Accumulation of lipid-laden foam cells in the arterial wall plays a central role in atherosclerotic lesion development, plaque progression, and late-stage complications of atherosclerosis. However, there are still fundamental gaps in our knowledge of the underlying mechanisms leading to foam cell formation in atherosclerotic arteries. Here, we investigated the role of receptor-independent macropinocytosis in arterial lipid accumulation and pathogenesis of atherosclerosis. Genetic inhibition of fluid-phase macropinocytosis in myeloid cells ( LysMCre ⁺ Nhe1 ^fl/fl ) and repurposing of a Food and Drug Administration (FDA)–approved drug that inhibits macrophage macropinocytosis substantially decreased atherosclerotic lesion development in low-density lipoprotein (LDL) receptor–deficient and Apoe ^−/− mice. Stimulation of macropinocytosis using genetic ( H-RAS ^G12V ) and physiologically relevant approaches promoted internalization of unmodified native (nLDL) and modified [e.g., acetylated (ac) and oxidized (ox) LDL] lipoproteins in both wild-type and scavenger receptor (SR) knockout ( Cd36 ^−/− / Sra ^−/− ) macrophages. Pharmacological inhibition of macropinocytosis in hypercholesterolemic wild-type and Cd36 ^−/− / Sra ^−/− mice identified an important role of macropinocytosis in LDL uptake by lesional macrophages and development of atherosclerosis. Furthermore, serial section high-resolution imaging, LDL immunolabeling, and three-dimensional (3D) reconstruction of subendothelial foam cells provide visual evidence of lipid macropinocytosis in both human and murine atherosclerotic arteries. Our findings complement the SR paradigm of atherosclerosis and identify a therapeutic strategy to counter the development of atherosclerosis and cardiovascular disease.

Impaired lymphatic drainage of the arterial wall results in intimal lipid accumulation and atherosclerosis. However, the mechanisms regulating lymphangiogenesis in atherosclerotic arteries are not well understood. Our studies identified elevated levels of matrix protein R-spondin 2 (RSPO2) in atherosclerotic arteries. In this study, we investigated the role of RSPO2 in lymphangiogenesis, arterial cholesterol efflux into lesion-draining lymph nodes (LNs) and development of atherosclerosis. The effect of RSPO2 on lymphangiogenesis was investigated using human lymphatic endothelial cells (LEC) in vitro and implanted Matrigel plugs in vivo. Cellular and molecular approaches, pharmacological agents, and siRNA silencing of RSPO2 receptor LGR4 were used to investigate RSPO2-mediated signalling in LEC. In vivo low-density lipoprotein (LDL) tracking and perivascular blockade of RSPO2–LGR4 signalling using LGR4-extracellular domain (ECD) pluronic gel in hypercholesterolemic mice were utilized to investigate the role of RSPO2 in arterial reverse cholesterol transport and atherosclerosis. Immunoblotting and imaging experiments demonstrated increased RSPO2 expression in human and mouse atherosclerotic arteries compared to non-atherosclerotic controls. RSPO2 treatment inhibited lymphangiogenesis both in vitro and in vivo. LGR4 silencing and inhibition of RSPO2–LGR4 signalling abrogated RSPO2-induced inhibition of lymphangiogenesis. Mechanistically, we found that RSPO2 suppresses PI3K-AKT-endothelial nitric oxide synthase (eNOS) signalling via LGR4 and inhibits activation of the canonical Wnt-β-catenin pathway. ApoE^−/− mice treated with LGR4-ECD developed significantly less atherosclerosis compared with control treatment. Finally, increased arterial lymphatic vessel density and improved lymphatic drainage of fluorescently labelled LDL to deep cervical LNs were observed in LGR4-ECD-treated mice. These findings demonstrate that RSPO2 inhibits lymphangiogenesis via LGR4 and downstream impairment of AKT-eNOS-nitric oxide signalling. These results may also inform new therapeutic strategies to promote lymphangiogenesis and improve cholesterol efflux from atherosclerotic arteries.

