Fig 2: Illustrated abstract. Schematic depiction of proposed speculative mechanism (created with BioRender.com). ApoE is more abundant in tissues from PAA patients compared to non-dilated A. pop. or other vascular diseases (AAA and PAOD). Treatment of VSMCs with ApoE leads to decreased proliferation and increased expression of contractile markers such as MYOCD, TAGLN, SMTN and CNN1on RNA and protein levels. Overall, this study suggest that ApoE might be involved in contractile phenotype restoration of popliteal VSMCs and thus counteract aneurysm formation (in individual patients).

Fig 3: Affymetrix Analysis, Pathway enrichment, comparative ApoE expression and PAA immunofluorescence. (A) Gene expression heatmap (top 25 hits, cutout from Figure S1C) with hierarchical clustering for PAA vs. popliteal artery based on lowest adjusted p-value (all > 0.05: Supplement Table S3) (annotations: Table S2). (B) Selected examples of KEGG pathway enrichment based on Affymetrix data (NES: normalized score shown in (F), * = p < 0.05). (C) RT-qPCR results of ApoE expression in PAA (red) compared to popliteal artery (light grey), AAA (dark grey) and (D) PAOD (grey) shown as 2^dCt values against the respective housekeeping gene (n = 11 AAA, n = 7 A. pop., n = 7 PAA; n = 6 PAOD, n = 7 PAA; mean +/− SEM; unpaired t-test, * p < 0.05). (E) PAA tissue sample (HE; scale bar 2 mm) and immunofluorescence with APOE (red) and SMA (green)-positive cells co-localization (scale bar 100 μm). (F) Exemplary comparative hallmarks pathway enrichment analysis of Affymetrix gene expression data and OLink Protein expression data for PAA vs. popliteal artery (PA) and PAA vs. AAA (all p-value < 0.0.5) (complete enrichment sets shown in Figure S4).

Fig 4: In vitro mRNA expression with delivery by protein-LNP complexes.a LNPs loaded with mRNA encoding luciferase were incubated with selected high-binding corona proteins (0.05 ng mRNA: 1 ng protein) prior to introduction to HepG2 cells seeded at 4.7 × 104 cells per cm2 (100 ng mRNA per well). The luminescence was measured as a proxy for mRNA expression to understand the effect of proteins on LNP delivery efficiency. Luminescence was normalized to the average of each no-corona LNP biological control for all in vitro studies. Created in BioRender. Voke, E. (2025) https://BioRender.com/58u4s4t. b Resulting luminescence of pre-incubations of individual proteins with LNPs showed no significant change in luminescence (mRNA expression) for ApoE, A2M, or a mixture of the proteins, while showing a significant decrease for VTN or CR, each relative to the no-corona LNP control. c Dose–response of protein concentrations for VTN incubated LNPs showed a significant decrease in mRNA expression compared to the no-corona LNP control. d Cell viability showed no statistical difference for protein incubations. N = 4 technical replicates, n = 3 biological replicates. Data points shown are biological replicates. All data are presented as mean values with standard deviation. Statistical analysis was performed by repeated measures one-way ANOVA test with Geisser–Greenhouse correction, followed by Dunnett’s multiple comparisons test where * and ** represent p ≤ 0.05 and p ≤ 0.01, respectively.

Fig 5: Uptake and lysosomal co-localization of protein-LNP complexes in HepG2 cells.a HepG2 cells internalizing LNPs loaded with Cy5-mRNA pre-incubated with high-binding corona proteins were visualized by confocal microscopy. Representative image of LNP + VTN incubations showing LNPs (Cy5; red), cell membrane (CellBrite membrane dye; green), and nuclei (Hoechst; blue). b Inset showing a magnified view of the region outlined by the red box in (a). c Quantification of Cy5 (LNP) signal per cell demonstrates differences in cell Cy5-mRNA uptake between select corona protein incubations (n = 4 technical replicates, n = 3 biological replicates). Each dot represents an individual field-of-view-level measurement, color-coded by biological replicate; larger, black-outlined dots indicate the mean value for each biological replicate. Statistical analysis was performed by a nested one-way ANOVA test followed by Dunnett’s multiple comparisons test. d, e Cy5 signal from HepG2 cells internalizing LNPs loaded with Cy5-mRNA pre-incubated with high-binding corona proteins was also quantified by flow cytometry. d Percentage of Cy5-positive cells and e mean fluorescence intensity show uptake trends consistent with microscopy. Data points shown are biological replicates (n = 3 technical replicates, n = 3 biological replicates). Error bars denote standard deviation. Statistical analysis was performed by repeated measures one-way ANOVA test with Geisser-Greenhouse correction, followed by Dunnett’s multiple comparisons test. To compare endosome entrapment for select protein incubations, co-localization of the Cy5 signal (LNP) and fluorescently labeled lysosomes (green) was analyzed. Representative image of LNP + ApoE incubation shows f LNPs (red), lysosomes (green) and nuclei (blue) fluorescently labeled. g Quantification of overlapping Cy5 (LNP) and lysosome signal per cell (n = 4 technical replicates, n = 4 biological replicates). Each dot represents an individual field-of-view-level measurement, color-coded by biological replicate; larger, black-outlined dots indicate the mean value for each biological replicate. Statistical analysis was performed by a nested one-way ANOVA test followed by Dunnett’s multiple comparisons test. For all statistical analyses performed, *, **, and *** represent p ≤ 0.05, 0.01, and 0.001, respectively. All image thresholding was applied uniformly across samples, and image channels were adjusted solely for visualization purposes.