Genetic or therapeutic neutralization of ALK1 reduces LDL transcytosis and atherosclerosis in mice - PubMed (original) (raw)

doi: 10.1038/s44161-023-00266-2. Epub 2023 May 11.

Hubertus Schleer 3, Hyojin Park 4 5, Erika Jang 6 7, Michael Boyer 1 2, Bo Tao 1 2, Ana Gamez-Mendez 1 2, Abhishek Singh 1 2, Ewa Folta-Stogniew 8, Xinbo Zhang 2 9, Lingfeng Qin 2, Xue Xiao 10, Lin Xu 10, Junhui Zhang 11, Xiaoyue Hu 12, Evanthia Pashos 13, George Tellides 14, Philip W Shaul 15, Warren L Lee 6 7, Carlos Fernandez-Hernando 2 9, Anne Eichmann 4 5, William C Sessa 16 17 18

Affiliations

Genetic or therapeutic neutralization of ALK1 reduces LDL transcytosis and atherosclerosis in mice

Sungwoon Lee et al. Nat Cardiovasc Res. 2023 May.

Abstract

Low-density lipoprotein (LDL) accumulation in the arterial wall contributes to atherosclerosis initiation and progression1. Activin A receptor-like type 1 (ACVRL1, called activin-like kinase receptor (ALK1)) is a recently identified receptor that mediates LDL entry and transcytosis in endothelial cells (ECs)2,3. However, the role of this pathway in vivo is not yet known. In the present study, we show that genetic deletion of ALK1 in arterial ECs of mice substantially limits LDL accumulation, macrophage infiltration and atherosclerosis without affecting cholesterol or triglyceride levels. Moreover, a selective monoclonal antibody binding ALK1 efficiently blocked LDL transcytosis, but not bone morphogenetic protein-9 (BMP9) signaling, dramatically reducing plaque formation in LDL receptor knockout mice fed a high-fat diet. Thus, our results demonstrate that blocking LDL transcytosis into the endothelium may be a promising therapeutic strategy that targets the initiating event of atherosclerotic cardiovascular disease.

© 2023. The Author(s).

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Conflict of interest statement

A pending patent has been filed (PCT/US2023/011339) from Genovac Antibody Discovery LLC and Yale University for mAbs used in the present study with H.S. and W.C.S. as coinventors. W.C.S. and E.P. are currently employees of Pfizer Inc. H.S. is currently an employee of Genovac Antibody Discovery LLC. All remaining authors declare no competing interests.

Figures

Fig. 1

Fig. 1. ALK1 expression is increased in human atherosclerotic arteries and ALK1 deletion prevents atherosclerosis via impaired LDL uptake.

a, ALK1 expression in arteries from a cohort of control and atherosclerotic (Athero.) patients. Cohorts contained 32 subjects in each group. b, Expression profile showing relative expression of ACVRL1, SCARB1 and LDLR in AC and PA regions. For the largest cluster 2, P = 9.346198 × 10−11. c,d, Representative images (c) and analysis (d) of whole aorta showing accumulation of neutral lipids by ORO staining of Alk1 f/f and Alk1 iΔaE mice injected with mPCSK9 virus and fed a WD for 12 weeks (n = 12 mice per group). Scale bar, 2 mm. P < 0.0001 for d. Values show mean ± s.e.m. e,f, Representative immunostaining (e) and analysis (f) of endogenous apoB content per CD31 length/section in the lesser curvature of Alk1 f/f Ldlr_−/_− and Alk1 iΔaEC Ldlr_−/_− mice fed a WD for 4 weeks. Three images were counted per mouse in six mice per group. Scale bar, 20 μm. P < 0.0001 for f. Values show mean ± s.e.m. g–i, Western blotting (g) analysis of apoB100 (h) and apoB48 (i) proteins in whole aorta lysates from Alk1 f/f Ldlr_−/_− and Alk1 iΔaEC Ldlr_−/_− mice fed a WD for 4 weeks (n = 3 mice per group). P = 0.0468 for h and P < 0.0001 for i, respectively. Values show mean ± s.e.m. j,k, Representative images (j) and analysis (k) of whole aorta showing accumulation of neutral lipids by ORO staining of Alk1 f/f;Ldlr_−/_− and Alk1 iΔaEC;Ldlr_−/_− mice fed a WD for 12 weeks (n = 11 mice per group). Scale bar, 2 mm. P < 0.0001 for k. Values show mean ± s.e.m. All P values were calculated by two-tailed, unpaired Student’s _t_-test. Source data

