Lipid infiltration promotes trans-differentiation of vascular smooth muscle cells into macrophage-like cells in early lesions of human coronary atherosclerosis

Fig. 1. Histological Analysis of Coronary Artery Lesions. Histological staining of coronary artery tissues from heart transplant recipients classified as initial lesions (n = 24), fatty streaks (n = 42), and advanced lesions (n = 48). (A1-A3) H&E, Masson’s trichtome and EVG staining shows diffuse intimal thickening in initial lesions with preserved elastic layers. (B1-3) Lipid accumulation and foam cells in fatty streaks. (C1-3) Plaque formation, elastic layer disruption, necrotic core, and collagen deposition in advanced lesions. Initial lesions display round cells and preserved spindle-shaped SMCs, fatty streaks show round cells with reduced spindle-shaped SMCs, and advanced lesions exhibit abundant round cells and fibrin/collagen deposition. Semi-quantitative analysis: D (tunica intimal thickness, 114.8 (IQR 75.8-157.8) vs. 221.0 (IQR 180.2-260.1) vs. 491.6 (IQR 388.2-587.6), µm), E (tunica medial thickness, 173.3 ± 65.5 vs. 159.8 ± 41.6 vs. 178.7 ± 36.7, µm), and F (intima-media ratio, 0.72 (IQR 0.56–0.89) vs. 1.41 (IQR 1.06–1.54) vs. 2.79 (IQR 2.52–3.05)) indicate that intimal thickness and intima-media ratio increase with lesion severity, while medial thickness shows no significant…

Fig. 2. Immunohistochemical Analysis of SMC Phenotypes, Lipid Infiltration and Macrophage-Related Markers. Immunohistochemical staining of coronary artery tissues from heart transplant recipients, classified into three groups: initial lesions (n = 24), fatty streaks (n = 42), and advanced lesions (n = 48), for α-SMA (1), smoothelin (2), CD248 (3), CD68 (4), FABP4 (5), and apoB (6). α-SMA is abundantly expressed in the intima and media across all lesion stages, indicating SMC predominance. Initial lesions show α-SMA in the middle intima layer with some CD248-positive cells. Fatty streaks and advanced lesions exhibit decreased α-SMA and increased CD248, indicating more secretory SMCs. Contractile antigen expression is reduced at the intima-media boundary in fatty streaks and minimal in advanced lesions, where the media is disrupted. CD68-positive cells are absent in initial lesions but abundant in advanced lesions. Anti-apoB staining demonstrates increased lipid load in the intima-media with lesion severity. Fatty streaks exhibit a significant rise in FABP4-positive SMCs in the intima, maintaining normal nuclear morphology. Advanced lesions show abundant FABP4-positive SMCs. FABP4 and apoB…

Fig. 3. Representative immunofluorescence staining of α-SMA (red) and FABP4 (green) in coronary lesions. Immunofluorescence staining for α-SMA (red), FABP4 (green), and nuclei (DAPI, blue) was performed on coronary artery specimens at progressive stages of atherosclerosis, including initial lesions (n = 24), fatty streaks (n = 42), and advanced lesions (n = 48). A stage-dependent increase in FABP4-positive smooth muscle cells (SMCs) was observed in the intima, while α-SMA expression showed a decreasing trend with lesion progression. Blue staining indicates DAPI-labeled nuclei. A wavy blue signal aligning with the internal elastic lamina (IEL), marked by white arrows, is observed in some images. This signal is presumed to reflect tissue autofluorescence or nonspecific background enhancement by DAPI and does not interfere with nuclear identification. Positive immunofluorescent signals are marked with white triangles. Semi-quantitative analysis of fluorescence intensity revealed a progressive decrease in α-SMA (D) and a corresponding increase in FABP4 (E) across the lesion stages. Data are shown as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. FABP4 = fatty acid-binding…

Fig. 4. Stage-dependent co-localization of ApoB with lipid-handling receptors. Representative immunofluorescence images showing lipid deposition and co-localization of ApoB with various lipid-handling receptors across different lesion stages: initial (1), fatty streak (2), and advanced (3). (A) Nile Red staining highlights neutral lipid accumulation. (B1-3) Co-staining of ApoB (red) and LOX-1 (green). (C1-3) ApoB (red) and LRP1 (green). (D1-3) ApoB (red) and CD36 (green). (E1-3) ApoB (red) and CD68 (yellow). (F1-3) ApoB (red) and FABP4 (green). (G) Merged image showing ApoB, FABP4, and CD68 in an advanced lesion. In early-stage lesions, ApoB predominantly co-localized with LOX-1, LRP1, and CD36 within the endothelial layer. As ApoB infiltration increased, co-localization extended to the intima–media junction and medial layer. In contrast, co-localization of ApoB with CD68 and FABP4 became prominent only in advanced lesions. Co-localization of ApoB (red) with endothelial markers CD31 (green, H1-3) and VE-cadherin (green, I1-3) was observed across lesion stages. DAPI staining (blue) was used to visualize cell nuclei. Panels E and F share the same ApoB images to demonstrate its…

Fig. 5. Correlation analysis. Correlation analysis between FABP4+ SMC density and intimal thickness (A), CD248+ cell density (B), foam cell density (C), and CD68+ cell density (D) in all patients showed strong positive correlations: intimal thickness (r = 0.76, P < 0.001), CD248+ cells (r = 0.86, P < 0.001), foam cells (r = 0.89, P < 0.001), and CD68+ cells (r = 0.86, P < 0.001). FABP4 = fatty acid-binding protein 4