Fig 1.
Analysis of morphologic changes in arteries of mini-pigs with induced atherosclerosis.
The α-SMA content of the arteries was detected by IHC staining of α-SMA; the collagen content of the plaques is represented by Sirius red staining visualized under polarized light. Representative images of plaques injected with either saline (n = 11), HMGB1 (n = 11), and TNF-α (n = 10) as indicated and stained with hematoxylin & eosin (H&E) (a-c [20× of A-C]), Masson’s trichrome (D-F), Masson’s pentachrome (G-I), α-SMA (J-L), and Sirius red (M-O) (100×). Black boxes in a, b, and c represent the areas further magnified in A-O corresponding to their respective columns. IHC area was calculated as (intima/plaque) × 100, %. Scale bars represent 100 μm. The intima-plaque ratio data are represented as the mean±SEM. *p<0.05, compared with the saline group.
Fig 2.
IHC analysis of the degree of inflammation and macrophages present in the arteries of mini-pigs with induced atherosclerosis.
Inflammation and macrophages present in arterial plaques was detected by Immunohistochemical (IHC) staining for RAGE, HMGB1, TNF-α, CD68, CD47, or CD206 in the saline (n = 11), HMGB1 (n = 11), and TNF-α (n = 10) groups. Representative images of IHC staining of RAGE (A), HMGB1 (B), TNF-α (C), CD68 (D), CD47 (E), and CD206 (F) in the mini-pig artery (amplification 100×). IHC area was calculated as (intima/plaque) × 100, %. Scale bars represent 100 μm. Quantitative data are represented as the mean±SEM. *p<0.05, compared with the saline group.
Fig 3.
IF analysis of macrophage infiltration in the arteries of mini-pigs with induced atherosclerosis.
The macrophage content of the plaques was detected by immunofluorescence (IF) using CD68, inducible nitric oxide synthase (iNOS; M1), and arginase-1 (Arg1; M2) antibodies. A) Representative images of M1/M2 macrophage immunofluorescence in the mini-pig artery of the saline, HMGB1, and TNF-α groups (n = 4 vessels/group) (amplification 400×). B) Measurement of the co-expression area of CD68-iNOS. C) Measurement of the co-expression area of CD68-Arg1. Relative area measurements were determined using a Zeiss LSM 700. Scale bars represent 100 μm. Data are represented as the mean±SEM. *p<0.05, compared with the saline group.
Fig 4.
Determination of apoptosis in the arterial plaques of mini-pigs with induced atherosclerosis using the TUNEL assay and co-IF stain of α-SMA and CD68.
The apoptosis content of the plaques was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and immunofluorescence (IF) using alpha-smooth muscle actin (α-SMA) and CD68 antibodies. Representative images of mini-pig arteries in the saline (n = 11), HMGB1 (n = 11), and TNF-α (n = 10) groups stained with (A) TUNEL and α-SMA or (B) TUNEL and CD68 (amplification 100×). Digital images of the vessels were scanned using a Zeiss LSM 700. Scale bars represent 100 μm.
Fig 5.
OCT of atheromatous characteristics and histologic classification based on AHA criteria.
(A) Optical coherence tomography (OCT) images of atheromatous characteristics of the arteries of mini-pigs with induced atherosclerosis from the saline (n = 11), HMGB1 (n = 11), and TNF-α (n = 10) groups. White arrows indicate location of plaques. (B) Histologic classification of plaques from each group according to the American Heart Association (AHA) criteria. Plaque characteristics were categorized into early (type I [blue], II [orange], and III [grey]) and advanced (type IV [yellow]).
Table 1.
Morphological parameters in histology, optical coherence tomography, and quantitative angiography.