Fig 2: FPR1-Gαi/Gαq signaling mediated FAM3D-facilitated intestinal epithelial MTTP upregulation and VSMC dedifferentiation(A–C) NanoBiT-G-protein dissociation assay of HEK293T cells overexpressing FPR1 with Gαi-Lg, Sm-Gβ5, and Gγ2 for NanoBiT-Gαi (A); Gαq-Lg, Sm-Gβ1, Gγ2, and RIC8A for NanoBiT-Gαq (B); or Gα13-Lg, Sm-Gβ1, and Gγ2 for NanoBiT-Gα13 (C). Transfected cells were stimulated with an increasing amount of FAM3D or fMLP for 3 min. n = 3.(D and E) BRET assays of HEK293T cells overexpressing FPR1-YFP and β-arrestin1-Rluc (D) or β-arrestin2-Rluc (E). The transfected cells were stimulated with an increasing amount of FAM3D or fMLP for 5 min. n = 3.(F) Schematic illustration of the experimental design for evaluating the role of Gαi/Gαq signaling in FAM3D-facilitated intestinal lipid absorption and VSMC dedifferentiation in Caco-2 cells and VSMCs, respectively. PTX, Gαi signaling inhibitor; YM254890, Gαq signaling inhibitor.(G) Representative images of western blotting and quantitative analysis of MTTP protein expression in Caco-2 cells infected with either Ad-FAM3D (1 × 108 PFU/mL) or Ad-GFP (1 × 108 PFU/mL) for 48 h, followed by treatment with or without PTX (10 ng/mL) or YM254890 (1 μM) for an additional 24 h. β-Actin was used as a loading control. n = 4; two-way ANOVA followed by Tukey’s test; ∗p < 0.05.(H) Quantitative analysis of MTTP activity in Caco-2 cells infected with either Ad-FAM3D (1 × 108 PFU/mL) or Ad-GFP (1 × 108 PFU/mL) for 48 h, followed by treatment with PTX (5 ng/mL) or YM254890 (0.5 μM) for an additional 24 h. n = 4; two-way ANOVA followed by Tukey’s test; ∗p < 0.05.(I) Representative images of western blotting and quantitative analysis of rat primary VSMC contractile protein (α-actin, calponin, and SM22α) expression after serum starvation for 12 h followed by stimulation with FAM3D (100 nM) for 24 h in the presence or absence of 2-h pretreatment with PTX (10 ng/mL) or YM254890 (1 μM). GAPDH was used as a loading control. n = 4; two-way ANOVA followed by Tukey’s test; ∗p < 0.05.(J) CCK-8 proliferation assays of rat primary VSMCs subjected to serum starvation for 12 h followed by stimulation with FAM3D (100 nM) for 24 h in the presence or absence of 2-h pretreatment with PTX (10 ng/mL) or YM254890 (1 μM). n = 4; two-way ANOVA followed by Tukey’s test; ∗p < 0.05.(K) Representative images of Transwell migration assays and quantitative analysis of migration in rat primary VSMCs subjected to serum starvation for 12 h followed by stimulation with FAM3D (100 nM) for 6 h in the presence or absence of PTX (10 ng/mL) or YM254890 (1 μM). Scale bar, 200 μm. n = 4; two-way ANOVA followed by Tukey’s test; ∗p < 0.05.All error bars represent standard error of the mean.

Fig 3: The elevation of circulating FAM3D correlates with the incidence of atherosclerosis in humans(A) Multiple conditional logistic regression and Cox proportional hazards regression analysis of the correlation of plasma FAM3D levels with the prevalence (N = 1,589/40,324) and incidence (N = 3,549/42,284) of coronary atherosclerosis from the UK Biobank. (Data sourced from https://proteome-phenome-atlas.com/) OR, odds ratio; HR, hazard ratio; CI, confidence interval.(B) Schematic illustration of the experimental design for evaluating the association between baseline plasma FAM3D levels (2012) and subsequent incidence of enhanced cIMT (2018). cIMT, carotid intima-media thickness.(C) Multiple conditional logistic regression between baseline plasma FAM3D levels (2012) and the subsequent incidence of enhanced cIMT (2018). Red line, fitted curve; shaded area, 95% CIs.(D) Sigmoid function modeling of FAM3D’s predictive value. Red line, fitted curve; shaded area, 95% CIs; vertical line, experimental FAM3D detection values.