Aims: Internalization of extracellular fluid and its solute by macropinocytosis requires dynamic reorganization of actin cytoskeleton, membrane ruffling, and formation of large endocytic vacuolar compartments, called macropinosomes, inside the cell. Although instigators of macropinocytosis, such as growth factors and phorbol esters, stimulate NADPH oxidase (Nox) activation and signal transduction mediators upstream of Nox assembly, including Rac1 and protein kinase C (PKC), are involved in macropinocytosis, the role of Nox enzymes in macropinocytosis has never been investigated. This study was designed to examine the role of Nox2 and the potential downstream redox signaling involved in macropinocytosis. Results: Phorbol myristate acetate activation of human and murine macrophages stimulated membrane ruffling, macropinosome formation, and subsequent uptake of macromolecules by macropinocytosis. Mechanistically, we found that pharmacological blockade of PKC, transcriptional knockdown of Nox2, and scavenging of intracellular superoxide anion abolished phorbol ester-induced macropinocytosis. We observed that Nox2-derived reactive oxygen species via inhibition of phosphatase and tensin homolog and activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway lead to activation of actin-binding protein cofilin, membrane ruffling, and macropinocytosis. Similarly, activation of macropinocytosis by macrophage colony-stimulating factor involves Nox2-mediated cofilin activation. Furthermore, peritoneal chimera experiments indicate that macropinocytotic uptake of lipids in hypercholesterolemic ApoE ^−/− mice was attenuated in Nox2 ^y/− macrophages compared with wild-type controls. Innovation and Conclusion: In summary, these findings demonstrate a novel Nox2-mediated mechanism of solute uptake via macropinocytosis, with broad implications for both general cellular physiology and pathological processes. The redox mechanism described here may also identify new targets in atherosclerosis and other disease conditions involving macropinocytosis. Antioxid. Redox Signal . 26, 902–916.

Background: Preeclampsia (PE) is the primary cause of perinatal maternal-fetal mortality and morbidity. The exact molecular mechanisms of PE pathogenesis are largely unknown. This study aims to identify the hub genes in PE and explore their potential molecular regulatory network.Methods: We downloaded the GSE148241, GSE190971, GSE74341, and GSE114691 datasets for the placenta and performed a differential expression analysis to identify hub genes. We performed Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO), Gene Set Enrichment Analysis (GSEA), and Protein–Protein Interaction (PPI) Analysis to determine functional roles and regulatory networks of differentially expressed genes (DEGs). We then verified the DEGs at transcriptional and translational levels by analyzing the GSE44711 and GSE177049 datasets and our clinical samples, respectively.Results: We identified 60 DEGs in the discovery phase, consisting of 7 downregulated genes and 53 upregulated genes. We then identified seven hub genes using Cytoscape software. In the verification phase, 4 and 3 of the seven genes exhibited the same variation patterns at the transcriptional level in the GSE44711 and GSE177049 datasets, respectively. Validation of our clinical samples showed that CADM3 has the best discriminative performance for predicting PEConclusion: These findings may enhance the understanding of PE and provide new insight into identifying potential therapeutic targets for PE.

Arterial accumulation of plasma-derived LDL and its subsequent oxidation contributes to atherosclerosis. Lymphatic vessel (LV)-mediated removal of arterial cholesterol has been shown to reduce atherosclerotic lesion formation. However, the precise mechanisms that regulate LV density and function in atherosclerotic vessels remain to be identified. The aim of this study was to investigate the role of native LDL (nLDL) and oxidized LDL (oxLDL) in modulating lymphangiogenesis and underlying molecular mechanisms. Western blotting and immunostaining experiments demonstrated increased oxLDL expression in human atherosclerotic arteries. Furthermore, elevated oxLDL levels were detected in the adventitial layer, where LV are primarily present. Treatment of human lymphatic endothelial cells (LEC) with oxLDL inhibited in vitro tube formation, while nLDL stimulated it. Similar results were observed with Matrigel plug assay in vivo. CD36 deletion in mice and its siRNA-mediated knockdown in LEC prevented oxLDL-induced inhibition of lymphangiogenesis. In addition, oxLDL via CD36 receptor suppressed cell cycle, downregulated AKT and eNOS expression, and increased levels of p27 in LEC. Collectively, these results indicate that oxLDL inhibits lymphangiogenesis via CD36-mediated regulation of AKT/eNOS pathway and cell cycle. These findings suggest that therapeutic blockade of LEC CD36 may promote arterial lymphangiogenesis, leading to increased cholesterol removal from the arterial wall and reduced atherosclerosis.