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Although simple in structure, lysophosphatidic acid (LPA) is a potent bioactive lipid that profoundly influences cellular signaling and function upon binding to G protein-coupled receptors (LPA1-6). The majority of circulating LPA is produced by the secreted enzyme autotaxin (ATX). Alterations in LPA signaling, in conjunction with changes in autotaxin (ATX) expression and activity, have been implicated in metabolic and inflammatory disorders including obesity, insulin resistance, and cardiovascular disease. This review summarizes our current understanding of the sources and metabolism of LPA with focus on the influence of diet on circulating LPA. Furthermore, we explore how the ATX-LPA pathway impacts obesity and obesity-associated disorders, including impaired glucose homeostasis, insulin resistance, and cardiovascular disease.

Background IL-6 classic signaling is linked to anti-inflammatory functions while the trans-signaling is associated with pro-inflammatory responses. Classic signaling is induced via membrane-bound IL-6 receptor (IL-6R) whereas trans-signaling requires prior binding of IL-6 to the soluble IL-6R. In both cases, association with the signal transducing gp130 receptor is compulsory. However, differences in the downstream signaling mechanisms of IL-6 classic- versus trans-signaling remains largely elusive. Methods In this study, we used flow cytometry, quantitative PCR, ELISA and immuno-blotting techniques to investigate IL-6 classic and trans-signaling mechanisms in Human Umbilical Vein Endothelial Cells (HUVECs). Results We show that both IL-6R and gp130 are expressed on the surface of human vascular endothelial cells, and that the expression is affected by pro-inflammatory stimuli. In contrast to IL-6 classic signaling, IL-6 trans-signaling induces the release of the pro-inflammatory chemokine Monocyte Chemoattractant Protein-1 (MCP-1) from human vascular endothelial cells. In addition, we reveal that the classic signaling induces activation of the JAK/STAT3 pathway while trans-signaling also activates the PI3K/AKT and the MEK/ERK pathways. Furthermore, we demonstrate that MCP-1 induction by IL-6 trans-signaling requires simultaneous activation of the JAK/STAT3 and PI3K/AKT pathways. Conclusions Collectively, our study reports molecular differences in IL-6 classic- and trans-signaling in human vascular endothelial cells; and elucidates the pathways which mediate MCP-1 induction by IL-6 trans-signaling.

Trimethylamine-N-oxide (TMAO) is a gut microbiota-derived metabolite and TNF-α is proinflammatory cytokine, both known to be associated with renal inflammation, fibrosis and chronic kidney disease. However, today there are no data showing the combined effect of TMAO and TNF-α on renal fibrosis-and inflammation. The aim of this study was to investigate whether TMAO can enhance the inflammatory and fibrotic effects of TNF-α on renal fibroblasts. We found that the combination of TNF-α and TMAO synergistically increased fibronectin release and total collagen production from renal fibroblasts. The combination of TMAO and TNF-α also promoted increased cell proliferation. Both renal proliferation and collagen production were mediated through Akt/mTOR/ERK signaling. We also found that TMAO enhanced TNF-α mediated renal inflammation by inducing the release of several cytokines (IL-6, LAP TGF-beta-1), chemokines (CXCL-6, MCP-3), inflammatory-and growth mediators (VEGFA, CD40, HGF) from renal fibroblasts. In conclusion, we showed that TMAO can enhance TNF-α mediated renal fibrosis and release of inflammatory mediators from renal fibroblasts in vitro. Our results can promote further research evaluating the combined effect of TMAO and inflammatory mediators on the development of kidney disease.

Endothelial dysfunction is an early driver of atherosclerosis, yet the direct impact of endogenous crystals such as cholesterol crystals and monosodium urate on endothelial activation remains incompletely understood. In this study, we examine how crystalline stimuli modulate human umbilical vein endothelial cells by assessing inflammatory signaling, mitochondrial respiration, and neutrophil recruitment. Using dose- and time-controlled experiments, we show that CC and MSU are internalized by endothelial cells, activating NF-κB and STAT3 signaling pathways and inducing a robust pro-inflammatory cytokine profile. Notably, CC caused marked mitochondrial dysfunction, evidenced by impaired respiratory capacity and loss of membrane potential, revealing a novel bioenergetic vulnerability in endothelial cells. Both direct crystal stimulation and exposure to crystal-primed conditioned media triggered endothelial adhesion molecule expression and promoted neutrophil adhesion, indicating that soluble mediators released upon crystal stimulation can propagate vascular inflammation. These findings demonstrate that crystalline stimuli are potent vascular danger signals capable of driving endothelial inflammation, mitochondrial impairment, and immune cell engagement, which are hallmarks of early atherogenesis. By elucidating these multifaceted endothelial responses, this study provides important mechanistic insights into how crystal-induced signals may contribute to vascular dysfunction and the early stages of atherogenesis.

