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. 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). 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. 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.

Vascular endothelial cells express glycoprotein 130 (gp130), which is utilized as a signaling receptor by cytokines in the interleukin-6 (IL-6) family. Several IL-6 family cytokines can be found in the circulatory system during physiological or pathological conditions, and may influence endothelial function and response. This study evaluated and compared the cellular and molecular responses induced by IL-6 family cytokines in human endothelial cells. A proteomic analysis showed that IL-6 family cytokines induce the release of a range of proteins from endothelial cells, such as C-C motif chemokine ligand 23, hepatocyte growth factor, and IL-6. Pathway analysis indicated that gp130-signaling in endothelial cells regulates several functions related to angiogenesis and immune cell recruitment. The present investigation also disclosed differences and similarities between different IL-6 family cytokines in their ability to induce protein release and regulate gene expression and intracellular signaling, in regards to which oncostatin M showed the most pronounced effect. Further, this study showed that soluble gp130 preferentially blocks trans-signaling-induced responses, but does not affect responses induced by classic signaling. In conclusion, IL-6 family cytokines induce both specific and overlapping molecular responses in endothelial cells, and regulate genes and proteins involved in angiogenesis and immune cell recruitment.

Background: Blockade of IL1RAP (interleukin 1 receptor associated protein) was recently shown to reduce atherosclerosis in mice, but the effect on human vascular cells is largely unknown. Targeting the IL1RAP coreceptor represents a novel strategy to block the IL1RAP‐dependent cytokines IL (interleukin)‐1, IL‐33, and IL‐36. In the present study, we aimed to evaluate the role of novel antibodies targeting IL1RAP to reduce the effects of IL‐1β, IL‐33, or IL‐36γ in human vascular cells. Methods: Expression of IL1RAP was observed in human atherosclerotic plaques by immunohistochemistry and microarray and in endothelial cells by flow cytometry. Endothelial cells were cultured with IL‐1β, IL‐33, or IL‐36γ cytokines with or without IL1RAP antibodies and analyzed with Olink proteomics, ELISA, Western blot, and real‐time quantitative polymerase chain reaction. The functional effect of IL1RAP antibodies on endothelial cells were analyzed with adhesion and permeability assays. Results: Olink proteomics showed inhibition of the inflammatory proteins LIF (leukemia inhibitory factor), OPG (osteoprotegerin), CCL4 (C‐C motif chemokine ligand 4), and MCP‐3 (monocyte chemoattractant protein 3) by IL1RAP‐blockade in endothelial cells after IL‐1β stimulation. In addition, the IL1RAP antibodies inhibited IL‐1β, and IL‐33 induced IL‐6 and IL‐8 secretion. Secretion of MCP‐1 (monocyte chemoattractant protein 1) was induced by IL‐1β, IL‐33, and IL‐36γ, and subsequently was inhibited by IL1RAP antibodies. Similar effects were found on mRNA expression level. Endothelial expression of the adhesion markers ICAM1 , VCAM1, and SELE were significantly reduced by IL1RAP antibodies, and neutrophil adhesion to endothelial cells induced by IL‐1β and IL‐33 was reduced by IL1RAP blockade. In human atherosclerotic lesions, IL1RAP expression correlated with markers of inflammation like IL6 , IL8, and MCP1 . Conclusions: IL1RAP‐targeting antibodies can reduce the expression of inflammatory cytokines and markers of adhesion in endothelial cells, which may be of importance for future putative targeted treatments against cardiovascular disease.

The nitrate ester-bearing 6-piperazinyl-purine analogue MK128 is a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor with cardioprotective, anti-inflammatory, and antiplatelet properties. However, structure-activity relationships of this compound class have not been systematically explored. This study aimed to elucidate the impact of spacer length on the antiplatelet activity of closely related piperazinyl-purine analogues and to identify the most active candidate for further evaluation. Antiplatelet activity of piperazinyl-purine analogues bearing short (MK128), intermediate (MK176), and long (MK118) spacers was evaluated by lumi-aggregometry and flow cytometry in isolated platelets and whole blood. Platelets were activated by proteinase-activated receptor-1 agonist SFLLRN-OH or collagen. Binding interactions with ROCK1/ROCK2 were examined using SelectScreen™ Kinase Profiling, in silico docking, and molecular dynamics simulations. MK128 exhibited the strongest inhibition of platelet aggregation and secretion in isolated platelets and whole blood, and rapidly promoted disaggregation of preformed aggregates. The rank order of potency in whole blood was MK128>MK176>MK118. All analogues inhibited ROCK1 and ROCK2 in cell-free assays, while broader kinase profiling demonstrated high selectivity. Docking studies suggested stable bidentate interactions between the purine core and hinge region, whereas molecular dynamics simulations identified spacer length-dependent differences in nitrate ester tail interactions. Reduced spacer flexibility in MK128 enabled productive interactions with catalytically relevant residues, providing a mechanistic explanation for its superior functional activity. Spacer length critically determines the antiplatelet effect of piperazinyl-purine analogues. The one-carbon spacer analogue MK128 demonstrated superior platelet and ROCK inhibition, making it a promising lead candidate for further evaluation in ischemic disease models.

Aortic smooth muscle cells (AoSMCs) are widely used in cardiovascular research for studies on inflammatory processes in atherosclerotic lesions. Usually, AoSMCs are cultured in two-dimensional (2D) cell culture models, although limitations exist compared to the three-dimensional (3D) environment, and differences between 2D and 3D conditions on inflammatory markers have not been explored. This study aims to elucidate differences in inflammatory biomarkers in 2D versus 3D cultured AoSMCs. After inflammatory treatment of 2D and 3D cultured cells, protein release and gene expression were measured by Olink proteomics/enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction, respectively. The 2D cultured cells showed generally higher release of inflammatory proteins, such as C-C motif chemokine 20 (CCL20), transforming growth factor alpha (TGF-α) and TNF superfamily member 12 (TWEAK), compared to the 3D model. In contrast, more matrix metallopeptidase-10 (MMP-10) and interleukin-8 (IL-8) were released from 3D cultured cells compared to 2D cultured cells. Elevated release of interleukin-1β in addition to transcriptional activation of inflammasome components was also observed in the 2D condition compared to the 3D condition. In conclusion, release of inflammatory proteins differs depending on the cell culture model, where 2D cultured AoSMCs release higher levels of several pro-inflammatory proteins compared to 3D cultured AoSMCs.