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Fatty acid metabolites, produced by cytochrome P450 enzymes and soluble epoxide hydrolase (sEH), regulate inflammation. Here, we report that the transforming growth factor β (TGF-β)-induced polarization of macrophages to a pro-resolving phenotype requires Alk5 and Smad2 activation to increase sEH expression and activity. Macrophages lacking sEH showed impaired repolarization, reduced phagocytosis, and maintained a pro-inflammatory gene expression profile. 11,12-Epoxyeicosatrienoic acid (EET) was one altered metabolite in sEH -/- macrophages and mimicked the effect of sEH deletion on gene expression. Notably, 11,12-EET also reduced Alk5 expression, inhibiting TGF-β-induced Smad2 phosphorylation by triggering the cytosolic translocation of the E3 ligase Smurf2. These findings suggest that sEH expression is controlled by TGF-β and that sEH activity, which lowers 11,12-EET levels and promotes TGF-β signaling by metabolizing 11,12-EET to prevent Alk5 degradation. Thus, an autocrine loop between sEH/11,12-EET and TGF-β1 regulates macrophage function., Competing Interests: The authors declare no competing financial interests., (© 2024 The Author(s).)

Tissue factor pathway inhibitor (TFPI) is an important regulator of coagulation and a link between inflammation and thrombosis. Here we investigated whether endothelial cell-driven oxidative post-translational modifications could have an impact on TFPI activity. We focused on S-sulfhydration, which is a hydrogen sulfide-dependent post-translational modification that, in endothelial cells, is regulated by the enzyme cystathionine γ-lyase (CSE). The study made use of human primary endothelial cells and blood from healthy individuals or subjects with atherosclerosis as well as from mice lacking endothelial CSE. TFPI was S-sulfhydrated in endothelial cells from healthy individuals and mice, while the loss of endothelial CSE expression/activity reduced its modification. Non-S-sulfhydrated TFPI was no longer able to interact with factor Xa, which facilitated the activation of tissue factor. Similarly, non-S-sulfhydratable TFPI mutants bound less protein S, while supplementation with hydrogen sulfide donors, preserved TFPI activity. Phenotypically, loss of TFPI S-sulfhydration increased clot retraction, suggesting that this post-translational modification is a new endothelial cell-dependent mechanism that contributes to the regulation of blood coagulation., Competing Interests: Declaration of competing interest There is no conflict of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Macrophages are plastic and heterogeneous immune cells that adapt pro- or anti-inflammatory phenotypes upon exposure to different stimuli. Even though there has been evidence supporting a crosstalk between coagulation and innate immunity, the way in which protein components of the hemostasis pathway influence macrophages remains unclear. We investigated the effect of thrombin on macrophage polarization. On the basis of gene expression and cytokine secretion, our results suggest that polarization with thrombin induces an anti-inflammatory, M2-like phenotype. In functional studies, thrombin polarization promoted oxLDL phagocytosis by macrophages, and conditioned medium from the same cells increased endothelial cell proliferation. There were, however, clear differences between the classical M2a polarization and the effects of thrombin on gene expression. Finally, the deletion and inactivation of secreted modular Ca 2+ -binding protein 1 (SMOC1) attenuated phagocytosis by thrombin-stimulated macrophages, a phenomenon revered by the addition of recombinant SMOC1. Manipulation of SMOC1 levels also had a pronounced impact on the expression of TGF-β-signaling-related genes. Taken together, our results show that thrombin induces an anti-inflammatory macrophage phenotype with similarities as well as differences to the classical alternatively activated M2 polarization states, highlighting the importance of tissue levels of SMOC1 in modifying thrombin-induced macrophage polarization.

Secreted modular calcium-binding protein 1 (SMOC1) is an osteonectin/SPARC-related matricellular protein, whose expression is regulated by microRNA-223 (miR-223). Given that platelets are rich in miR-223, this study investigated the expression of SMOC1 and its contribution to platelet function. Human and murine platelets expressed SMOC1, whereas platelets from SMOC1+/- mice did not present detectable mature SMOC1 protein. Platelets from SMOC1+/- mice demonstrated attenuated responsiveness to thrombin (platelet neutrophil aggregate formation, aggregation, clot formation, Ca2+ increase, and β3 integrin phosphorylation), whereas responses to other platelet agonists were unaffected. SMOC1 has been implicated in transforming growth factor-β signaling, but no link to this pathway was detected in platelets. Rather, the SMOC1 Kazal domain directly bound thrombin to potentiate its activity in vitro, as well as its actions on isolated platelets. The latter effects were prevented by monoclonal antibodies against SMOC1. Platelets from miR-223-deficient mice expressed high levels of SMOC1 and exhibited hyperreactivity to thrombin that was also reversed by preincubation with monoclonal antibodies against SMOC1. Similarly, SMOC1 levels were markedly upregulated in platelets from individuals with type 2 diabetes, and the SMOC1 antibody abrogated platelet hyperresponsiveness to thrombin. Taken together, we have identified SMOC1 as a novel thrombin-activating protein that makes a significant contribution to the pathophysiological changes in platelet function associated with type 2 diabetes. Thus, strategies that target SMOC1 or its interaction with thrombin may be attractive therapeutic approaches to normalize platelet function in diabetes., (© 2021 by The American Society of Hematology.)

Background: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H 2 S n ), that exert their biological actions via cysteine S -sulfhydration of target proteins. This study set out to map the " S -sulfhydrome" (ie, the spectrum of proteins targeted by H 2 S n ) in human endothelial cells., Methods: Liquid chromatography with tandem mass spectrometry was used to identify S -sulfhydrated cysteines in endothelial cell proteins and β3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction., Results: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H 2 S n donor, SG1002. The endothelial cell " S -sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on β3 integrin in detail we found that S -sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin S -sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S -sulfhydration impaired interactions between β3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H 2 S n generation, impaired flow-induced dilatation, and failure to detect β3 integrin S -sulfhydration, all of which were rescued after the administration of an H 2 S n supplement., Conclusions: Vascular disease is associated with marked changes in the S -sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H 2 S n supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

The AMP-activated protein kinase (AMPK) is an energy sensing kinase that is activated by a drop in cellular ATP levels. Although several studies have addressed the role of the AMPKα1 subunit in monocytes and macrophages, little is known about the α2 subunit. The aim of this study was to assess the consequences of AMPKα2 deletion on protein expression in monocytes/macrophages, as well as on atherogenesis. A proteomics approach was applied to bone marrow derived monocytes from wild-type mice versus mice specifically lacking AMPKα2 in myeloid cells (AMPKα2 ∆MC mice). This revealed differentially expressed proteins, including methyltransferases. Indeed, AMPKα2 deletion in macrophages increased the ratio of S-adenosyl methionine to S-adenosyl homocysteine and increased global DNA cytosine methylation. Also, methylation of the vascular endothelial growth factor and matrix metalloproteinase-9 (MMP9) genes was increased in macrophages from AMPKα2 ∆MC mice, and correlated with their decreased expression. To link these findings with an in vivo phenotype, AMPKα2 ∆MC mice were crossed onto the ApoE -/- background and fed a western diet. ApoExAMPKα2 ∆MC mice developed smaller atherosclerotic plaques than their ApoExα2 fl/fl littermates, that contained fewer macrophages and less MMP9 than plaques from ApoExα2 fl/fl littermates. These results indicate that the AMPKα2 subunit in myeloid cells influences DNA methylation and thus protein expression and contributes to the development of atherosclerotic plaques.