Assessing the impact of an antigen-specific antibody response on atherosclerosis development in mice.

The antibody immune response plays a critical role in atherosclerosis. Here, we present a protocol for assessing the impact of an antigen-specific germinal center antibody response on atherosclerosis development, using a pro-atherogenic mouse model deficient for the production of germinal-center-derived antibodies. We describe steps for bone marrow transfer from donor mice into irradiated recipient mice. We then detail immunization of mouse chimeras with atheroprotective malondialdehyde low-density lipoprotein during high-fat diet feeding and atherosclerosis burden analysis. For complete details on the use and execution of this protocol, please refer to Martos-Folgado et al. (2022).1.

MDA-LDL vaccination induces athero-protective germinal-center-derived antibody responses.

Atherosclerosis is a chronic inflammatory disease of the arteries that can lead to thrombosis, infarction, and stroke and is the leading cause of mortality worldwide. Immunization of pro-atherogenic mice with malondialdehyde-modified low-density lipoprotein (MDA-LDL) neo-antigen is athero-protective. However, the immune response to MDA-LDL and the mechanisms responsible for this athero-protection are not completely understood. Here, we find that immunization of mice with MDA-LDL elicits memory B cells, plasma cells, and switched anti-MDA-LDL antibodies as well as clonal expansion and affinity maturation, indicating that MDA-LDL triggers a bona fide germinal center antibody response. Further, Prdm1fl/flAicda-Cre+/kiLdlr-/- pro-atherogenic chimeras, which lack germinal center-derived plasma cells, show accelerated atherosclerosis. Finally, we show that MDA-LDL immunization is not athero-protective in mice lacking germinal-center-derived plasma cells. Our findings give further support to the development of MDA-LDL-based vaccines for the prevention or treatment of atherosclerosis.

ALDH4A1 is an atherosclerosis auto-antigen targeted by protective antibodies.

Cardiovascular disease (CVD) is the leading cause of mortality in the world, with most CVD-related deaths resulting from myocardial infarction or stroke. The main underlying cause of thrombosis and cardiovascular events is atherosclerosis, an inflammatory disease that can remain asymptomatic for long periods. There is an urgent need for therapeutic and diagnostic options in this area. Atherosclerotic plaques contain autoantibodies1,2, and there is a connection between atherosclerosis and autoimmunity3. However, the immunogenic trigger and the effects of the autoantibody response during atherosclerosis are not well understood3-5. Here we performed high-throughput single-cell analysis of the atherosclerosis-associated antibody repertoire. Antibody gene sequencing of more than 1,700 B cells from atherogenic Ldlr-/- and control mice identified 56 antibodies expressed by in-vivo-expanded clones of B lymphocytes in the context of atherosclerosis. One-third of the expanded antibodies were reactive against atherosclerotic plaques, indicating that various antigens in the lesion can trigger antibody responses. Deep proteomics analysis identified ALDH4A1, a mitochondrial dehydrogenase involved in proline metabolism, as a target antigen of one of these autoantibodies, A12. ALDH4A1 distribution is altered during atherosclerosis, and circulating ALDH4A1 is increased in mice and humans with atherosclerosis, supporting the potential use of ALDH4A1 as a disease biomarker. Infusion of A12 antibodies into Ldlr-/- mice delayed plaque formation and reduced circulating free cholesterol and LDL, suggesting that anti-ALDH4A1 antibodies can protect against atherosclerosis progression and might have therapeutic potential in CVD.

Aging-Associated miR-217 Aggravates Atherosclerosis and Promotes Cardiovascular Dysfunction.

OBJECTIVE: microRNAs are master regulators of gene expression with essential roles in virtually all biological processes. miR-217 has been associated with aging and cellular senescence, but its role in vascular disease is not understood. Approach and Results: We have used an inducible endothelium-specific knock-in mouse model to address the role of miR-217 in vascular function and atherosclerosis. miR-217 reduced NO production and promoted endothelial dysfunction, increased blood pressure, and exacerbated atherosclerosis in proatherogenic apoE-/- mice. Moreover, increased endothelial miR-217 expression led to the development of coronary artery disease and altered left ventricular heart function, inducing diastolic and systolic dysfunction. Conversely, inhibition of endogenous vascular miR-217 in apoE-/- mice improved vascular contractility and diminished atherosclerosis. Transcriptome analysis revealed that miR-217 regulates an endothelial signaling hub and downregulates a network of eNOS (endothelial NO synthase) activators, including VEGF (vascular endothelial growth factor) and apelin receptor pathways, resulting in diminished eNOS expression. Further analysis revealed that human plasma miR-217 is a biomarker of vascular aging and cardiovascular risk. CONCLUSIONS: Our results highlight the therapeutic potential of miR-217 inhibitors in aging-related cardiovascular disease.