Blocking NNMT Slashes Atherosclerosis by Reshaping Macrophage NAD Metabolism

**Why this matters:** Macrophage accumulation in arterial plaques is a central driver of atherosclerosis, yet the metabolic signals that control lesional macrophage numbers remain incompletely understood. This study places NAD metabolism — long studied in aging and metabolic disease — at the heart of plaque macrophage biology, opening a potentially druggable pathway.

**What was studied:** The team began with a GWAS in the Hybrid Mouse Diversity Panel (HMDP) atherosclerosis model, which pointed to a chromosome 9 locus containing *Nnmt*. NNMT methylates nicotinamide (NAM) to produce N-methylnicotinamide, thereby removing NAM from the pool available for NAD synthesis via the salvage pathway. To test causality, the researchers used an Ionis ASO (50 mg/kg/week i.p.) in APOE-Leiden.CETP hyperlipidemic mice fed a Western diet for 16 weeks. They then dissected tissue-specific contributions using siRNA targeting liver/adipose, and hematopoietic contributions via bone marrow transplantation (BMT) from *Nnmt* knockout (KO) or CD38-deficient donors into irradiated hyperlipidemic recipients. Macrophage proliferation and apoptosis were quantified in vivo by EdU incorporation and TUNEL staining of aortic sections, and confirmed in cultured bone-marrow-derived macrophages (BMDMs) from heterozygous KO mice.

**Key results:** Global NNMT-ASO treatment reduced aortic root lesion area 5–10 fold in both male and female mice without significant changes in plasma lipid profiles, arguing against a lipid-mediated mechanism. Liver- and adipose-targeted siRNA knockdown — the tissues with highest NNMT expression — had little or no effect on lesion size, and feeding mice exogenous NAM likewise did not reduce atherosclerosis, ruling out systemic NAM elevation as the mediator. BMT from *Nnmt* KO donors significantly reduced lesional macrophage proliferation (EdU⁺/CD68⁺ cells), increased macrophage apoptosis (TUNEL⁺/CD68⁺ cells), and reduced plaque area compared with WT donors. Matching results were obtained with CD38-deficient BMT donors. In cultured BMDMs, heterozygous *Nnmt* KO cells showed elevated NAD/NADH ratios, reduced Ki-67 and EdU positivity, and increased TUNEL positivity relative to wild-type cells, directly linking higher intracellular NAD to the anti-proliferative, pro-apoptotic phenotype.

**Implications:** The findings establish a mechanistic chain: NNMT activity in macrophages depletes the NAM available for salvage NAD synthesis → lower NAD suppresses pro-apoptotic and anti-proliferative signaling → more macrophages accumulate in plaques → larger lesions. CD38 acts in parallel to consume NAD, and its inhibition recapitulates NNMT loss-of-function. This suggests that strategies raising macrophage NAD — whether through NNMT inhibition, CD38 inhibition, or NAD precursor supplementation targeted to macrophages — could reduce atherosclerosis burden independently of lipid lowering.

**Caveats:** All experiments were conducted in mice on a highly artificial hyperlipidemic transgenic background, and translation to human cardiovascular disease requires validation. The ASO used achieves only global (not cell-type-specific) knockdown, and full KO studies relied on heterozygous reductions in macrophages, suggesting the effect size in humans with naturally varying NNMT activity may be more modest. Long-term safety of systemic NNMT or CD38 inhibition — given their broad roles in methylation and NAD biology — has not been assessed.