This study aimed to explore the value of synchrotron-based phase-contrast microcomputed tomography (micro-CT) in pulmonary vascular pathobiology. The microanatomy of the lung is complex with intricate branching patterns. Tissue sections are therefore difficult to interpret. Recruited intrapulmonary bronchopulmonary anastomoses (IBAs) have been described in several forms of pulmonary hypertension, including alveolar capillary dysplasia with misaligned pulmonary veins (ACD/MPV). Here, we examine paraffin-embedded tissue using this nondestructive method for high-resolution three-dimensional imaging. Blocks of healthy and ACD/MPV lung tissue were used. Pulmonary and bronchial arteries in the ACD/MPV block had been preinjected with dye. One section per block was stained, and areas of interest were marked to allow precise beam-alignment during image acquisition at the X02DA TOMCAT beamline (Swiss Light Source). A ×4 magnifying objective coupled to a 20-µm thick scintillating material and a sCMOS detector yielded the best trade-off between spatial resolution and field-of-view. A phase retrieval algorithm was applied and virtual tomographic slices and video clips of the imaged volumes were produced. Dye injections generated a distinct attenuation difference between vessels and surrounding tissue, facilitating segmentation and three-dimensional rendering. Histology and immunohistochemistry post-imaging offered complementary information. IBAs were confirmed in ACD/MPV, and the MPVs were positioned like bronchial veins/venules. We demonstrate the advantages of using synchrotron-based phase-contrast micro-CT for three-dimensional characterization of pulmonary microvascular anatomy in paraffin-embedded tissue. Vascular dye injections add additional value. We confirm intrapulmonary shunting in ACD/MPV and provide support for the hypothesis that MPVs are dilated bronchial veins/venules.

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 one lumen were defined as plexiform. Prior radiopaque dye injection in some samples facilitated 3D-rendering. Four distinct types of plexiform lesions were identified: (1) localized within or derived from monopodial branches (supernumerary arteries), often with 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 re-canalization. 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, peri-bronchial vessels or the vasa vasorum. Collaterals, by-passing 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.

Pulmonary arterial hypertension (PAH) is a lethal condition for which there is no effective curative pharmacotherapy. PAH is characterized by vasoconstriction, wall thickening of pulmonary arteries, and increased vascular resistance. Versican is a chondroitin sulfate proteoglycan in the vascular extracellular matrix that accumulates following vascular injury and promotes smooth-muscle cell proliferation in systemic arteries. Here, we investigated whether versican may play a similar role in PAH. Paraffin-embedded lung sections from patients who underwent lung transplantation to treat PAH were used for immunohistochemistry. The etiologies of PAH in the subjects involved in this study were idiopathic PAH, scleroderma, and congenital heart disease (atrial septal defect) with left-to-right shunt. Independent of the underlying etiology, increased versican immunostaining was observed in areas of medial thickening, in neointima, and in plexiform lesions. Western blot of lung tissue lysates confirmed accumulation of versican in patients with PAH. Double staining for versican and CD45 showed only occasional colocalization in neointima of high-grade lesions and plexiform lesions. In vitro, metabolic labeling with [(35)S]sulfate showed that human pulmonary artery smooth-muscle cells (hPASMCs) produce mainly chondroitin sulfate glycosaminoglycans. In addition, hypoxia, but not cyclic stretch, was demonstrated to increase both versican messenger RNA expression and protein synthesis by hPASMCs. Versican accumulates in vascular lesions of PAH, and the amount of versican correlates more with lesion severity than with underlying etiology or inflammation. Hypoxia is a possible regulator of versican accumulation, which may promote proliferation of pulmonary smooth-muscle cells and vascular remodeling in PAH.