Fig. 2

Fig. 2. A selective, anti-ALK1 monoclonal antibody (mAb2) binds ALK1 and blocks LDL uptake and transcytosis but not BMP9 signaling.

a, Western blot showing mAb2 (20 μg ml−1) not blocking BMP9 (10 ng ml−1)-mediated SMAD 1/5 phosphorylation. The negative control is nonimmune IgG (20 μg ml−1) and the positive control to quench BMP9 signaling is ALK1-Fc (1 μg ml−1). The experiment was repeated three times. Ctrl, control. b, SPR analysis of binding of mAb2 to ALK1-Fc. RU, relative units. c,d, SPR analysis of binding of mAb2 to ALK1-Fc saturated with BMP9 (c) or BMP10 (d). Two consecutive injections were delivered to the Fc-ALK1 surface (n = 2). e, Analysis of DiI-LDL uptake into HUVECs incubated with increasing concentrations of IgG or mAb2 (n = 3). P < 0.0001. Values show mean ± s.e.m. f, Comparison of DiI-LDL uptake into either HUVECs treated with mAb2 or ALK1-silenced HUVECs treated with mAb2 (20 μg ml−1) (n = 6). P < 0.0001. Values show mean ± s.e.m. g, Relative DiI-LDL transcytosis events measured by TIRF microscopy between IgG- and mAb2-treated HCAECs. IgG or mAb2 was used at 20 μg ml−1. The experiment was repeated twice and the representative dots were combined from the two independent experiments. P = 0.0004. Values show mean ± s.e.m. h, DiI-LDL uptake into Ldlr_−/_− MLECs treated with the mAb2. IgG or mAb2 used at 20 μg ml−1 (n = 4). P < 0.0001. Values show mean ± s.e.m. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test for e and f and by two-tailed, unpaired Student’s _t_-test for g and h. Source data

Fig. 3

Fig. 3. Efficient inhibition of apoB accumulation and atherosclerosis progression by mAb2.

a,b, Representative images (a) and analysis of neutral lipids (b) by ORO staining in whole aorta. Scale bar, 2 mm. P < 0.0001. Values show mean ± s.e.m. c–e, Representative histological images (c) and analysis of neutral lipids by ORO in aortic roots (d) and BCAs (e). Scale bars, 500 μm for aortic root and 100 μm for BCA. P = 0.0025 for d and P = 0.0011 for e. Values show mean ± s.e.m. f,g, Confocal images (f) and analysis of apolipoprotein B (apoB) content per CD31 length per section (g) in the lesser curvature region of the aortic arch: red: apoB; green: CD31; blue: nuclei. Scale bar, 20 μm. P = 0.0004 for g. Values show mean ± s.e.m. h,i, Confocal images (h) and analysis of macrophage infiltration (i) in BCA sections: green: CD68; blue: nuclei. Scale bar, 100 μm. P = 0.0160 for i. Values show mean ± s.e.m. j–l, Representative histological images (j) and analysis for necrotic core (k) and collagen content (l) by trichrome staining in BCAs. Scale bar, 100 μm. P < 0.0001 for both k and l. All samples are from Ldlr_−/_− mice treated with PBS, IgG or mAb2 fed a WD for 12 weeks. Mice were injected with 250 μg of IgG or mAb2 twice weekly during the 12-week feeding period. Aortic root and BCAs were sectioned at 6 μm (n = 12 mice). Values show mean ± s.e.m. P values were calculated by two-way ANOVA with Tukey’s multiple comparisons test. Source data

Fig. 4

Fig. 4. A combination of mAb2 with dietary lipid lowering synergistically reduces plaque formation and mAb2 treatment alone inhibits atherosclerosis progression during sustained hyperlipidemia.

