A Morphomolecular Approach to Alveolar Capillary Dysplasia
2022; Elsevier BV; Volume: 192; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2022.05.004
ISSN1525-2191
AutoresJan C. Kamp, Lavinia Neubert, Maximilian Ackermann, Helge Stark, Edith Plucinski, Harshit Shah, Sabina Janciauskiene, Anke K. Bergmann, Gunnar Schmidt, Tobias Welte, Axel Haverich, Christopher Werlein, Peter Braubach, Florian Laenger, Nicolaus Schwerk, Karen M. Olsson, Jan Fuge, Da‐Hee Park, Jonas C. Schupp, Marius M. Hoeper, Mark Kuehnel, Danny Jonigk,
Tópico(s)Neonatal Respiratory Health Research
ResumoAlveolar capillary dysplasia (ACD) is a rare lung developmental disorder leading to persistent pulmonary arterial hypertension and fatal outcomes in newborns. The current study analyzed the microvascular morphology and the underlying molecular background of ACD. One ACD group (n = 7), one pulmonary arterial hypertension group (n = 20), and one healthy con1trol group (n = 16) were generated. Samples of histologically confirmed ACD were examined by exome sequencing and array-based comparative genomic hybridization. Vascular morphology was analyzed using scanning electron microscopy of microvascular corrosion casts. Gene expression and biological pathways were analyzed using two panels on inflammation/kinase-specific genes and a comparison analysis tool. Compartment-specific protein expression was analyzed using immunostaining. In ACD, there was an altered capillary network, a high prevalence of intussusceptive angiogenesis, and increased activity of C-X-C motif chemokine receptor 4 (CXCR4), hypoxia-inducible factor 1α (HIF1A), and angiopoietin signaling pathways compared with pulmonary arterial hypertension/healthy controls. Histologically, there was a markedly increased prevalence of endothelial tyrosine kinase receptor (TEK/TIE2)+ macrophages in ACD, compared with the other groups, whereas the CXCR4 ligand CXCL12 and HIF1A showed high expression in all groups. ACD is characterized by dysfunctional capillaries and a high prevalence of intussusceptive angiogenesis. The results indicate that endothelial CXCR4, HIF1A, and angiopoietin signaling as well as TIE2+ macrophages are crucial for the induction of intussusceptive angiogenesis and vascular remodeling. Future studies should address the use of anti-angiogenic agents in ACD, where TIE2 appears as a promising target. Alveolar capillary dysplasia (ACD) is a rare lung developmental disorder leading to persistent pulmonary arterial hypertension and fatal outcomes in newborns. The current study analyzed the microvascular morphology and the underlying molecular background of ACD. One ACD group (n = 7), one pulmonary arterial hypertension group (n = 20), and one healthy con1trol group (n = 16) were generated. Samples of histologically confirmed ACD were examined by exome sequencing and array-based comparative genomic hybridization. Vascular morphology was analyzed using scanning electron microscopy of microvascular corrosion casts. Gene expression and biological pathways were analyzed using two panels on inflammation/kinase-specific genes and a comparison analysis tool. Compartment-specific protein expression was analyzed using immunostaining. In ACD, there was an altered capillary network, a high prevalence of intussusceptive angiogenesis, and increased activity of C-X-C motif chemokine receptor 4 (CXCR4), hypoxia-inducible factor 1α (HIF1A), and angiopoietin signaling pathways compared with pulmonary arterial hypertension/healthy controls. Histologically, there was a markedly increased prevalence of endothelial tyrosine kinase receptor (TEK/TIE2)+ macrophages in ACD, compared with the other groups, whereas the CXCR4 ligand CXCL12 and HIF1A showed high expression in all groups. ACD is characterized by dysfunctional capillaries and a high prevalence of intussusceptive angiogenesis. The results indicate that endothelial CXCR4, HIF1A, and angiopoietin signaling as well as TIE2+ macrophages are crucial for the induction of intussusceptive angiogenesis and vascular remodeling. Future studies should address the use of anti-angiogenic agents in ACD, where TIE2 appears as a promising target. Persistent pulmonary hypertension of the newborn is a rare clinical condition resulting from the failed transition from in utero to post-partum circulation, with an annual incidence rate of 30.1 cases per million children.1van Loon R.L. Roofthooft M.T. Hillege H.L. ten Harkel A.D. van Osch-Gevers M. Delhaas T. Kapusta L. Strengers J.L. Rammeloo L. Clur S.A. Mulder B.J. Berger R.M. Pediatric pulmonary hypertension in the Netherlands: epidemiology and characterization during the period 1991 to 2005.Circulation. 