Fig 2: Retention of apoB lipoproteins in intimal hyperplasia developed during low cholesterol levels. Carotid injury was performed in wild‐type mice fed chow diet to induce intimal hyperplasia under normal cholesterol levels. Three weeks later, hypercholesterolemia was induced by injection of PCSK9 virus and a switch of diet to western HFD. Four days after virus injection (3 days on HFD), carotid arteries were harvested, sectioned and immunostained for apoB (red), VSMCs (α‐actin, green) and cell nuclei (DAPI, blue). (A) Quantification of apoB‐positive area in intimal hyperplasia (IH) and media of the injured region and the proximal uninjured region as indicated. Graph bars show median values. Data was analyzed using Mann–Whitney rank sum test. P < 0.05 is regarded significant, n = 6 in each group. (B) Tissue sections from the injured region (upper panels) and uninjured proximal region (lower panels) from a representative carotid artery. White frames in overview pictures (right panels) depict the enlarged areas shown in left panels. Red=apoB, green=VSMCs (α‐actin), blue=nuclei (DAPI). Lumen is facing upwards. IH=intimal hyperplasia, M=media.

Fig 3: 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 (α‐actin), blue=nuclei (DAPI).

Fig 4: 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 5: cf-mRNAs are enriched and protected in EVs.a Expression of protein markers (CD9, ApoA1, Ago2, and ApoB) using immunoprecipitation across plasma fractions. Ladder was cropped from full gel to show size reference. b RT-qPCR analysis of selected genes using supernatants eluted from CD9, ApoB, or IgG immunocaptures from FR14 and FR58. P-value is derived from the Tukey’s test (ns = not significant, P > 0.05; *P < 0.05, **P < 0.01, and ****P < 0.0001) with three technical replicates of FR14 and FR58 of plasma pooled from three healthy individuals. c A box plot of negative raw cycle threshold (- raw ct) of individual genes with RNase and/or detergent treatment using qRT-PCR. RNA isolated from combined FR14 and FR58 using three healthy individuals and three technical replicates of control RNA were treated with RNase and/or detergent. Data are analyzed using RStudio and reported as means ± SD of three independent samples.