Distinct types of plexiform lesions identified by synchrotron-based phase-contrast micro-CT

Abstract

In pulmonary arterial hypertension, plexiform lesions are associated with severe arterial obstruction and right ventricular failure. Exploring their structure and position is crucial for understanding the interplay between hemodynamics and vascular remodeling. The aim of this research was to use synchrotron-based phase-contrast micro-CT to study the three-dimensional structure of plexiform lesions. Archived paraffin-embedded tissue samples from 14 patients with pulmonary arterial hypertension (13 idiopathic, 1 with known BMPR2-mutation) were imaged. Clinical data showed high-median PVR (12.5 WU) and mPAP (68 mmHg). Vascular lesions with more than 1 lumen were defined as plexiform. Prior radiopaque dye injection in some samples facilitated 3-D rendering. Four distinct types of plexiform lesions were identified: 1) localized within or derived from monopodial branches (supernumerary arteries), often with a connection to the vasa vasorum; 2) localized between pulmonary arteries and larger airways as a tortuous transformation of intrapulmonary bronchopulmonary anastomoses; 3) as spherical structures at unexpected abrupt ends of distal pulmonary arteries; and 4) as occluded pulmonary arteries with recanalization. By appearance and localization, types 1-2 potentially relieve pressure via the bronchial circulation, as pulmonary arteries in these patients were almost invariably occluded distally. In addition, types 1-3 were often surrounded by dilated thin-walled vessels, often connected to pulmonary veins, peribronchial vessels, or the vasa vasorum. Collaterals, bypassing completely occluded pulmonary arteries, were also observed to originate within plexiform lesions. In conclusion, synchrotron-based imaging revealed significant plexiform lesion heterogeneity, resulting in a novel classification. The four types likely have different effects on hemodynamics and disease progression.

Keywords: imaging; lung; plexiform lesion; pulmonary arterial hypertension; synchrotron.

Figure 1.

Normal lung imaged with synchrotron-based phase-contrast micro-CT. A: a bronchovascular bundle with a pulmonary artery (PA) and a bronchiole (Br) is seen to the right. The resolution allows for individual layers of the vascular wall to be discerned (white arrowhead). To the left, a vein (V) that collects oxygenated blood is shown, and below the vein two alveolar ducts (AD). Supplemental Video S1 scans through the 3-D volume from which the 2-D image in A has been captured. B: a cross section of a bifurcating muscular pulmonary artery (PA) and a bronchiole (Br). C: a longitudinal virtual section of an alveolar duct (AD) is shown. The white arrowheads mark small vessels within the wall of the alveolar duct. D: a pulmonary vein (PV), positioned within a septa (S) as expected. Scale bars in A–D = 200 µm. 2-D, two-dimensional; 3-D; three-dimensional.

Figure 2.

A–D: four virtual imaging planes of one plexiform lesion to illustrate the limitation of studying these lesions in 2-D. *The center of the plexiform lesion in all four images. A: no larger parent vessel is visualized. B: next to the plexiform lesion a completely occluded artery is seen (white arrowhead). C: the pulmonary artery next to the lesion is shown to be patent more proximally and then completely occluded (both dichotomous branches are occluded). The white arrowhead marks the internal elastic lamina and the area of complete occlusion. D: a 90° branch (white arrowhead) which exits the pulmonary artery proximal to the obstruction and appears to communicate with the plexiform lesion. The scale bar in D (200 µm) is valid for A–D. E and F: the radiopaque dye by histology and synchrotron tomography respectively. In E, hematoxylin-eosin staining of the BMPR2 lung injected with green dye in the pulmonary arteries is shown. In F, the same imaging plane has been localized in a tomography volume to illustrate the high attenuation of the dye, seen in white. The scale bar in F (200 µm) is valid for E and F.

Figure 3.

Schematic illustration of the four different plexiform lesion types. Type 1: in monopodial branches, often with a connection to the vasa vasorum (in red). Type 2: between pulmonary arteries (orange) and airways (blue) as a tortuous transformation of intrapulmonary bronchopulmonary anastomoses (IBA), connecting the pulmonary artery with peribronchiolar vessels (red). Type 3: at unexpected abrupt ends of distal pulmonary arteries/arterioles. Often, small dilated vessels are seen leaving type 3 lesions (red). Type 4: completely occluded pulmonary arteries with recanalization, or incomplete blockage.

Figure 4.

Vessel size by lesion type. Distribution of vessel diameter measured proximal to each identified plexiform lesion, in micrometers. Type 4 measured at the site of the lesion. Lines represent mean and standard deviation (GraphPad Prism 8).

Figure 5.