2011; 124: 1755-1764Crossref PubMed Scopus (211) Google Scholar The fetal circulation is characterized by high pulmonary vascular resistance and low pulmonary blood flow.2Roth W. Bucsenez D. Bläker H. Berger I. Schnabel P.A. Misalignment of pulmonary vessels with alveolar capillary dysplasia: association with atrioventricular septal defect and quadricuspid pulmonary valve.Virchows Arch. 2006; 448: 375-378Crossref PubMed Scopus (16) Google Scholar Physiologically, this constellation reverses within minutes after delivery with a decrease in pulmonary vascular resistance and increased pulmonary blood flow. Failure of this circulatory adaptation results in persistent pulmonary hypertension of the newborn, which, in turn, leads to right-to-left shunting of blood through the patent ductus arteriosus or the foramen ovale and causes severe hypoxemia.3Martinho S. Adão R. Leite-Moreira A.F. Brás-Silva C. Persistent pulmonary hypertension of the newborn: pathophysiological mechanisms and novel therapeutic approaches.Front Pediatr. 2020; 8: 342Crossref PubMed Scopus (17) Google ScholarAlveolar capillary dysplasia (ACD) is a rare underlying disease of persistent pulmonary hypertension of the newborn which frequently manifests concomitantly with a suspected misalignment of the pulmonary veins (MPV).4Cater G. Thibeault D.W. Beatty Jr., E.C. Kilbride H.W. Huntrakoon M. Misalignment of lung vessels and alveolar capillary dysplasia: a cause of persistent pulmonary hypertension.J Pediatr. 1989; 114: 293-300Abstract Full Text PDF PubMed Scopus (75) Google Scholar In the current classification of children's interstitial lung diseases, ACD is assigned to group A2, related to lung developmental disorders.5Deutsch G.H. Young L.R. Deterding R.R. Fan L.L. Dell S.D. Bean J.A. Brody A.S. Nogee L.M. Trapnell B.C. Langston C. Pathology Cooperative Group Albright E.A. Askin F.B. Baker P. Chou P.M. Cool C.M. Coventry S.C. Cutz E. Davis M.M. Dishop M.K. Galambos C. Patterson K. Travis W.D. Wert S.E. White F.V. ChILD Research Co-operativeDiffuse lung disease in young children: application of a novel classification scheme.Am J Respir Crit Care Med. 2007; 176: 1120-1128Crossref PubMed Scopus (336) Google Scholar Affected infants experience respiratory distress and cyanosis immediately or within the first weeks after birth,6Michalsky M.P. Arca M.J. Groenman F. Hammond S. Tibboel D. Caniano D.A. Alveolar capillary dysplasia: a logical approach to a fatal disease.J Pediatr Surg. 2005; 40: 1100-1105Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar whereas later presentations are less frequent.7Ito Y. Akimoto T. Cho K. Yamada M. Tanino M. Dobata T. Kitaichi M. Kumaki S. Kinugawa Y. A late presenter and long-term survivor of alveolar capillary dysplasia with misalignment of the pulmonary veins.Eur J Pediatr. 2015; 174: 1123-1126Crossref PubMed Scopus (23) Google Scholar,8Kodama Y. Tao K. Ishida F. Kawakami T. Tsuchiya K. Ishida K. Takemura T. Nakazawa A. Matsuoka K. Yoda H. Long survival of congenital alveolar capillary dysplasia patient with NO inhalation and epoprostenol: effect of sildenafil, beraprost and bosentan.Pediatr Int. 2012; 54: 923-926Crossref PubMed Scopus (13) Google Scholar In contrast to other severe children's interstitial lung disease entities manifesting during early infancy, such as surfactant-related disorders, the leading symptom is pulmonary hypertension, and radiological signs, such as diffuse ground glass opacities, are often missing. In most cases, ACD is a fatal developmental disorder of the pulmonary vasculature, and the only option for survival is lung transplantation, which is extremely challenging in newborns.9Towe C.T. White F.V. Grady R.M. Sweet S.C. Eghtesady P. Wegner D.J. Sen P. Szafranski P. Stankiewicz P. Hamvas A. Cole F.S. Wambach J.A. Infants with atypical presentations of alveolar capillary dysplasia with misalignment of the pulmonary veins who underwent bilateral lung transplantation.J Pediatr. 