Abstract Body: Based on a genome-wide CRISPR/Cas9 knockout (KO) screen, we previously identified transgelin (Sm22) as a novel gene involved in hepatic LDL endocytosis. Sm22-KO hepatocytes showed reduced LDL uptake due to slower LDL receptor (LDLR) internalization. Furthermore, Sm22-KO mice presented with modestly increased plasma LDL and a disproportionately augmented atherosclerosis. Sm22 is highly expressed in vascular smooth muscle cells (VSMC) at the arterial wall and is used as a VSMC marker. We hypothesize Sm22 may play a role in atherogenesis independently of hyperlipidemia. Aim: To study whether Sm22 modulates atherogenesis beyond its effects on LDL metabolism. Methods: 16 Sm22/Ldlr-double KO (DKO) (8 females/8 males) and 16 Ldlr-KO (8 females/8 males) were fed with high fat diet (42% cal. from fat) for 12 weeks. Weight and plasma lipids were monitored before and throughout the study. At the end, we collected blood for lipid measurements and aortas for in-situ atherosclerotic plaque assessment and gene expression analysis. Results: No changes in final body weight were observed between DKO and Ldlr-KO (p>0.12). Additionally, plasma total cholesterol (TC) and triglycerides (TG) showed no differences between DKO and Ldlr-KO mice throughout the study (p>0.21) with higher levels in males compared to females (p<0.01). Notably, DKO mice showed a 50% increase in atherosclerotic plaque area (p=0.007). Remarkably, when considering sex as a variable, DKO male mice showed an 81% increase in atherosclerosis compared to Ldlr-KO males (p=0.0001), but no correlation with TC or TG was observed in male mice (r=0.16, p=0.58 and r=0.01, p=0.98, respectively). In contrast, no changes in atherosclerosis were observed in females (p=0.51). Gene expression analysis in plaque-free arterial tissue from male mice revealed that DKO mice had a 40% drop in the expression of myosin heavy chain 11 (Myh11) (p=0.03), a marker of contractile VSMC. Conclusions: Besides being a VSMC marker, our data suggest that Sm22 plays a key role in atherogenesis independently of plasma lipids. Our data indicate that males may be more susceptible, or females more resistant, to Sm22-driven atherosclerosis, likely due to the influence of sex hormones. In males, Sm22 deficiency may be linked to a less differentiated VSMC phenotype. Understanding the sex-associated differences will require further investigation; however, our data highlight the importance of including male and female mice in atherosclerosis studies.