a,b, Plasma total cholesterol (a) and triglyceride (b) levels measured at endpoint of 16 weeks: 12 weeks of WD followed by 4 weeks of normal chow diet (n = 10 mice per group for IgG or mAb2 injected and n = 3 for diet alone). P < 0.0001 for both a and b. Values show mean ± s.e.m. c,d, Representative images (c) and analysis of whole aorta (d) showing accumulation of neutral lipids by ORO staining of Ldlr_−/_− mice injected with IgG or mAb2 and fed a WD for 12 weeks followed by normal diet for 4 weeks (n = 10 mice per group). Scale bar, 2 mm. P < 0.0001 for d. Values show mean ± s.e.m. e,f, Representative ORO-stained images (e) and quantification of neutral lipid content (f) of BCAs (n = 3 for PBS- and n = 6 for IgG- and mAb2-treated BCA). Scale bar, 100 μm. P < 0.0001 for f. Values show mean ± s.e.m. g,h, Plasma total cholesterol (g) and triglyceride (h) levels after 16 weeks of WD and, IgG or mAb2 was injected during the last 4 weeks (n = 12 mice per group). P = 0.4902 for g and P = 0.7228 for h. Values show mean ± s.e.m. i,j, Representative images (i) and analysis of whole aorta (j) showing accumulation of neutral lipids by ORO staining of Ldlr_−/_− mice fed a WD for 16 weeks and IgG or mAb2 injected during the last 4 weeks (n = 12 mice per group). Scale bar, 2 mm. P < 0.0001 for j. Values show mean ± s.e.m. k,l, Representative ORO-stained images (k) and quantification of neutral lipid content (l) of BCAs (n = 6). Scale bar, 100 μm. P < 0.0001 for j. Value shows mean ± s.e.m. All BCAs were sectioned at 6 μm. P values were calculated by two-way ANOVA with Tukey’s multiple comparisons test for a–f and by two-tailed, unpaired Student’s _t_-test for g–l. Source data

Extended Data Fig. 1

Extended Data Fig. 1. ALK1 expression is increased in atherosclerotic aorta ECs and BmxCre induces ALK1 deletion in arterial endothelial cells without changing right ventricle pressure.

a, Representative images of human left main artery specimens sections stained for H&E (Scale bar: 600μm) and for ALK1 with CD31 (Scale bar: 20μm). Specimens collected from individual patient was categorized into Non-Athero, Mild, Moderate, and Severe depending on the severity (thickness) of plaques. b-d, Representative confocal images (b), and analysis of ALK1 (c) and VE-cad intensity (d). Two images were counted per mouse in three mice per group for a total of n = 6. Scale bar: 20μm. P < 0.0001 for c and P = 0.0140 for d. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. Image J software was used to quantify the ALK1 and VE-cad intensity. e, Representative images of Alk1 fl/fl BmxCre ERT2 mTmG mouse adult retinal vessels, 4 months after tamoxifen injection. White: ALK1, green: GFP for mTmG, red: isolectin B4. Scale bar: 200μm. n = 3. f, Relative ALK1 expression from isolated aortic ECs of Alk1 f/f and Alk1 iΔaE mice, 4 months after tamoxifen injection. n = 5. P = 0.0032. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. g, Representative confocal images of Alk1 fl/fl BmxCre ERT2 mTmG mouse thoracic aorta versus jugular vein vessels, 4 months after tamoxifen injection. White: ALK1, green:GFP for mTmG, red: VE-cadherin. Scale bar: 20μm. n = 3. Data are mean ± s.e.m. P values were calculated by two-tailed unpaired t-test. h, Right Ventricular Systolic Pressure (RVSP) of male Alk1 f/f and Alk1 iΔaEC mice at 6 weeks after tamoxifen injection, showing no difference between control and ALK1 deleted group. n = 4 male mice per group. P = 0.5918. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. Source data

Extended Data Fig. 2

Extended Data Fig. 2. Arterial EC ALK1 deficiency reduces the atherosclerosis progression.