Fig 4: FAM3D upregulates MTTP expression in intestinal epithelial cells through FPR1(A) Schematic illustration of intestinal TC and TG uptake and transport. CE, cholesterol ester; TAG, triacylglycerol; MAG, monoacylglycerol; FA, fatty acid; NPC1L1, Niemann-Pick C1-like 1; SR-BI, scavenger receptor class B, type I; CD36, cluster of differentiation 36; ABCG5/8, ATP-binding cassette subfamily G members 5/8; ACAT2, acyl-CoA: acyltransferase 2; MAGT, monoacylglycerol acyltransferases; DGAT, diacylglycerol acyltransferases; MTTP, microsomal triglyceride transfer protein; CM, chylomicron; ER, endoplasmic reticulum.(B) RT-qPCR analysis of MTTP and ACAT2 mRNA expression in jejunal epithelial cells isolated from 8-week-old male WT and Fam3d−/− mice infected with AAV8-PCSK9D377Y (2 × 1011 vg/mouse) and maintained on an HFD feeding for 12 weeks. GAPDH was used as an internal control. n = 5–7; unpaired Student’s t test; ∗p < 0.05.(C) Representative images of western blotting and quantitative analysis in jejunal epithelial cells isolated from WT and Fam3d−/− mice suffering from atherosclerosis induction for 12 weeks. β-Actin was used as a loading control. n = 5; unpaired Student’s t test; ∗p < 0.05.(D) Representative images of western blotting and quantitative analysis in Caco-2 cells infected with either adenovirus overexpressing FAM3D (Ad-FAM3D, 1 × 108 plaque-forming unit [PFU]/mL) or GFP control (Ad-GFP, 1 × 108 PFU/mL) for 48 h. β-Actin was used as a loading control. n = 4; unpaired Student’s t test; ∗p < 0.05.(E) Quantitative analysis of MTTP activity in jejunal epithelial cells isolated from 8-week-old WT and Fam3d−/− mice suffering from atherosclerosis induction for 12 weeks. n = 6; unpaired Student’s t test; ∗p < 0.05.(F) Quantitative analysis of MTTP activity in Caco-2 cells infected with either Ad-FAM3D (1 × 108 PFU/mL) or Ad-GFP (1 × 108 PFU/mL) for 48 h. n = 9; unpaired Student’s t test; ∗p < 0.05.(G) Representative images of western blotting and quantitative analysis in Caco-2 cells infected with either Ad-FAM3D (1 × 108 PFU/mL) or Ad-GFP (1 × 108 PFU/mL) in the presence of CsH (1 μM) or WRW4 (1 μM) treatment for 48 h. β-Actin was used as a loading control. n = 4; two-way ANOVA followed by Tukey’s test; ∗p < 0.05.(H) Quantitative analysis of MTTP activity in Caco-2 cells infected with either Ad-FAM3D (1 × 108 PFU/mL) or Ad-GFP (1 × 108 PFU/mL) for 48 h, followed by treatment with CsH (0.5 μM) or WRW4 (0.5 μM) for an additional 24 h. n = 5; two-way ANOVA followed by Tukey’s test; ∗p < 0.05; ns, not significant.All error bars represent standard error of the mean.

Fig 5: Targeting FAM3D ameliorates atherosclerosis by lowering blood lipids and suppressing vascular remodeling(A) Schematic illustration of the experimental design for evaluating the therapeutic role of two FAM3D mAbs (642 and 643) in atherosclerosis. Eight-week-old male Ldlr−/− mice were maintained on an HFD for 6 weeks to establish atherosclerosis prior to FAM3D mAb treatment initiation. Mice then received weekly intraperitoneal injections of either control IgG or two FAM3D mAbs (10 mg/kg body weight, once a week) for 6 consecutive weeks while maintaining HFD feeding. i.p., intraperitoneal; qw, once a week.(B and C) Quantitative analysis of plasma TC (B) and TG (C) levels in Ldlr−/− mice. n = 8–11; one-way ANOVA followed by Tukey’s test; ∗p < 0.05; ns, 642 group vs. IgG group, not significant.(D and E) Quantitative analysis of TG (D) and TG (E) in plasma lipoprotein fractions (VLDL/CM, LDL, and HDL) separated by FPLC from Ldlr−/− mice, n = 5.(F) Representative images and quantitative analysis of en face oil red O staining in the whole aortas from Ldlr−/− mice. Scale bar, 5 mm. n = 8–11; one-way ANOVA followed by Tukey’s test; ∗p < 0.05.(G) Representative images and quantitative analysis of oil red O staining of cross-sections of the aortic roots from Ldlr−/− mice. Scale bar, 500 μm. n = 8–11; one-way ANOVA followed by Tukey’s test; ∗p < 0.05.(H) Quantitative analysis of fecal cholesterol (%) and FFA (μg/mg) in Ldlr−/− mice. n = 6; one-way ANOVA followed by Tukey’s test; ∗p < 0.05.(I) Quantitative analysis of plasma TG levels in Ldlr−/− mice. Mice were starved for 16 h and subsequently gavaged with olive oil (10 mL/kg body weight) to measure plasma TG at the indicated time points (0, 0.5, 1, 2, and 4 h). n = 5–6; one-way ANOVA followed by Tukey’s test; ∗p < 0.05.(J) Representative images of western blotting and quantitative analysis of MTTP protein expression in jejunal epithelial cells isolated from Ldlr−/− mice. β-Actin was used as a loading control. n = 5; one-way ANOVA followed by Tukey’s test; ∗p < 0.05.(K) Representative images and quantitative analysis of H&E-stained cross-sections of carotid arteries from 8-week-old C57BL6/J male mice treated with either control IgG or two FAM3D mAbs (10 mg/kg body weight, once per week) for 5 consecutive weeks and subjected to partial carotid artery ligation for 4 weeks. Scale bar, 100 μm. n = 8; two-way ANOVA followed by Tukey’s test; ∗p < 0.05; ns, not significant.All error bars represent standard error of the mean.