Cellular and Matrix Organisation of the Human Aortic Valve Interleaflet Triangles

Figure 1. The aortic root includes all the tissue from the beginning of the left ventricle outflow tract in the left ventricle to the sinotubular junction at the beginning of the ascending aorta. Within these boundaries lie the 3 interleaflet triangles, the 3 sinuses of Valsalva, the left and right coronary ostia, the 3 leaflets, and the 3 commissures. From Ref. [5].

Figure 2. (A,B) Open view of 2 human aortic valves (AVs) showing the leaflets, the ILTs and the aortic-mitral fibrous curtain (dashed line) and (C) a pulmonary valve (PV) for comparison. LN: left to non ILT; LR: left-to-right ILT; and NR: non-to-right ILT.

Figure 3. Representative immunoperoxidase staining of CD31 (A) showing surface endothelial cells and microvessels in the porcine N-R ILT wall. (B) shows vWF (von Willebrand Factor) staining of surface endothelium in porcine N-R ILT wall. Representative SEMs of porcine ILTs (C) with magnified zones 1–3. Scale bar is 250 µm in (A); 100 µm in (B); 500 µm in (C), and 50 µm in magnified panels of (C). Red arrow in A shows surface endothelial cells, blue arrows show vascular endothelium, and white arrow in C1 shows direction of endothelial cell orientation.

Figure 4. Representative longitudinal sections of human N-R ILT sections showing positive cellular staining for α-smooth muscle actin (A) with magnified inset (A’) and smooth muscle myosin heavy chain (B) with magnified inset (B’) showing positive cells running parallel to the surface and to varying heights within the ILTs. Scale bar is 1mm in (A’,B’).

Figure 5. TEM of the different cell types observed in the porcine ILTs: fibroblasts (A) showing abundant rough endoplasmic reticulum (rER), cell surface filopodia (F), and an absence of myofilaments and caveolae; myofibroblasts (B) showing few rER, some peripheral myofilaments (M), a few focal densities (blue arrows), and few filopodia; smooth muscle cells (C) showing very little rER, prominent and abundant surface and cytoplasmic focal densities (blue arrows), caveolae under the cell surface (red arrows), cytoplasm full of myofilaments (M), and no filopodia. Scale bars are 1 μm in (A), 800 nm in (B), and 2 µm in (C).

Figure 6. Representative elastic van Gieson (EVG) staining of longitudinally sectioned human ILTs, showing purple coloured elastin fibres running below the luminal surface, magnified images below illustrating elastin fibres, and 2 distinct layers of loosely packed and densely packed collagen fibres (pink): (A) L-N, (B) L-R, and (C) N-R ILTs (scale bar = 2.5 mm) with (A’–C’) showing respective magnified images (scale bar = 250 µm). (D) shows the mean thicknesses of the 3 ILTs. ** p < 0.01; **** p < 0.001.

Figure 7. Representative alcian blue and Picrosirius red staining of longitudinally sectioned human ILTs showing the distribution of collagen (red) and proteoglycans (blue): (A) L-N, (B) L-R, and (C) N-R ILTs (scale bar = 2.5 mm) with (A’–C’) showing respective magnified images (scale bar = 500 µm). (D) shows the mean thicknesses of the 3 ILTs. * p < 0.05; ** p < 0.01.

Figure 8. Representative troponin I staining of longitudinal sections of human ILTs showing the extent of myocardial and adipose support: (A) L-N, (B) L-R, and (C) N-R ILTs (scale bar = 2.5 mm) with (A’) showing respective magnified image of panel in (A) (scale bar = 500 µm). (D) shows staining for leptin.

Figure 9. Representative longitudinal sections of an N-L human ILT showing positive neurofilament staining with nerve bundles marked with a pinpoint (A) and at higher magnification (A’). Graph showing the number of nerve bundles in each ILT (B). Colocalisation of acetylcholine (red) and tyrosine hydroxylase (green) in the same nerve bundle (C). Colocalisation of VIP (red) and tyrosine hydroxylase (green) in the same nerve bundle (C). Scale bars are 2.5 mm in (A), 250 µm in (A’), and 20 µm in (C,D). * p < 0.05; ns: not significant.