a, LDL receptor protein levels from livers of Alk1 f/f and Alk1 iΔaEC mice received AAV9-mPCSK9 and fed a Western diet for 12 weeks. n = 6 mice per group. b-d, Analysis of weight (b), plasma total cholesterol (c) and triglycerides (d) levels from Alk1 f/f and Alk1 iΔaEC mice received AAV9-mPCSK9 through retro-orbital sinus delivery and fed a Western diet for 12 weeks. n = 12 mice per group. P = 0.6235 for b, P = 0.9366 for c, and P = 0.1535 for d. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. e,f, Lipoprotein profiles of plasma total cholesterol (e) and triglyceride (f) from Alk1 f/f and Alk1 iΔaEC mice received AAV9-mPCSK9 and fed a Western diet for 12 weeks. n = 6 mice per group. g-i, Representative ORO-stained images (g), and quantification of neutral lipid content of aortic roots (h) and brachiocephalic arteries (i). Scale bars: 500μm and 100μm, for aortic root and brachiocephalic arteries, respectively. n = 6. P = 0.0058 for h and P = 0.0002 for i. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. j,k, Confocal images (j) and analysis (k) of brachiocephalic arterial sections for macrophage infiltration, green: CD68, red: SMA, blue: nuclei. Scale bar: 100μm. P < 0.0001 for k. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. All sectioned samples are prepared from Alk1 f/f and Alk1 iΔaEC mice received AAV9-mPCSK9 and fed a Western diet for 12 weeks. n = 6. All aortic root and brachiocephalic arteries were sectioned at 6μm. Image J software was used to quantify the samples. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. Source data

Extended Data Fig. 3

Extended Data Fig. 3. 3 Arterial EC specific ALK1 deletion does not affect blood pressure or vascular tone and reduces DiI-LDL uptake.

a,b, Telemetric systolic (a) and diastolic (b) BP of Alk1 f/f Ldlr_−/_− and Alk1 iΔaEC Ldlr_−/_− mice monitored over 4 consecutive days at 12 weeks after tamoxifen injection, showing no difference between control and ALK1 deleted group. n = 5 male mice per group. c-e, Representative confocal images (c), and analysis of ALK1 (d) and VE-cad intensity (e). Two images were counted per mouse in three mice per group for a total of n = 6. Scale bar: 20μm. P < 0.0001 for d and P = 0.7105 for e. Values show mean ± S.E.M. Image J software was used to quantify the ALK1 and VE-cad intensity. f-h, Relative VE-cad (f), PECAM-1 (g), and ALK1 (h) expression from isolated aortic ECs of Alk1 f/f Ldlr_−/_− and Alk1 iΔaEC Ldlr_−/_− mice fed a Western diet for 6 weeks. n = 3. P = 0.3571 for f, P = 0.8423 for g, and P = 0.0175 for h. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. i, Isolectin B4 staining of retinal vessels of Alk1 f/f Ldlr_−/_− and Alk1 iΔaEC Ldlr_−/_− mice fed a Western diet for 12 weeks. n = 6. Scale bar: 400μm. j,k, Representative en face immunofluorescence (j) analysis (k) of injected DiI-LDL in the ascending aorta of Alk1 f/f Ldlr_−/_− and Alk1 iΔaEC Ldlr_−/_− mice. Three images were counted per mouse in two mice per group for a total of n = 6. Scale bar: 20μm. P = 0.0002 for k. Values show mean ± S.E.M. All P values were calculated by two-tailed unpaired t-test. Source data

Extended Data Fig. 4

Extended Data Fig. 4. Arterial EC ALK1 deficiency reduces the progression of atherosclerosis without changing lipid metabolism.

a,b, Plasma total cholesterol (a) and triglyceride (b) level from Alk1 f/f Ldlr_−/_− and Alk1 iΔaEC Ldlr_−/_− mice fed a Western diet for 4 weeks. n = 10. P = 0.9957 for a and P = 0.0936 for b. Values show mean ± S.E.M. c-f, Analysis of weight (c), plasma total cholesterol (d), triglycerides (e), and blood glucose (f) levels from Alk1 f/f Ldlr_−/_− and Alk1 iΔaEC Ldlr_−/_− mice fed a Western diet for 12 weeks. n = 10 mice per group. P = 0.6485 for c, P = 0.0932 for d, P = 0.6875 for e, and P = 0.8998 for f. Values show mean ± S.E.M. g-i, Representative ORO-stained images (g), and quantification of neutral lipid content of aortic roots (h) and brachiocephalic arteries (i). Scale bars: 500μm and 100μm, for aortic root and brachiocephalic arteries, respectively. P < 0.0001 for h and P < 0.0001 for i. Values show mean ± S.E.M. j,k, Confocal images (j) and analysis (k) of brachiocephalic artery sections for apoB content, red: apoB, green: CD31, blue: nuclei. Scale bar: 20μm. P < 0.0001 for k. Values show mean ± S.E.M. l,m, Confocal images (l) and analysis (m) of brachiocephalic arterial sections for macrophage infiltration, green: CD68, blue: nuclei. Scale bar: 100μm. P < 0.0001 for m. Values show mean ± S.E.M. n-p, Trichrome staining (n) and analysis of brachiocephalic artery sections for plaque necrosis (o) and collagen content (p). Scale bar: 100μm. P < 0.0001 for both o and p. Values show mean ± S.E.M. All samples are prepared from Alk1 f/f Ldlr_−/_− and Alk1 iΔaEC Ldlr_−/_− mice fed a Western diet for 12 weeks. All aortic root and brachiocephalic arteries were sectioned at 6μm. n = 7 mice per group. Image J software was used to quantify the samples. All P values were calculated by two-tailed unpaired t-test. Source data