A–D: the four types of plexiform lesions from four different patients with IPAH. PA indicates pulmonary artery, and the asterisks mark the lesions. The arrowheads in A and B indicate the monopodial branch leaving the parent artery. In D, the arrowhead indicates the open lumen distal to the obstruction. Supplemental Videos S2–S5 scan through the four different lesions in A–D. The scale bar in D (200 µm) is valid for A–D. E–H: all four types of plexiform lesions from the dye-injected lung from the BMPR2-mutation carrier. PA marks open lumen proximal to the plexiform lesion in all four images and the asterisks mark the lesions. The arrowhead in E indicates the monopodial branch leaving the parent artery. Scale bars in E–H = 200 µm. IPAH, idiopathic pulmonary arterial hypertension.

Figure 6.

Segmentation and 3-D rendering of dye-injected vessels (the injected dye is white in the tomograms and orange in the 3-D renderings) to illustrate the four types of plexiform lesions. Insets show 2-D cross sections of the same lesions. Black asterisk, plexiform lesion; PA, pulmonary artery. A: type 1 lesion. The black arrowhead marks the monopodial branch connecting the PA to the lesion, and white asterisks connections to the vasa vasorum. White arrowheads mark the adventitia. B: type 2 lesion. The white asterisk marks the intrapulmonary bronchopulmonary anastomoses (IBA), proximal to the occlusion of the PA. The lesion connects with the peribronchiolar vascular network surrounding the airway (3-D reconstructed in blue). White arrowheads mark collateral flow to the poorly perfused area distal to the obstruction. C: type 3 lesion. The white asterisk shows a pulmonary vein that connects with the lesion. D: type 4 lesion within the PA. The cross section shown in the inset is captured approximately where the black asterisk is positioned, to illustrate how it would look in a 2 D histological slide. Supplemental Videos S6–S9 show the same lesions as in A–D. As images A through D are 2-D images of 3-D projections, regular scale bars are inapplicable. For reference, the lumen diameter of parent vessels are indicated by dashed lines: A = 359 µm; B = 228 µm; C = 137 µm; D = 251 µm. 2-D, two-dimensional; 3-D; three-dimensional.

Figure 7.

Demonstration of how manual 3-D reconstructions of non-dye injected tissue and comparisons of imaging and histology and/or immunohistochemistry can make pulmonary vascular lesions easier to interpret. A: the type 1 plexiform lesion in Fig. 2, A–D is shown in cross section, in a view that shows both the patent parent pulmonary artery (PA) and a nearby airway (A). B: the PA above the imaging plane and a monopodial branch that enters the plexiform lesion have been manually reconstructed in red. At the site marked by the white asterisk, the passage for the blood to pass becomes very narrow (stenosis) and distal from this point the vessels within and around the plexiform lesion were thin-walled and dilated. C: the dilated thin-walled vessels have been reconstructed in blue. Black asterisk marks the center of the plexiform lesion in D–F. D and E: histology of sections that match the imaging plane seen in A–C. D: Elastin van Gieson staining with elastic fibers in black. E: Alcian blue/Periodic acid-Schiff staining, with acidic mucins in bright blue. F: Immunohistochemistry to localize lymphatic vessels (podoplanin in brown). G–I: Showing the same lesion but in a different imaging plane. From this angle, it is clear that the pulmonary artery is occluded distal from the plexiform lesion (white arrowhead in G). Asterisks in H and I mark the narrow passage within the plexiform lesion and the arrowhead in I marks a site where the dilated thin-walled vessels in blue appear to communicate with the vasa vasorum. As images A–C and G–I show 3-D projections, regular scale bars are inapplicable. The size of the vessel shown in images A–I can be gauged by the scale bar in F (200 µm). 2-D, two-dimensional; 3-D; three-dimensional.

Figure 8.

Vascular connections from plexiform lesions. Black asterisks mark plexiform lesions. A and B: a type 3 lesion, with and without 3-D rendering of the vasculature. In B, a pulmonary vein (white asterisk) can be seen to drain the lesion, possibly through connections larger than regular capillaries. C: a type 2 lesion that connects with the peribronchial microvessels (white arrowheads) which then drains to bronchial veins (white asterisk). D: a 3-D rendered type 2 lesion next to an airway reconstructed in blue. The white arrowhead marks a distal complete occlusion of the PA, and the white asterisks mark veins that drain the plexiform lesion. E: dilated thin-walled vessels originating from a type 1 lesion that follow the occluded monopodial branch (white asterisk) distally while separate vessels supply a nearby alveolar duct (white arrowhead). F: thin-walled collateral vessels from the same type 2 lesion as in D can be seen to reenter (white arrowhead) the lumen distal to complete occlusion. The insets, viewed from left (occluded) to right (patent), show the collateral entering (white arrowhead) the PA. As images A–F are 2-D images of 3-D projections, regular scale bars are inapplicable. Dashed lines indicate a measurement at a given point. In A and B, the lumen diameter of the nearby vein is 120 µm; the lumen diameter of the parent vessel is given for D and F = 173 µm; the parent vessel diameter is given for C = 144 µm and E = 228 µm. 2-D, two-dimensional; 3-D; three-dimensional.