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.

Aims/hypothesis Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are two peptides that function to promote insulin secretion. Dipeptidyl peptidase-4 (DPP-4) inhibitors increase the bioavailability of both GLP-1 and GIP but the dogma continues to be that it is the increase in GLP-1 that contributes to the improved glucose homeostasis. We have previously demonstrated that pancreatic rather than intestinal GLP-1 is necessary for improvements in glucose homeostasis in mice. Therefore, we hypothesise that a combination of pancreatic GLP-1 and GIP is necessary for the full effect of DPP-4 inhibitors on glucose homeostasis. Methods We have genetically engineered mouse lines in which the preproglucagon gene (Gcg) is absent in the entire body (GcgRA^ΔNull) or is expressed exclusively in the intestine (GcgRA^ΔVilCre) or pancreas and duodenum (GcgRA^ΔPDX1Cre). These mice were used to examine oral glucose tolerance and GLP-1 and GIP responses to a DPP-4 inhibitor alone, or in combination with incretin receptor antagonists. Results Administration of the DPP-4 inhibitor, linagliptin, improved glucose tolerance in GcgRA^ΔNull mice and control littermates and in GcgRA^ΔVilCre and GcgRA^ΔPDX1Cre mice. The potent GLP-1 receptor antagonist, exendin-[9–39] (Ex9), blunted improvements in glucose tolerance in linagliptin-treated control mice and in GcgRA^ΔPDX1Cre mice. Ex9 had no effect on glucose tolerance in linagliptin-treated GcgRA^ΔNull or in GcgRA^ΔVilCre mice. In addition to GLP-1, linagliptin also increased postprandial plasma levels of GIP to a similar degree in all genotypes. When linagliptin was co-administered with a GIP-antagonising antibody, the impact of linagliptin was partially blunted in wild-type mice and was fully blocked in GcgRA^ΔNull mice. Conclusions/interpretation Taken together, these data suggest that increases in pancreatic GLP-1 and GIP are necessary for the full effect of DPP-4 inhibitors on glucose tolerance.

Anja Jaeschke, April Haller, James G. Cash, Christopher Nam, Emily Igel, Anton J.M. Roebroek, David Y. Hui

Elevated cholesterol poses cardiovascular risks. The glucocorticoid receptor (GR) harbors a still undefined role in cholesterol regulation. Here, we report that a coding SNP in the gene encoding the GR, rs6190 , is associated with increased cholesterol in women according to UK Biobank and All of Us (NIH) datasets. In SNP-genocopying mice, we found that the SNP enhanced hepatic GR activity to transactivate Pcsk9 and Bhlhe40 , negative regulators of LDL and HDL receptors, respectively. In mice, the SNP was sufficient to elevate circulating cholesterol across all lipoprotein fractions and the risk and severity of atherosclerotic lesions on the proatherogenic hAPOE * 2/ * 2 background. The SNP effect on atherosclerosis was blocked by in vivo liver knockdown of Pcsk9 and Bhlhe40 . Also, corticosterone and testosterone were protective against the mutant GR program in cholesterol and atherosclerosis in male mice, while the SNP effect was additive to estrogen loss in females. Remarkably, we found that the mutant GR program was conserved in human hepatocyte-like cells using CRISPR-engineered, SNP-genocopying human induced pluripotent stem cells. Taken together, our study leverages a nonrare human variant to uncover a GR-dependent mechanism contributing to atherogenic risk, particularly in women.

Phospholipase A₂ (PLA₂) enzymes influence inflammatory bowel disease in both positive and negative manners depending on the type of PLA₂ that is expressed. This study explored the influence of the abundantly expressed Group 1B PLA₂ (PLA2G1B) on ulcerative colitis. Wild-type C57BL/6J mice and Pla2g1b^−/− mice were treated with dextran sulfate sodium (DSS) for 5 days to induce epithelial injury, followed by another 5 days without DSS for recovery. The Pla2g1b^−/− mice displayed significantly less body weight loss, colitis pathology, and disease activity indexes compared to the wild-type mice. The differences in colitis were not due to differences in the colonic lysophospholipid levels, but higher numbers of stem and progenitor cells were found in the intestines of Pla2g1b^−/− mice compared to the wild-type mice. The DSS-treated Pla2g1b^−/− mice also showed higher expressions of genes that are responsible for epithelial repair and lower expressions of proinflammatory cytokine genes in the colon, as well as reduced inflammatory cytokine levels in the plasma. In vitro experiments revealed the PLA2G1B stimulation of inflammatory cytokine expression by myeloid cells. PLA2G1B inactivation protects against DSS-induced colitis in mice by increasing the intestinal stem cell reservoir for epithelial repair and reducing myeloid cell inflammation in the diseased colon. Thus, PLA2G1B may be a target for colitis management.

The human APOE gene is polymorphic with three major alleles that encode apolipoprotein (apo) E2, apoE3, and apoE4. Both apoE2 and apoE4 are associated with increased atherosclerosis risk. This study utilized human APOE2, APOE3, and APOE4 gene replacement mice and single-cell RNA sequencing approach to delineate the mechanisms underlying this association. The human APOE2, APOE3, and APOE4 mice were fed a Western-type high fat–cholesterol diet for 16 weeks. Hyperlipidemia and significant atherosclerosis were observed in APOE2 mice but not in APOE3 or APOE4 mice. However, increased vascular inflammation was observed in both APOE2 and APOE4 mice. Single-cell RNA sequencing followed by cluster analysis identified 25 major cell types in the aorta that include various immune cell types, endothelial cells, and various vascular mural cell subsets. Results showed that cells from the APOE2 mice were enriched with genes associated with intracellular lipid accumulation and inflammation, whereas cells from the APOE4 mice displayed elevated oxidative- and/or endoplasmic reticulum-stress and inflammatory response. Thus, apoE2 accelerates atherosclerosis by inducing diet-induced hyperlipidemia and inflammation, while apoE4 does not induce hyperlipidemia but enhances inflammation that may prime the vasculature for atherosclerosis development. The distinct mechanisms by which apoE2 and apoE4 promote atherosclerosis underscore the importance of including apoE genotype information in the design of therapeutics for atherosclerosis intervention.