Sprouting and intussusceptive angiogenesis in postpneumonectomy lung growth: mechanisms of alveolar neovascularization

Abstract

In most rodents and some other mammals, the removal of one lung results in compensatory growth associated with dramatic angiogenesis and complete restoration of lung capacity. One pivotal mechanism in neoalveolarization is neovascularization, because without angiogenesis new alveoli can not be formed. The aim of this study is to image and analyze three-dimensionally the different patterns of neovascularization seen following pneumonectomy in mice on a sub-micron-scale. C57/BL6 mice underwent a left-sided pneumonectomy. Lungs were harvested at various timepoints after pneumonectomy. Volume analysis by microCT revealed a striking increase of 143 percent in the cardiac lobe 14 days after pneumonectomy. Analysis of microvascular corrosion casting demonstrated spatially heterogenous vascular densitities which were in line with the perivascular and subpleural compensatory growth pattern observed in anti-PCNA-stained lung sections. Within these regions an expansion of the vascular plexus with increased pillar formations and sprouting angiogenesis, originating both from pre-existing bronchial and pulmonary vessels was observed. Also, type II pneumocytes and alveolar macrophages were seen to participate actively in alveolar neo-angiogenesis after pneumonectomy. 3D-visualizations obtained by high-resolution synchrotron radiation X-ray tomographic microscopy showed the appearance of double-layered vessels and bud-like alveolar baskets as have already been described in normal lung development. Scanning electron microscopy data of microvascular architecture also revealed a replication of perialveolar vessel networks through septum formation as already seen in developmental alveolarization. In addition, the appearance of pillar formations and duplications on alveolar entrance ring vessels in mature alveoli are indicative of vascular remodeling. These findings indicate that sprouting and intussusceptive angiogenesis are pivotal mechanisms in adult lung alveolarization after pneumonectomy. Various forms of developmental neoalveolarization may also be considered to contribute in compensatory lung regeneration.

Fig. 1

Compensatory lung growth in the cardiac lobe. Left pneumonectomy in mice results in compensatory growth of the right lung. The cardiac lobe in particular extends dramatically into the left pleural cavity enabling restoration of the vital capacity within 21 days. a The cardiac lobe fills out the left pleural cavity almost completely, already 14 days after surgery. b Finite element reconstructions of the cardiac lobe originating from microCT data reveal an increase of 143 percent in volume 14 days after pneumonectomy. Reconstruction created by Nenad Filipovic, University of Kragujevac, Serbia. c, d Contrary to the vasculature of control cardiac lobes (c), low power SEM images of microvascular corrosion casts showing heterogeneous vascular densities and distributions 7 days after surgery

Fig. 2

Spatial growth pattern of cardiac lobe. a The spatial heterogeneity of different lung growth can be seen in SEM-images of microvascular corrosion casts (above) and anti-PCNA-staining (below): perivascular lung growth located mainly in central parts of the lung (left panel) and subpleural lung growth (right panel) containing numerous anastomoses with pleural vessels. b High resolution synchrotron radiation tomography revealed the compact zones of vascular growth upon cross-section. Bar = 100 μm

Fig. 3

Sprouting and intussusceptive angiogenesis on pleural plexus. The pleural surface of the cardiac lobe is underlaid by a highly ramified capillary plexus originating from and draining to pulmonary vessel branches. a Coverage of approximately 15–25 % of the pleural surface. Bar = 50 μm. b 7 days after pneumonectomy this plexus increases in density, focally resulting in coverage of up to 85 %. Bar = 50 μm. c Numerous small caliber holes with diameters of between 1 and 5 μm as hallmarks of pillar formation (white arrows) show the high intussusceptive angiogenesis activity. Bar = 25 μm. d In less densely packed areas sprouts (red arrowheads) as well as pillars are evident. Bar = 20 μm

