Fig 2: Retention of apoB lipoproteins following immunization with idiotypic antibody that blocks the binding of lipoproteins to proteoglycan GAG-chains. (A) Schematic illustration of the study design. Carotid injury was performed in wild-type mice and immunized weekly three times with an idiotypic proteoglycan GAG-chain binding antibody (chP3R99) or control idiotypic antibody (control). Four weeks after surgery, hypercholesterolemia was induced by injection of PCSK9 virus and a switch of diet to western HFD. At 5 weeks (7 days on HFD), the carotid arteries were harvested, sectioned and immunostained. (B) Quantification of apoB-positive area. Graph bars show median values. Data was analyzed using Mann–Whitney rank sum test. P < 0.05 is regarded significant, n = 4–5 in each group. (C) Representative tissue sections from the chP3R99 antibody-treated (upper panel) and the control antibody-treated (lower panel) groups. White frames in overview pictures (right panels) depict the enlarged areas shown in left panels. Lumen is facing upwards. Red=apoB, green=VSMCs (a-actin), blue=nuclei (DAPI).

Fig 3: LDL binding to the vessel wall in vitro following treatment with Site B peptide or enzymatic digestion of proteoglycan GAG chains. Tissue sections of carotid arteries from wild-type mice with intimal hyperplasia were incubated with human LDL. Bound LDL was detected using anti-apoB antibody. (A) LDL binding to tissue sections pre-incubated with positively charged SiteB peptide (white circles) or neutrally charged SiteB KE peptide (gray circles). Black circles=no pre-treatment. White triangles=no LDL incubation (buffer only). (B) LDL binding to tissue sections pre-treated with the GAG-degrading enzymes chondroitinase (white circles) or heparinase (white dot circles). Black circles=no pre-treatment. White triangles=no LDL incubation (buffer only). Data was analyzed using Mann–Whitney rank sum test. Graph bars show median values. P < 0.05 is regarded significant, n = 5–8 in each group. (C) Multi-immunostaining for biglycan (red, left panels) and perlecan (green, right panels) of tissue sections from the injured region (upper panels) and the uninjured region (lower panels) 3 weeks after carotid injury in a wild-type mouse. Nuclei are stained with DAPI (blue).

Fig 4: Vascular retention of apoB-containing lipoproteins following carotid injury in mice with and without mild hypercholesterolemia. (A) Schematic illustration of the common carotid artery (CCA) after carotid injury, depicting the injured region with intimal hyperplasia and the uninjured region without intimal hyperplasia. Dark green=intimal hyperplasia. ICA=internal carotid artery, ECA=external carotid artery, TA=thyroid artery. (B) Morphology of the vessel wall in wild-type mice 3 weeks after carotid injury. Carotid injury was performed in wild-type mice (n = 6). Three weeks after surgery the carotid arteries were harvested, sectioned and multi-immunostained for VSMCs (a-actin, green), endothelium (CD31, red), leukocytes (CD18, cyan), and DAPI (blue). Representative pictures from the injured region. White line in merged panel depicts the internal elastic lamina. IH=intimal hyperplasia, M=media. (C and D) Carotid injury was performed on APOB100Tg/Tg mice (mild hypercholesterolemia) and wild-type mice (normal cholesterol levels), both fed chow diet. Three weeks after surgery, carotid arteries were harvested, sectioned and multi-immunostained for apoB, VSMCs (a-actin), and cell nuclei (DAPI). (C) Quantification of apoB-positive area in intimal hyperplasia (IH) and media in the injured region and the proximal uninjured region as indicated. Data was analyzed using Mann–Whitney rank sum test. Graph bars show median values. P < 0.05 is regarded significant, n = 6 in each group. (D) Representative pictures of the injured region (upper panels) and corresponding uninjured proximal region (lower panels) from an APOB100Tg/Tg mouse (left panels) and a wild-type mouse (right panels). Red=apoB, green=VSMCs, blue=nuclei, IH=intimal hyperplasia. (E) Representative pictures of a carotid artery of an APOB100Tg/Tg mouse, 3 weeks after surgery, multi-stained for apoB (red), VSMCs (green), leukocytes (cyan) and nuclei (DAPI). White line depicts the internal elastic lamina. IH=intimal hyperplasia, M=media, * =lumen.