Extended Data Fig. 5

Extended Data Fig. 5. Selective, anti-ALK1 mAb screening and evaluation.

a, A representative experiment showing how mAbs were screened. HUVEC were stimulated with BMP-9 (30 min) and the levels of p-SMAD1/5 were used as a readout for BMP-9 signaling. BMP9: 10 ng/ml, IgG: 20 μg/ml, ALK1-Fc: 1 μg/ml, supernatant concentration: 20 μg/ml. b, Western blot analysis showing that seven purified mAbs did not block BMP-9 stimulated p-SMAD1/5 signaling in HUVEC. BMP9: 10 ng/ml, IgG: 20 μg/ml, ALK1-Fc: 1 μg/ml, antibodies: 20 μg/ml. c, mAbs 2 and 3 reduced DiI-LDL uptake in HUVEC. IgG or mAb (20 μg/ml of each) was pre-incubated. Three independent experiments were performed. DiI-LDL fluorescence was quantified by Image J software. P < 0.0001 and Values show mean ± S.E.M. P values were calculated by two-way ANOVA with Tukey’s multiple comparisons test. d,e, Western blot (d) and quantification (e) showing that incubation with mAb for different time points (0.5-8hrs) did not impact BMP-9 / p-SMAD1/5 signaling. BMP9: 10 ng/ml, ALK1-Fc: 1 μg/ml. n = 3 for two controls and ALK1-Fc, and n = 6 for BMP9 treated samples. f,g, Western blot analysis (f) and quantification (g) showing that increasing doses of mAb2 (0.5-20μg/ml) does not impact BMP-9 signaling. BMP9: 10 ng/ml, ALK1-Fc: 1 μg/ml. n = 3 for two controls and ALK1-Fc, and n = 6 for BMP9 treated samples. h,i, Western blot (h) and quantification (i) showing that mAb2 does not block short term BMP-9 signaling. BMP9: 10 ng/ml, mAb2: 20 μg/ml. n = 3 for 0.5 min. and n = 6 for 5, 30, 60 min. BMP9 treatment. j,k, Western blot analysis (j) and quantification (k) showing that increasing doses of BMP10 (0.5-12.5 ng/ml) does not impact p-SMAD1/5 signaling in HUVEC pre-incubated with mAb2 (20 μg/ml for 60 min.). n = 3 for 0 and n = 6 for 0.5, 2.5, and 12.5 ng/ml of BMP10 treatment. l,m, Western blot analysis (l) and quantification (m) showing that increasing doses of mAb2 (1-20 μg/ml) does not impact BMP-10 signaling. BMP10: 2.5 ng/ml for 30 min. n = 3 for control and ALK1-Fc treatment and n = 6 for 0.5, 1, 5, 10, and 20 μg/ml mAb2 treatment. All experiments were repeated at least three times. Values show mean ± S.E.M. P values were calculated by two-way ANOVA with Tukey’s multiple comparisons test. Source data

Extended Data Fig. 6

Extended Data Fig. 6. LDL uptake is regulated by ALK1, SR-BI, or LDLR.

a,b, Representative confocal images (a), and quantification (b) of DiI-LDL uptake upon individual or pairwise silencing of ALK1, SR-BI, or LDLR in HUVEC. Two images were counted per sample in three separate experiments for a total of n = 6. Scale bar: 20μm. P < 0.0001 for b. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. c, Western blot showing knockdown efficiency of each ALK1, SR-BI, or LDLR. Three separate experiments were performed to confirm the knockdown. Source data

