Excessive Adventitial and Perivascular Vascularisation Correlates with Vascular Inflammation and Intimal Hyperplasia

Figure 1. Immuno- and electron microscopy phenotyping of MPO+ clusters reveals positive staining for myeloperoxidase and macrophage appearance of the cells. (A) Immunohistochemical staining for a pan-leukocyte marker CD45, myeloid cell markers CD11b and myeloperoxidase (MPO), macrophage markers F4/80 and CD68, T cell marker CD3, B cell marker CD19, and vascular smooth muscle cell marker SM-MHC. Note the positive staining of MPO+ clusters (indicated by red arrows) for MPO, F4/80, and CD3. In contrast, the adjacent lymph node (indicated by blue arrows) is totally positive for CD45 and CD3. ×200 magnification (scale bar: 100 µm); (B) Verification immunofluorescence staining for MPO. Note the positive MPO+ clusters and a negative adjacent blood vessel (left image). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI). ×400 magnification (scale bar: 25 µm); and (C) EM-BSEM visualisation of MPO+ clusters. Note the similarity of the cells within the MPO+ clusters (indicated by green arrows) to macrophages (indicated by red arrows) but not neutrophils (indicated by blue arrows). The right MPO+ clusters image (×2500 magnification) is a close-up of the left image (×500 magnification).…

Figure 2. Ultrastructural examination of MPO+ clusters and nerve bundles. (A) Backscattered scanning electron microscopy examination of the different types of nerve bundles (top) and MPO+ clusters (bottom). Rat aortic adipose tissue. Red and blue contours demarcate ×1000 and ×2500 close-ups, respectively. ×250, ×1000, and ×2500 magnification (scale bars: 200 µm, 50 µm, and 20 µm, respectively; accelerating voltage: 15 kV); (B) Immunohistochemical staining of MPO+ clusters (indicated by black arrows) for a macrophage marker CD68, myeloid cell marker myeloperoxidase, and neural tissue markers S100B and NeuN. Rat aortic adipose tissue. Note that MPO+ clusters are positively stained for myeloperoxidase and CD68 while being negatively stained for S100B and NeuN. ×200 magnification (scale bar: 100 µm); and (C) Immunofluorescence anti-CD68 and anti-MPO staining of the human coronary artery adipose tissue. Note the positive red staining within the MPO+ clusters and negative staining around them. DAPI nuclear counterstaining. ×400 magnification (scale bar: 25 µm). DAPI nuclear counterstaining. MPO—myeloperoxidase, DAPI—4′,6-diamidino-2-phenylindole.

Figure 3. Balloon-induced IH is associated with the expansion of VV and development of MPO+ clusters. (A) Representative images showing VV and MPO+ clusters in rats with or without IH in the abdominal aortas 5 weeks upon the balloon angioplasty. Red and blue contours demarcate the close-ups of the areas with representative VV and MPO+ clusters, respectively. Red and blue arrows indicate VV and MPO+ clusters, respectively. Haematoxylin and eosin staining, ×200 magnification (scale bar: 100 µm) and ×400 magnification (scale bar: 50 µm); (B) Quantitative analysis of number, area, and density of VV in rats from the experiment in A. Each dot represents an aortic section from one rat. Whiskers indicate range, box bounds indicate 25th–75th percentiles, center lines indicate median. p-values provided above boxes, Mann–Whitney U-test; and (C) Quantitative analysis of number, area, and density of MPO+ clusters in rats from the experiment in A. Each dot represents an aortic section from one rat. Whiskers indicate range, box bounds indicate 25th–75th percentiles, center lines indicate median. p-values provided above boxes, Mann–Whitney U-test. IH—intimal hyperplasia, VV—vasa vasorum, and…

Figure 4. Number and density of immune cells are associated with the development of IH and expansion of MPO+ clusters. (A) Quantitative analysis of number and density of CD45+ cells in the tunica adventitia of rats with or without IH in the abdominal aortas 5 weeks upon the balloon angioplasty (from the experiment in Figure 3A). Each dot represents an aortic section from one rat. Whiskers indicate range, box bounds indicate 25th–75th percentiles, center lines indicate median. p-values provided above boxes, Mann–Whitney U-test; (B) Correlation plots indicate a strong correlation of the number of CD45+ cells (top) and their density (bottom) in the tunica adventitia with both measures of intimal hyperplasia (neointima-to-intima ratio, left and percent stenosis, right). Spearman’s rank correlation coefficient; and (C) Correlation plots showing a moderate and statistically significant correlation between the density of CD45+ cells in the tunica adventitia and both number (left) and area (right) of MPO+ clusters. Spearman’s rank correlation coefficient. MPO—myeloperoxidase, VV—vasa vasorum, and IH—intimal hyperplasia.

Figure 5. Endothelial dysfunction enhances sprouting of VV and growth of MPO+ clusters. (A) Representative images showing adventitial VV and MPO+ clusters in rats which underwent balloon angioplasty of the abdominal aorta and optional intravenous administration of CPPs, a trigger of endothelial dysfunction (900 µL of particles per injection, equal to 600 µg calcium) or equal volume of physiological saline for 5 days, followed by euthanasia upon 5 weeks. Red and blue arrows indicate VV and MPO+ clusters, respectively. EM-BSEM, ×500 magnification (scale bar: 100 µm, accelerating voltage: 10 kV); (B) Quantitative analysis of number and area of adventitial VV and MPO+ clusters in rats from the experiment in A. Each dot represents a cross-section of the epoxy resin-embedded aorta. Whiskers indicate range, box bounds indicate 25th–75th percentiles, center lines indicate median. p-values provided above boxes, Mann–Whitney U-test; (C) Representative images showing perivascular VV and MPO+ clusters in rats from the experiment in A. Red and blue arrows indicate VV and MPO+ clusters, respectively. EM-BSEM, ×500 magnification (scale bar: 100 µm, accelerating voltage: 10 kV); and (D) Quantitative…

Figure 6. Saphenous veins utilised for CABG surgery frequently suffer from pre-implantation stenosis which correlates with the amount of VV. (A) Above: representative images showing pairwise collected conduits for CABG surgery (IMAs, top and SVs, bottom) without (left) or with (right) IH. Note a larger amount of IH in SVs (bottom) than in IMAs (top). EM-BSEM, ×42–×60 magnification (scale bar: 500 µm and 1 mm, accelerating voltage: 15 kV). Below: Quantitative analysis of 30 pairwise collected IMAs and SVs. Bar graph, p-values provided above the bars, Pearson’s chi-squared test with Yates’s correction for continuity; (B) Above: VV pattern in bypass grafts (IMAs, top and SVs, bottom) without (left) or with (right) IH. Note the higher VV number in both IMAs and SVs with IH (right). Red arrows indicate VV. EM-BSEM, ×500 magnification (scale bar: 100 µm, accelerating voltage: 15 kV). Below: correlation of VV count and VV density (A/P area per 1 VV, µm2) with IH expressed as neointima-to-intima ratio, Spearman’s rank correlation coefficient; and (C) Quantitative analysis of the samples from (A,B). Each dot represents a cross-section of the epoxy resin-embedded blood vessel (one IMA and one SV per…