Macrophage Metabolic Pathways and Cellular Crosstalk in Atherosclerosis Regression

In human atherosclerosis, a positive-feedback cycle in lesional macrophages (Ms) involving efferocytosis (clearance of apoptotic cells [ACs]) and inflammation resolution is impaired, promoting clinically dangerous "unstable" plaques with thin fibrous caps. When LDL is lowered, however, the cycle is "reawakened", plaques stabilize ("regress"), and CAD risk is lowered, but the mechanisms remain largely unknown. Based on data from our lab and others, our overarching hypothesis is that resolution pathways in regressing lesional Ms are acti- vated by efferocytosis-induced metabolic changes, promoting cap thickening/plaque stabilization by cell-auton- omous pathways and by crosstalk with collagen-producing stromal cells involved in cap formation (capSCs). This hypothesis is directly linked to the central PPG theme on M-cell autonomous and M-stromal cell crosstalk mechanisms that affect plaque stability, and our approach is highly dependent on our collaborations with Drs. Tall and Zhang, the Cores, and shared models. We will explore integrated, efferocytosis-induced pathways that promote cap thickening in regression and are functional in human Ms. Aim 1 explores how AC-methionine drives DNA-methyltransferase 3A (DNMT3A) to methylate/repress Dusp4, which enhances pro-resolving signal- ing and further amplifies efferocytosis. DNMT3A loss-of-function (LoF) variants are a common cause of clonal hematopoiesis (CH), but mechanisms are poorly understood. Our published and preliminary studies suggest a new mechanism. Aim 2 explores how AC-nucleotides drive efferocytosis-induced M proliferation (EIMP), which expands the pool of resolving Ms to promote regression. EIMP requires another pathway in which efferocytosis induces lactate (EIL). Aim 1 will explore 3 hypotheses related to the DNMT3A pathway: (1) methylation/repres- sion of Dusp4 by DNMT3A is a key step in resolution in effero-Ms in vitro and in athero-regression (Tall/Zhang); (2) DNMT3A LoF impairs athero-regression in DNMT3A-CH mice and correlates with unstable plaques in hu- mans with DNMT3A-CH (Tall/Zhang/Core C). We will also test a mechanism-based nanotherapy in DNMT3A- CH mice using lesion-targeted pro-resolving nanoparticles; and (3) crosstalk between effero-Ms undergoing the DNMT3A pathway and smooth muscle cell-derived cells (SDCs) promotes regression by enhancing SDC con- version to fibromyocytes (FbMs) to promote cap thickening (Tall/Zhang/Reilly/Core B). Aim 2 will investigate the following hypotheses related to EIMP/EIL: (1) EIMP expands the pool of TGF-producing Ms, which pro- motes cap thickening by enhancing SDC-to-FbM conversion, and EIMP is impaired in unstable vs. stable human plaques (Tall/Zhang/Reilly/Cores B/C); (2) the EIMP and DNMT3A pathways are linked, as the DNMT3A path- way enhances EIMP via ERK (Zhang); and (3) EIL promotes capSCs to stabilize plaques by enabling EIMP and possibly through a direct effect of lactate on SDC-to-FbM conversion, and EIL is associated with plaque stability in humans (Tall/Zhang/Reilly/Core C). We reason that studying these pathways in the overall PPG framework will provide new insights into mechanisms of plaque stabilization and how this insight may inform new therapies.