2018; 194: 158-164.e1Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar,10Nakajima D. Oda H. Mineura K. Goto T. Kato I. Baba S. Ikeda T. Chen-Yoshikawa T.F. Date H. Living-donor single-lobe lung transplantation for pulmonary hypertension due to alveolar capillary dysplasia with misalignment of pulmonary veins.Am J Transplant. 2020; 20: 1739-1743Crossref PubMed Scopus (4) Google ScholarTo date, >200 ACD cases have been documented. Approximately 60% of all reported cases have been associated with distinct heterozygous variants of forkhead box F1 (FOXF1) gene (located at 16q24.1), its transcriptional enhancer (located 257 kb 5′ to FOXF1), or other genetic alterations.11Stankiewicz P. Sen P. Bhatt S.S. Storer M. Xia Z. Bejjani B.A. et al.Genomic and genic deletions of the FOX gene cluster on 16q24.1 and inactivating mutations of FOXF1 cause alveolar capillary dysplasia and other malformations.Am J Hum Genet. 2009; 84: 780-791Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar,12Szafranski P. Gambin T. Dharmadhikari A.V. Akdemir K.C. Jhangiani S.N. Schuette J. et al.Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.Hum Genet. 2016; 135: 569-586Crossref PubMed Scopus (62) Google Scholar Histopathologic features of ACD include the following: i) the presence of reduced and dysplastic alveoli; ii) a suspected MPV (ie, abnormal placement of pulmonary artery and vein in the same bronchiovascular bundle); iii) a rarefaction in pulmonary capillarization; iv) widened or thickened alveolar septa with concomitant apposition deficiency of the alveolar capillaries to the alveolar epithelia; v) medial hypertrophy of the small pulmonary arteries; vi) dilation of the pulmonary veins; and vii) incidence of thrombi in dilated veins or hypertrophied arteries.13Edwards J.J. Murali C. Pogoriler J. Frank D.B. Handler S.S. Deardorff M.A. Hopper R.K. Histopathologic and genetic features of alveolar capillary dysplasia with atypical late presentation and prolonged survival.J Pediatr. 2019; 210: 214-219.e2Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 14Pucci A. Zanini C. Ferrero F. Arisio R. Valori A. Abbruzzese P. Forni M. Misalignment of lung vessels: diagnostic role of conventional histology and immunohistochemistry.Virchows Arch. 2003; 442: 597-600Crossref PubMed Google Scholar, 15Miranda J. Rocha G. Soares H. Vilan A. Brandão O. Guimarães H. Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV): a case series.Case Rep Crit Care. 2013; 2013: 327250PubMed Google ScholarData on the molecular mechanisms leading to ACD, as well as data on the origin of the misaligned pulmonary veins in ACD, are still limited and partially controversial. In a recent work, Norvik et al16Norvik C. Westöö C.K. Peruzzi N. Lovric G. van der Have O. Mokso R. Jeremiasen I. Brunnström H. Galambos C. Bech M. Tran-Lundmark K. Synchrotron-based phase-contrast micro-CT as a tool for understanding pulmonary vascular pathobiology and the 3-D microanatomy of alveolar capillary dysplasia.Am J Physiol Lung Cell Mol Physiol. 2020; 318: L65-L75Crossref PubMed Scopus (20) Google Scholar analyzed the vascular microanatomy of ACD cases with suspected MPV using Synchrotron-based phase-contrast microcomputed X-ray tomography. They confirmed the existence of intrapulmonary bronchopulmonary shunts, which have previously also been reported by other groups.13Edwards J.J. Murali C. Pogoriler J. Frank D.B. Handler S.S. Deardorff M.A. Hopper R.K. Histopathologic and genetic features of alveolar capillary dysplasia with atypical late presentation and prolonged survival.J Pediatr. 