We recently described four distinct types of plexiform lesions in human idiopathic and familial pulmonary arterial hypertension (PAH) [1], visualising the three-dimensional lesion structure using synchrotron-based phase-contrast micro-computed tomography (SPµCT). Two types, 1 and 2, are shunt-type lesions that connect pulmonary arteries to the bronchial circulation: type 1 to the vasa vasorum, and type 2 to peribronchial vessels. Type 3 lesions are found peripherally in the lung as spherical structures abruptly terminating the distal pulmonary artery/arteriole, and type 4 lesions are characterised by recanalisation of an occluded artery/arteriole. Our observation of type 1 and type 2 lesions in PAH supports previous work that demonstrated intrapulmonary bronchopulmonary anastomoses (IBAs) connected to plexiform lesions in human PAH, suggesting that shunting of blood can occur within lesions in the setting of supra-systemic pulmonary arterial pressure [2]. Further haemodynamic studies of distinct subtypes of plexiform lesions have been hampered by the lack of available animal models with plexiform lesions representative of the full range of lesion types found in human disease. Plexiform lesions have previously been described in the Sugen5416/hypoxia rat model of pulmonary hypertension when time until sacrifice following hypoxia is extended to 13-14 weeks. Initially plexiform lesions were identified within the pulmonary artery, as well as in the form of aneurysm-like lesions projecting outside the vessel lumen [3], and recently the latter type was shown to form in supernumerary arteries [4]. However, neither study observed plexiform lesions communicating with the bronchial circulation, possibly because of methodological limitations of the histological analysis.Here, we set out to further characterise the range of plexiform lesion types in the prolonged Sugen5416/ hypoxia rat model using SPµCT combined with injections of a radiopaque dye. As previously suggested by our group and others [5], SPµCT grants superior three-dimensional imaging of biological structures with low and homogenous attenuation and is an emerging tool in the fields of pulmonary vascular physiology and digital pathology.The well-established Sugen5416/hypoxia model, combining vascular endothelial growth factor receptor 2 inhibitor injections with a second hit of hypoxia, was used as initially described [6], but sacrifice was delayed until 15 weeks post injections. No animals died during the study period. Following removal of the heart-lung block, the main pulmonary artery was injected with a radiopaque green dye (CDI's Tissue Marking Dye; Cancer Diagnostics, Durham, NC, USA), diluted with an equal volume of tap water to ensure low viscosity. Super-resolution x-ray radiographies of paraffin-embedded lobes were acquired using a laboratory setup as previously described [7], confirming the presence of contrast agent (radiopaque green dye) and thus enabling tracing of corresponding areas between individuals for subsequent high-resolution SPµCT imaging, ...

Pulmonary vasodilator therapy is still often an off-label treatment for pulmonary hypertension in children. The aim of this nationwide register-based study was to assess patient characteristics and strategies for pulmonary vasodilator therapy in young Swedish children. Prescription information for all children below seven years of age at treatment initiation, between 2007-2017, was retrieved from the National Prescribed Drug Register and medical information was obtained by linkage to other registers. All patients were categorized according to the WHO classification of pulmonary hypertension. In total, 233 patients had been prescribed pulmonary vasodilators. The treatment was initiated before 1 year of age in 61% (N=143). Sildenafil was most common (N=224 patients), followed by bosentan (N=29), iloprost (N=14), macitentan (N=4), treprostinil (N=2) and riociguat (N=2). Over the study period the prescription rate for sildenafil tripled. Monotherapy was most common, 87% (N=203), while 13% (N=20) had combination therapy. Bronchopulmonary dysplasia (N=82, 35%) and/or congenital heart defects (CHD; N=156, 67%) were the most common associated conditions. 8% (N=18) of the patients had Down syndrome. Cardiac catheterization had been performed in 39% (N=91). Overall mortality was 13% (N=30) during the study period. This study provides an unbiased overview of national outpatient use of pulmonary vasodilator therapy in young children. Few cases of idiopathic pulmonary arterial hypertension were found, but a large proportion of pulmonary hypertension associated with CHD or bronchopulmonary dysplasia. Despite treatment, mortality was high and additional pediatric studies are needed for a better understanding of underlying pathologies and evidence of treatment effects.