Extended Data Fig. 7

Extended Data Fig. 7. mAb2 concentration is reduced to half in 4 days and does not inhibit BMP-9 signaling or vascular tone in vivo.

a,b, Representative en face images (a) and quantification (b) of DiI-LDL in Ldlr_−/_− mouse aorta injected with either IgG or increasing doses of mAb2 (100-500 μg/mouse). One image was counted per mouse in five mice per group for a total of n = 5. Scale bar: 20μm. Image J software was used to quantify the samples. P < 0.0001 for b. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. c,d, Representative en face immunofluorescence images (c) and quantification (d) of endogenous p-SMAD1/5 localization in aortic arch of Ldlr_−/_− mice injected with PBS, IgG, or mAb2. P = 0.8607 for d. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. Scale bar: 20μm. e, Serum antibody concentrations were measured using an ELISA assay. Anti-ALK mAb concentration change over seven days after single injection (250 μg) to Ldlr_−/_− mice fed a Western diet for 7 days. Values show mean ± S.E.M. f-h, Representative confocal images (f), and analysis of ALK1 (g) and VE-cad intensity (h). Two images were counted per mouse in three mice per group for a total of n = 6. Scale bar: 20μm. P = 0.7636 for g and P = 0.7679 for h. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. Image J software was used to quantify the ALK1 and VE-cad intensity. i-k, Relative VE-cad (i), PECAM-1 (j), and ALK1 (k) expression from isolated aortic ECs of Ldlr_−/_− mice fed a Western diet for 6 weeks with IgG or mAb2 treatment. n = 3. P = 0.8225 for i, P = 0.2299 for j, and P = 0.9459 for k. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. l, Isolectin B4 staining of retinal vessels of Ldlr_−/_− mice fed a Western diet for 12 weeks with either IgG or mAb2 treatment. n = 6. Source data

Extended Data Fig. 8

Extended Data Fig. 8. Selective anti-ALK mAb2 attenuates the initiation of atherosclerosis in mice.

a-c, mAb2 did not impact the weight (a) or plasma lipids (total cholesterol (b) and triglycerides (c)) of Ldlr_−/_− mice fed a Western diet for 6 weeks. n = 7 for PBS treatment and n = 12 for IgG and mAb2 treated mice. P = 0.7654 for a, P = 0.8498 for b, and P = 0.2501 for c. Values show mean ± S.E.M. d,e, Individual aorta images (d) and quantification (e) of neutral lipids from Ldlr_−/_− mice fed a Western diet for 6 weeks. Total 250 μg of IgG or mAb2 was injected twice weekly for 6 weeks duration. n = 7 for PBS treatment and n = 12 for IgG and mAb2 treated mice. Scale bar: 2 mm. Image J software was used to quantify the ORO area. P < 0.0001 for e. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. f-h, Representative images (f), and quantification showing ORO staining of aortic root (g) and brachiocephalic arterial sections (h). Scale bars: 500μm and 100μm, for aortic root and brachiocephalic artery, respectively. P < 0.0001 for both g and h. Values show mean ± S.E.M. i,j, Representative confocal images (i) and analysis (j) showing apoB (red) content per CD31 length/section of brachiocephalic artery. Scale bar: 20μm. P < 0.0001 for j. Values show mean ± S.E.M. k,l, Confocal images (k) and analysis (l) showing macrophage infiltration, green:CD68. Scale bar: 100μm. All samples are from Ldlr_−/_− mice treated with PBS, IgG, or mAb2 and western diet for 6 weeks. Total 250 μg of IgG or mAb2 was injected twice per week for 6 weeks. Aortic root and brachiocephalic arteries were sectioned at 6μm. P = 0.0034 for l. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. Source data

Extended Data Fig. 9

Extended Data Fig. 9. Selective anti-ALK mAb2 attenuates the progression of atherosclerosis in mice and mAb2 operates independently of LDL receptor in vivo.

a-d, Analysis of weight (a), plasma total cholesterol (b), triglyceride (c), and blood glucose level (d) from Ldlr_−/_− mice fed a western diet for 12 weeks with IgG or mAb2 treatment. Bi-weekly injections of 250 μg per injection. n = 12. P = 0.0912 for a, P = 0.5239 for b, P = 0.8829 for c, and P = 0.9913 for d. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. e,f, Relative mRNA (e) and protein LDLR (f) expression level from the livers of LDLR WT, KO, and Het mice fed a Western diet for 6 weeks with antibody treatment. n = 5. P < 0.0001 for e. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. g, Relative sterol-sensing gene, SREBF2, expression from the livers of LDLR WT, KO, and Het mice fed a Western diet for 6 weeks with antibody treatment. n = 3. P = 0.7954. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. Source Data.

