction such as permeability or inflammation remain elusive. We investigated the role of catecholamines and other vasopressors on Toll-like receptor agonists-induced microvascular endothelial permeability and inflammation.SettingUniversity research laboratory/cell research.SubjectsHuman pulmonary microvascular endothelial cells from multiple donors.InterventionConfluent monolayers of human pulmonary microvascular endothelial cells were treated with Toll-like receptor agonists (lipopolysaccharide, Poly[I:C], or tripalmitoyl-S-glyceryl cysteine) in the presence or absence of epinephrine, norepinephrine, vasopressin, and angiotensin-II. Permeability was inferred from transendothelial resistance, measured using electrical cell impedance sensing, where decreased transendothelial resistance is consistent with increased permeability. Cell-cell junction molecule expression was assessed via immunofluorescence microscopy and flow cytometry. We quantified cytokines in supernatants of Toll-like receptor agonist-treated human pulmonary microvascular endothelial cells.Measurements and main resultsEpinephrine and norepinephrine both ameliorate lipopolysaccharide, polyinosinic:polycytidylic acid, or tripalmitoyl-S-glyceryl cysteine-induced reductions in transendothelial resistance, a surrogate for endothelial permeability. In contrast, the noncatecholaminergic agents, vasopressin, and angiotensin-II did not affect Toll-like receptor agonists-induced reductions in transendothelial resistance. β1- and β2-adrenergic receptor antagonists reduced the effects of the catecholamines on transendothelial resistance, whereas α-adrenergic receptor antagonists did not. We observed that epinephrine and norepinephrine induced actin cytoskeletal rearrangement and normalized the membrane expression of proteins involved with adherens-junctions (vascular endothelial-cadherin) and tight-junctions (zona occludens-1). Despite having a substantial effect on endothelial permeability, epinephrine and norepinephrine did not affect human pulmonary microvascular endothelial cell survival or production of interleukin-8, interleukin-6, or monocyte chemoattractant protein-1 (CCL-2) induced by Toll-like receptor agonists, suggesting that these functions are regulated separately from permeability.ConclusionsOur findings demonstrate that treatment with epinephrine or norepinephrine strongly reduces endothelial permeability induced by agonists of multiple Toll-like receptors (Toll-like receptor-2, Toll-like receptor-3, Toll-like receptor-4) in vitro. Our studies suggest that both β1- and β2-adrenergic receptors mediate the stabilizing effects of epinephrine and norepinephrine on the endothelial barrier.

Project description:BackgroundPrevious experiments revealed phospholipid scramblase 4 (PLSCR4) mRNA to be significantly increased in a lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) model of human pulmonary microvascular endothelial cells (HPMECs); however, the effect of PLSCR4 and its mechanism have not been reported to date. The PLSCR family is thought to mediate the transmembrane movement of phospholipids (PS), and has been found to be involved in pyroptosis through combing with gasdermin D (GSDMD). We therefore speculated that PLSCR4 may contribute to cell death via pyroptosis.MethodsTo investigate the effect and mechanism of PLSCR4 in ARDS, we constructed an in vitro model of LPS-induced ARDS in HPMECs transfected with PLSCR4 small interfering RNA (siRNA) or scramble siRNA (sc siRNA). After 4 h of LPS stimulation, western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), tracer flux assays, and fluorescence assays were used to study the relationship between PLSCR4 and pyroptosis with regards to their impact on ARDS. We also established an ARDS mouse model which was pretreated with a liquid complex of PLSCR4 siRNA/sc siRNA-lipofectamine 2000 through the fundus venous plexus. Finally, we used DNA pull-down and protein profiling to study the potential transcription factor of PLSCR4.ResultsIt was found that when the expression of PLSCR4 was elevated, the concentration of interleukin 1 beta (IL-1?) and IL-18 decreased, along with barrier damage (P<0.05). Furthermore, HPMEC injury was reduced with more distribution of PS and N-terminal cleavage product (GSDMD-NT) of GSDMD on the external side of cell membrane. However, the pyroptosis-relevant proteins of GSDMD and caspase-1 were not obviously changed (P<0.05); we further found that when PLSCR4 was depressed, the lung injury was aggravated in the mice. In the DNA pull-down assay, P62280 remarkably increased, which suggested that P62280 might be the transcription factor for PLSCR4.ConclusionsPLSCR4 alleviated pyroptosis by transporting PS to the outside of the membrane, blocking the formation of pyroptosis pores composed of GSDMD. Moreover, P62280 might be the transcription factor of PLSCR4. These insults may provide useful insights into the clinical treatment of ARDS.

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ERK1/2 Has Divergent Roles in LPS-Induced Microvascular Endothelial Cell Cytokine Production and Permeability.

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