Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation.

Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche.

Glycolytic side pathways regulating macrophage inflammatory phenotypes and functions.

Macrophages are crucial effector cells of the innate immune system and have important roles in the initiation and resolution of inflammation as well as in tissue homeostasis. To fulfill these diverse roles, macrophages exhibit metabolic flexibility to quickly adapt to the needs of the effector functions required, as well as to the microenvironment. This metabolic flexibility is exemplified by proinflammatory macrophages, which upregulate glycolysis to both initiate and sustain the process of inflammation. Upregulation of glycolysis does not only represent a fast means of ATP generation. It also fuels glycolytic side pathways that are crucial for an effective inflammatory response by influencing the cell's redox balance as well as by providing building blocks and substrates for epigenetic reprogramming. The aim of this short review is to explore how three of these pathways - the pentose phosphate pathway, the glycerol phosphate shuttle, and the serine synthesis pathway - help macrophages sustain their proinflammatory phenotype and functions.

Cardiotoxicity of cyclophosphamide's metabolites: an in vitro metabolomics approach in AC16 human cardiomyocytes.

Cyclophosphamide is a widely used anticancer and immunosuppressive prodrug that unfortunately causes severe adverse effects, including cardiotoxicity. Although the exact cardiotoxic mechanisms are not completely understood, a link between cyclophosphamide's pharmacologically active metabolites, namely 4-hydroxycyclophosphamide and acrolein, and the toxicity observed after the administration of high doses of the prodrug is likely. Therefore, the objective of this study is to shed light on the cardiotoxic mechanisms of cyclophosphamide and its main biotransformation products, through classic and metabolomics studies. Human cardiac proliferative and differentiated AC16 cells were exposed to several concentrations of the three compounds, determining their basic cytotoxic profile and preparing the next study, using subtoxic and toxic concentrations for morphological and biochemical studies. Finally, metabolomics studies were applied to cardiac cells exposed to subtoxic concentrations of the aforementioned compounds to determine early markers of damage. The cytotoxicity, morphological and biochemical assays showed that 4-hydroxycyclophosphamide and acrolein induced marked cardiotoxicity at µM concentrations (lower than 5 µM), being significantly lower than the ones observed for cyclophosphamide (higher than 2500 µM). Acrolein led to increased levels of ATP and total glutathione on proliferative cells at 25 µM, while no meaningful changes were observed in differentiated cells. Higher levels of carbohydrates and decreased levels of fatty acids and monoacylglycerols indicated a metabolic cardiac shift after exposure to cyclophosphamide's metabolites, as well as a compromise of precursor amino acids used in the synthesis of glutathione, seen in proliferative cells' metabolome. Overall, differences in cytotoxic mechanisms were observed for the two different cellular states used and for the three molecules, which should be taken into consideration in the study of cyclophosphamide cardiotoxic mechanisms.

The Secretome of Human Neonatal Mesenchymal Stem Cells Modulates Doxorubicin-Induced Cytotoxicity: Impact in Non-Tumor Cells.

Doxorubicin (Dox) is one of the most widely used treatments for breast cancer, although limited by the well-documented cardiotoxicity and other off-target effects. Mesenchymal stem cell (MSC) secretome has shown immunomodulatory and regenerative properties, further potentiated under 3D conditions. This work aimed to uncover the effect of the MSC-derived secretome from 3D (CM3D) or 2D (CM2D) cultures, in human malignant breast cells (MDA-MB-231), non-tumor breast epithelial cells (MCF10A) and differentiated AC16 cardiomyocytes, co-treated with Dox. A comprehensive proteomic analysis of CM3D/CM2D was also performed to unravel the underlying mechanism. CM3D/CM2D co-incubation with Dox revealed no significant differences in MDA-MB-231 viability when compared to Dox alone, whereas MCF10A and AC16 viability was consistently improved in Dox+CM3D-treated cells. Moreover, neither CM2D nor CM3D affected Dox anti-migratory and anti-invasive effects in MDA-MB-231. Notably, Ge-LC-MS/MS proteomic analysis revealed that CM3D displayed protective features that might be linked to the regulation of cell proliferation (CAPN1, CST1, LAMC2, RANBP3), migration (CCN3, MMP8, PDCD5), invasion (TIMP1/2), oxidative stress (COX6B1, AIFM1, CD9, GSR) and inflammation (CCN3, ANXA5, CDH13, GDF15). Overall, CM3D decreased Dox-induced cytotoxicity in non-tumor cells, without compromising Dox chemotherapeutic profile in malignant cells, suggesting its potential use as a chemotherapy adjuvant to reduce off-target side effects.