Extended Data Fig. 10

Extended Data Fig. 10. mAb2 treatment in lipid lowering condition further reduces the progression of atherosclerosis.

a, Schematic diagram showing experimental procedure. Samples are prepared from Ldlr_−/_− mice fed a Western diet for 12 weeks with another 4 weeks of normal diet. The mice received IgG or mAb2 during the last 4 weeks, twice weekly with 250μg per injection. Control mice were sacrificed after 12 weeks of Western diet while IgG or mAb2 treated mice were sacrificed after 16 weeks. b,c, Analysis of weight (b)and blood glucose (c) level from Ldlr_−/_− mice fed a Western diet for 12 weeks with another 4 weeks of normal diet to lower lipid. The mice received twice weekly injections of 250 μg per injection during the last 4 weeks. n = 10. P = 0.9012 for b and P = 0.6414 for c. Values show mean ± S.E.M. d,e, Representative confocal images (d), and analysis (e) of brachiocephalic artery sections for apoB content, red: apoB, green: CD31, blue: nuclei. Scale bar: 20μm. P < 0.0001 for e. Values show mean ± S.E.M. f,g, Confocal images (f) and analysis (e) of brachiocephalic arterial sections for macrophage infiltration, green: CD68, blue: nuclei. Scale bar: 100μm. P = 0.0006 for g and Values show mean ± S.E.M. h-j, Trichrome staining (h) of brachiocephalic artery sections for plaque necrosis (i) and collagen content (j). Scale bar: 100μm. P < 0.0001 for i and P = 0.0018 for j. Values show mean ± S.E.M. k-m, H&E staining (k) of brachiocephalic artery sections for plaque necrosis (l) and fibrous cap thickness (m). Scale bar: 100μm. All aortic root and brachiocephalic arteries were sectioned at 6μm. n = 6 mice per group. Image J software was used to quantify the samples. P = 0.0031 for l and P = 0.0484 for m. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. Source data

Extended Data Fig. 11

Extended Data Fig. 11. Therapeutic treatment of mAb2 reduces atherosclerosis progression.

a, Schematic diagram showing experimental procedure. Samples are prepared from Ldlr_−/_− mice fed a Western diet for 16 weeks. The mice received IgG or mAb2 during the last 4 weeks, twice weekly with 250μg per injection. b,c, Analysis of weight (b) and blood glucose (c) level from Ldlr_−/_− mice fed a Western diet for 16 weeks. The mice received twice weekly injections of 250 μg per injection during the last 4 weeks. n = 12. P = 0.9319 for b and P = 0.9673 for c. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. d,e, Representative confocal images (d), and analysis (e) of brachiocephalic artery sections for apoB content, red: apoB, green: CD31, blue: nuclei. Scale bar: 20μm. P < 0.0001 for e. Values show mean ± S.E.M. f,g, Confocal images (f) and analysis (g) of brachiocephalic arterial sections for macrophage infiltration, green: CD68, blue: nuclei. Scale bar: 100μm. P < 0.0001 for g. Values show mean ± S.E.M. h-j, Trichrome staining (h) of brachiocephalic artery sections for plaque necrosis (i) and collagen content (j). Scale bar: 100μm. P = 0.6276 for i and P = 0.0003 for j. Values show mean ± S.E.M. k-m, H&E staining (k) of brachiocephalic artery sections for plaque necrosis (l) and fibrous cap thickness (m). Scale bar: 100μm. All aortic root and brachiocephalic arteries were sectioned at 6μm. n = 6 mice per group. Image J software was used to quantify the samples. P = 0.8491 for l and P = 0.0062 for m. Values show mean ± S.E.M. All P values were calculated by two-tailed unpaired t-test. Source data

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