Background The formation and accumulation of cholesterol crystals (CC) at the lesion site is a hallmark of atherosclerosis. Although studies have shown the importance of vascular smooth muscle cells (VSMCs) in the disease atherosclerosis, little is known about the molecular mechanism behind the uptake of CC in VSMCs and their role in modulating immune response. Methods Human aortic smooth muscle cells were cultured and treated with CC. CC uptake and CC mediated signaling pathway and protein induction were studied using flow cytometry, confocal microscopy, western blot and Olink proteomics. Conditioned medium from CC treated VSMCs was used to study neutrophil adhesion, ROS production and phagocytosis. Neutrophil extracellular traps (NETs) formations were visualized using confocal microscopy. Results VSMCs and macrophages were found around CC clefts in human carotid plaques. CC uptake in VSMCs are largely through micropinocytosis and phagocytosis via PI3K–AkT dependent pathway. The uptake of CC in VSMCs induce the release inflammatory proteins, including IL-33, an alarming cytokine. Conditioned medium from CC treated VSMCs can induce neutrophil adhesion, neutrophil reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. IL-33 neutralization in conditioned medium from CC treated VSMCs inhibited neutrophil ROS production and NETs formation. Conclusion We demonstrate that VSMCs due to its vicinity to CC clefts in human atherosclerotic lesion can modulate local immune response and we further reveal that the interaction between CC and VSMCs impart an inflammatory milieu in the atherosclerotic microenvironment by promoting IL-33 dependent neutrophil influx and NETs formation.

Background The Caspase activation and recruitment domain 8 (CARD8) protein is a component of innate immunity as a negative regulator of NF- ĸB, and has been associated with regulation of proteins involved in inflammation. Expression of CARD8 mRNA and protein has been identified in human atherosclerotic lesions, and the truncated T30A variant (rs2043211) of CARD8 has been associated with lower C-reactive (CRP) and MCP-1 levels in myocardial infarction patients. The present study examines the role of a genetic variation in the CARD8 gene in relation to a selection of markers of inflammation. Methods In a cross-sectional study of young healthy individuals (18.0–25.9 yrs, n  = 744) the association between the rs2043211 variant in the CARD8 gene and protein markers of inflammation was assessed. Genotyping of the CARD8 C10X (rs2043211) polymorphism was performed with TaqMan real time PCR on DNA from blood samples. Protein levels were studied via Olink inflammation panel ( https://olink.com/ ). Using linear models, we analyzed men and two groups of women with and without estrogen containing contraceptives separately, due to previous findings indicating differences between estrogen users and non-estrogen using women. Genotypes were analyzed by additive, recessive and dominant models. Results The minor (A) allele of the rs2043211 polymorphism in the CARD8 gene was associated with lower levels of CCL20 and IL-6 in men (CCL20, Additive model: p  = 0.023; Dominant model: p  = 0.016. IL-6, Additive model: p  = 0.042; Dominant model: p  = 0.039). The associations remained significant also after adjustment for age and potential intermediate variables. Conclusions Our data indicate that CARD8 may be involved in the regulation of CCL20 and IL-6 in men. No such association was observed in women. These findings strengthen and support previous in vitro data on IL-6 and CCL20 and highlight the importance of CARD8 as a factor in the regulation of inflammatory proteins. The reason to the difference between sexes is however not clear, and the influence of estrogen as a possible factor important for the inflammatory response needs to be further explored.