2019; 210: 214-219.e2Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar In addition, they found suspected misaligned pulmonary veins in the designated position of bronchial veins/venules and hypothesized that these were not pulmonary, but bronchial, veins/venules. Recently, functional relevance of bronchopulmonary shunts for continued perfusion has been demonstrated in several other pulmonary diseases,17Ackermann M. Tafforeau P. Wagner W.L. Walsh C.L. Werlein C. Kühnel M.P. Länger F.P. Disney C. Bodey A.J. Bellier A. Verleden S.E. Lee P.D. Mentzer S.J. Jonigk D.D. The bronchial circulation in COVID-19 pneumonia.Am J Respir Crit Care Med. 2022; 205: 121-125Crossref PubMed Scopus (9) Google Scholar expanding the hypothesis by Norvik et al16Norvik C. Westöö C.K. Peruzzi N. Lovric G. van der Have O. Mokso R. Jeremiasen I. Brunnström H. Galambos C. Bech M. Tran-Lundmark K. Synchrotron-based phase-contrast micro-CT as a tool for understanding pulmonary vascular pathobiology and the 3-D microanatomy of alveolar capillary dysplasia.Am J Physiol Lung Cell Mol Physiol. 2020; 318: L65-L75Crossref PubMed Scopus (20) Google Scholar to additional diseases.This study further analyzed the vascular morphology of ACD and used molecular analyses to gain new insights into this rare but fatal disease.Materials and MethodsStudy Groups and HistopathologyAs ACD represents one entity out of the spectrum of pulmonary hypertensive disease, three study groups were generated: one ACD group, one group of patients diagnosed with idiopathic pulmonary arterial hypertension (PAH), and one group of healthy controls. Of the ACD group, all samples were formalin fixed, paraffin embedded (FFPE) and come from infants with a diagnosis of ACD with or without MPV. Samples were collected in the period 2005 to 2020 and were taken from the archives of the Institute of Pathology at Hannover Medical School (Hannover, Germany) and retrospectively reviewed. For the PAH group, FFPE material was selected from n = 20 lung explants; and for the control group, FFPE material was used from human pulmonary down-sizing specimens from n = 16 donors (ie, healthy tissues from lung grafts that were oversized in regard to the recipients' thorax, and therefore, were surgically down sized during transplantation). Every sample that was used as a control was diagnosed as healthy via histopathologic assessment before sampling. Sagittal sections were taken bilaterally from all lung lobes. Approximately 1-μm thick sections were cut and stained with hematoxylin and eosin, periodic acid–Schiff, and elastic van Gieson dyes. Diagnosis of ACD required the presence of widened or thickened alveolar septa with concomitant apposition deficiency of the alveolar capillaries to the alveolar epithelia and rarefaction in pulmonary capillarization as well as signs of pulmonary arterial hypertension, such as medial hypertrophy of the small pulmonary arteries. Cases with and without MPV were differentiated according to the presence of suspected misaligned pulmonary veins within the bronchiovascular bundle. This study was approved by the local ethics committee at Hannover Medical School (ethics vote number 2702-2015). All subjects or their relatives gave written informed consent.Genetic AnalysisDNA was isolated from FFPE blocks using the Maxwell RSC DNA FFPE Kit (Promega Corp., Madison, WI). DNA content was measured using the NanoDrop one microvolume UV-vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA), guaranteeing a minimum of 500 ng DNA in each sample. Whole exome sequencing and array-based comparative genomic hybridization were performed to analyze genetic variants that have been suggested to play a role in ACD in former studies to better characterize the study group. Genes analyzed were dedicator of cytokinesis 8 (DOCK8), epithelial splicing regulatory protein 1 (ESRP1), FOXF1, myosin phosphatase rho-interacting protein (MPRIP), plexin B2 (PLXNB2), solute carrier family 50 member 1 (SLC50A1), zinc finger myeloid, nervy, and deaf-type containing 11 (ZMYND11), and signaling receptor and transporter of retinol (STRA6).Vascular MorphologyMicrovascular architecture was analyzed via scanning electron micrographs of native lung tissue and microvascular corrosion casts using a Philips XL30 microscope (Philips, Eindhoven, the Netherlands) at 15 keV and 21 μA. Microvascular corrosion casts were generated according to a standardized protocol previously described by us.18Ackermann M. Houdek J.P. Gibney B.C. Ysasi A. Wagner W. Belle J. Schittny J.C. Enzmann F. Tsuda A. Mentzer S.J. Konerding M.A. Sprouting and intussusceptive angiogenesis in postpneumonectomy lung growth: mechanisms of alveolar neovascularization.Angiogenesis. 