Fig. 4

Transmission electron micrographs. a Transcapillary pillar formation indicates the occurrence of intussusceptive angiogenesis during compensatory lung growth which can be seen as protrusive interendothelial junctions. Pil denotes pillar, al alveolus, am alveolar membrane, ec endothelial cell; Bar = 10 μm b Semi-thin sections reveal the close spatial relationship between alveolar macrophages (black stars) and type II pneumocytes (red arrowheads), indicated in the subpleural region (P _L : pleura). Bar = 50 μm c, d Appearance of monocytes, alveolar macrophages, and type II pneumocytes indicates their proangiogenic relevance for alveolar morphogenesis. ATII, type II pneumocytes; mo monocyte; c capillary; MΦ; alveolar macrophage. c Bar = 15 μm; d Bar = 30 μm

Fig. 5

Corrosion casts of new alveolar septum formation. a In the midline of the alveolar cavity, an elevated vessel (dotted red line) starts to upfold, whereby another double-layered vessel crosses under it. b The septal vessel continues to elevate tightened dumbbell-shaped by two vessel at the end (white arrows). c A septal ridge (red dotted line) can be seen in the middle of the alveolar cavity whose local influxes (white arrows) are increasing by pillar formation (yellow stars) and vascular duplications. d Upfolding of a new alveolar septum (red dotted line) starts towards the alveolar entrance ring (AER). A second double-layered ring (orange dotted line) is developing by intussusceptive angiogenesis (yellow stars). e In the developed new alveoli with AERs (red dotted lines), an elevation and formation of new septa (red lines) is present in the midline. f Vascular remodeling modifies and expands the alveolar basket by the occurrence of pillars, typical hallmarks of intussusceptive angiogenesis (circle)

Fig. 6

Synchrotron radiation tomographic microscopy. a Three-dimensional evaluation of microvascular corrosion casts by SRXTM illustrating the alveolar basket structure accompanied by the limiting AER vessels (blue arrowheads) and the elevated ridge (asterisk). Bar = 15 μm. b A typical example of double-layered vessels (red arrow) during alveolarization. Bar = 20 μm. c Analysis of central areas of the cardiac lobe revealed an increased density of intussusceptive holes (red arrowheads) indicative of the occurrence of intussusceptive angiogenesis around larger vessel structures. Bar = 60 μm d In lung alveolarization a ridge (dashed line) can be seen in the midline of the alveolar basket accompanied by double capillary layers (blue arrows) enabling the lifting-off of the inter-airspace septum. The rapid expansion by intussusceptive angiogenesis (red arrowheads) allows the pacing of isotropic lung growth after pneumonectomy. Bar = 10 μm (see movies in supplemental material)

Fig. 7

Vascular remodeling of the alveolar vascular ring. a Microvascular corrosion casts and SEM of vascular rings at the alveolar opening depict the incidence of remodeling due to intussusceptive angiogenesis. b The appearance of transluminal pillars on the interconnections between the alveolar entry rings indicates the occurrence of intussusceptive angiogenesis at the site of the alveolar opening. Bar = 15 um. c Elongation of such transluminal pillars results in a vessel duplication of ring blood vessels. Bar = 15 um. d High-resolution Synchrotron radiation tomographic microscopy demonstrates a vascular duplication (red arrowheads). Bar = 20 μm

Fig. 8

Schematic illustration of alveolar neovascularization (a) as seen in microvascular corrosion cast replicas with sprouting angiogenesis (b), which is frequently seen subpleurally and in compact growth zones. Sprouts are evident as blind ends or protrusions (arrowheads). c Alveolar intussusceptive angiogenesis, recognizable by the presence of numerous small caliber holes with diameters between 1 and 5 μm as hallmarks of pillar formation (arrows). d The formation of new alveolar septa is accompanied by the occurrence of parallely orientated intussusceptive pillars (arrows)—visisble in the cast as holes—ensuring a rapid expansion of the alveolar microvascular network. e Vascular remodeling also occurs on the AER vessels (arrowhead). Intussusceptive pillars may merge and split up the primary vessel