Background Endothelial dysfunction profoundly compromises the barrier function that precludes trans-endothelial entry of low-density lipoprotein cholesterol (LDL-C) into the vessel wall. LDL-C retention in the vessel wall is atherogenic and its flux involves several mechanisms including LDL-receptor (LDL-R) mediated transcytosis, a process that is facilitated by inflammatory stressors. In this study, we aimed to investigate the role of interleukin-6 (IL-6) in regulating LDL-R and LDL-C uptake by vascular endothelial cells. Method We used commercially available Human umbilical vein endothelial cells (HUVECs) in this study. Flow cytometry, western blotting, qRT-PCR and ELISA were used to investigate expression of LDL-R and Mylip/IDOL. LDL-C uptake and free cholesterol levels in HUVECs was assessed using flowcytometry and mass-spectrometry respectively. Results We show that HUVECs treated with a combination of IL-6 and soluble IL-6 receptor (sIL-6R) result in a significant reduction in surface expression of LDL-R, an effect that is reversed by soluble gp130Fc – an antagonist of IL-6 trans-singling. Using pharmacological inhibitors and gene silencing techniques, we demonstrate that IL-6 trans-signaling induced downregulation of LDL-R is attained through lysosomal degradation mediated by the E3 ubiquitin ligase Mylip. Conversely, HUVECs treated with IL-6 in combination with sIL-6R exhibit markedly increased uptake of native LDL-C which is also inhibited by sgp130Fc, the actin inhibitor Cytochalasin D and the macropinocytosis inhibitor EIPA. Although stimulation of HUVECs upregulated the expression of scavenger receptors CD36 and CXCL16, their contribution to native LDL-C uptake turned out to be negligible. Conclusion Collectively, this study highlights the role of IL-6 in the regulation of LDL-R expression and cholesterol homeostasis in vascular ECs. IL-6 trans-signaling downregulates LDL-R yet increases LDL-C uptake via an LDL-R–independent, actin-dependent macropinocytosis pathway.

The Caspase activation and recruitment domain 8 (CARD8) protein is a component of innate immunity and overexpression of CARD8 mRNA was previously identified in atherosclerosis. However, very little is known about the regulation of CARD8 in endothelial cells and atherosclerosis. The aim of this study was to investigate CARD8 in the regulation of cytokine and chemokine expression in endothelial cells. Sections of human atherosclerotic lesions and non-atherosclerotic arteries were immunostained for CARD8 protein. Expression of CARD8 was correlated to mediators of inflammation in atherosclerotic lesions using Biobank of Karolinska Endarterectomies microarray data. The CARD8 mRNA was knocked-down in human umbilical vein endothelial cells (HUVECs) in vitro, followed by quantitative RT-PCR analysis and OLINK Proteomics. Endothelial and smooth muscle cells in arterial tissue expressed CARD8 and CARD8 correlated with vWF , CD163 and the expression of inflammatory genes, such as CXCL1 , CXCL6 and PDGF-A in plaque. Knock-down of CARD8 in HUVECs significantly altered proteins involved in inflammatory response, such as CXCL1, CXCL6, PDGF-A, MCP-1 and IL-6. The present study suggest that CARD8 regulate the expression of cytokines and chemokines in endothelial cells and atherosclerotic lesions, suggesting that CARD8 plays a significant role in endothelial activation.

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Understanding how uropathogenic Escherichia coli (UPEC) modulates the immune response in the kidney is essential to prevent UPEC from reaching the bloodstream and causing urosepsis. The purpose of this study was to elucidate if renal fibroblasts can release IL-1β during a UPEC infection and to investigate the mechanism behind the IL-1β release. We found that the UPEC strain CFT073 induced an increased IL-1β and LDH release from renal fibroblasts, but not from renal epithelial cells. The UPEC-induced IL-1β release was found to be NLRP3, caspase-1, caspase-4, ERK 1/2, cathepsin B and serine protease dependent in renal fibroblasts. We also found that the UPEC virulence factor α-hemolysin was necessary for IL-1β release. Conditioned medium from caspase-1, caspase-4 and NLRP3-deficient renal fibroblasts mediated an increased reactive oxygen species production from neutrophils, but reduced UPEC phagocytosis. Taken together, our study demonstrates that renal fibroblasts, but not renal epithelial cells, release IL-1β during a UPEC infection. This suggest that renal fibroblasts are vital immunoreactive cells and not only structural cells that produce and regulate the extracellular matrix.