2014; 17: 541-551Crossref PubMed Scopus (51) Google ScholarGene ExpressionRNA was isolated using the Maxwell 16 LEV RNA FFPE Purification Kit (Promega Corp.). RNA content was measured using the Qubit RNA IQ Assay (Thermo Fisher Scientific), guaranteeing a minimum of 200 ng RNA in each sample. Samples were analyzed using two commercial panels on inflammation- and kinome-specific genes (536 and 255 target genes, respectively) and the nCounter Analysis System (NanoString Technologies, Seattle, WA), which is optimized for FFPE-based experiments. Counts were normalized using the nSolver analysis software version 3.0 (NanoString Technologies). Normalization included positive normalization (geometric mean), negative normalization (arithmetic mean), and reference normalization (geometric mean). Established reference genes (glyceraldehyde-3-phosphate dehydrogenase, glucuronidase β, hypoxanthine phosphoribosyltransferase 1, phosphoglycerate kinase 1, and tubulin β class I) were designated as reference genes for standardization processes. Assembling of parameters from both panels was conducted using the nSolver software while defining a multi–reporter library file (RLF) experiment and centering on 28 genes, including expression of the reference genes that were present in both panels. Further analyses on the ascertained log2 mRNA counts were performed using R software version 3.4.4 (R Foundation for Statistical Computing, Vienna, Austria; https://cran.r-project.org/bin/windows/base) and the nCounter Advanced Analysis module version 1.1.5. Absolute gene expression values were analyzed and compared with those of healthy control samples. FFPE lung tissue from healthy down-sized lung transplants (ie, healthy lung tissue that was donated but not used for transplantation) was used as control. Two samples were obtained from each patient for the mRNA expression analysis. Because of technical problems, only 12 of 14 obtained ACD samples could be used for the analysis. However, as these failures occurred in samples from different patients, at least one sample repeat was included for each patient. U-tests were used for pairwise comparisons between groups. Kruskal-Wallis tests were used for multigroup comparisons, providing false discovery rates. Statistical analysis was concluded by correction for multiple testing (Holm Bonferroni method). False discovery rate values <0.05 were considered statistically significant.Biological PathwaysBiological pathway analysis was performed using the comparison analysis module from the Ingenuity Pathway Analysis tool (Qiagen Inc., Venlo, the Netherlands) to complement our gene expression data by predictions on the activation/inhibition of biological functions. The relative gene expression signature of each ACD sample was compared with the median gene expression of the control samples. All significantly regulated genes (false discovery rate, <0.05) of these comparisons were used as input parameters. The analysis was performed with default settings. As described before, the Ingenuity Pathway Analysis tool calculates z scores as a quantitative confidence estimation that a biological function is either activated (positive z score) or inactivated (negative z score), as well as P values.19Neubert L. Borchert P. Stark H. Hoefer A. Vogel-Claussen J. Warnecke G. Eubel H. Kuenzler P. Kreipe H.H. Hoeper M.M. Kuehnel M. Jonigk D. Molecular profiling of vascular remodeling in chronic pulmonary disease.Am J Pathol. 2020; 190: 1382-1396Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Significant differences in the predicted regulation of biological functions were determined via pairwise U-tests and multigroup Kruskal-Wallis tests. P < 0.05 was considered statistically significant.Protein ExpressionParaffin sections (2 μm thick) of all groups were used for immunostaining. After deparaffinization twice with xylene for 10 minutes, rehydration was performed using the decreasing concentration of ethanol. The sections were subjected to heat-induced epitope retrieval in respective antibody buffer and stained with primary antibodies, according to the manufacturer's protocol using ZytoChem Plus HRP Polymer Kit (Zytomed Systems, Berlin, Germany) and 3, 3′-diaminobinzidine solution. The staining Eukitt mounting medium was used as an adhesive and a sealant. Images were taken after automated whole-slide imaging using the APERIO CS2 scanner (Leica Biosystems, Wetzlar, Germany) and ImageScope software version 12.3.3.5048 (Leica Biosystems). Pediatric healthy lung tissue as well as lung explants from patients diagnosed with PAH were used as controls. Appropriate markers were selected on the basis of the disease-specific mRNA profile and putatively involved biological pathways. Details about all antibodies utilized are depicted in Table 1. Immunostaining patterns of ACD samples were compared with appropriate control samples.Table 1Target Antibodies Used for ImmunohistochemistryTarget of antibodyProduct nameHost speciesPretreatmentDilutionManufacturerCXCL12SDF1 (CXCL12) Polyclonal Antibody (AP20632PU-N)RabbitSodium citrate buffer1:200Origene (Rockville, MD)HIF1AAnti–HIF-1 α antibody (ESEE122; ab8366)MouseTris-EDTA buffer1:1000Abcam (Cambridge, UK)TIE2 (TEK)Anti-TIE2, C-terminal antibody (SAB4502942)RabbitSodium citrate buffer1:500Sigma Aldrich (St. Louis, MO)BMPR2Anti-BMPR2 antibody (1F12; ab130206)RabbitSodium citrate buffer1:5000Abnova (Taipei, Taiwan)WNT10bAnti-Wnt10b antibody (ab70816)RabbitTris-EDTA buffer1:500AbcamVEGFR1Recombinant Anti-VEGF Receptor 1 antibody (Y103; ab32152)MouseTris-EDTA buffer1:50AbcamSTAT3Recombinant Anti-STAT3 (phosphorylated Y705) antibody (EPR23968-52; ab267373)MouseTris-EDTA buffer1:50AbcamBMPR2, bone morphogenetic protein receptor type 2; HIF1A, hypoxia-inducible factor 1α; TIE2 (TEK), TEK receptor tyrosine kinase; VEGFR1, vascular endothelial growth factor 1; WNT10b, WNT family member 10b. Open table in a new tab ResultsStudy GroupsPatient characteristics are shown in Supplemental Table S1. The ACD group was composed of five male and two female patients, and the median age at sampling was 1 month (interquartile range, 25/75; 0.58 to 8.5 months). Samples were obtained from four autopsies and two biopsies, and one sample was obtained from a lung explant of a patient who underwent lung transplantation. Pathogenic variants were not identified. The PAH group was composed of 3 male and 17 female patients, and the median age at sampling was 25.5 years (interquartile range, 25/75; 16.25 to 37.25 years). Clinical information regarding the control group is not available, to respect legal regulations and the anonymity of donors.Morphologic AnalysisIn all cases, the diagnosis of ACD/PAH was confirmed histologically by three experienced pulmonary pathologists (L.N., F.L., and D.D.J.). As shown in Figure 1, the histopathologic workup revealed typical features of ACD in all cases (ie, rarefied pulmonary capillaries, thickened alveolar septa with malapposed capillaries, medial hypertrophy of the small pulmonary arteries, and pulmonary venous dilation), whereas suspected misaligned pulmonary veins were present in n = 4 cases. Scanning electron microscopy of microvascular corrosion casts revealed multiple angiomatoid-like expansions of the vascular plexus with widened, deformed, blunt-ended capillaries and without functional capillary network or any perceptible vessel hierarchy. In addition, multiple intussusceptive pillars were observed in loco typico, indicating early stages of intussusceptive angiogenesis. High-magnification scanning electron microscopy of native lung tissue showed thickened alveolar septa and protrusions within the alveolar wall, as well as malapposed and flattened capillaries.Gene ExpressionAs depicted in Figure 2, 163 of 791 genes showed a differential expression on the mRNA level compared with controls: n = 96 in ACD, and n = 94 in PAH. Of these, 26 genes showed similar expression in both entities: increased expression was found for activin A receptor type 2B (ACVR2B), ataxia telangiectasia-related serine/threonine kinase (ATR), aurora kinase A (AURKA), calcium/calmodulin-dependent protein kinase IV (CAMK4), casein kinase 1G3 and 2A1 (CSNK1G3/2A1), capped intron-containing pre-mRNA–like kinases 2 and 4 (CLK2/4), ephrin type-A receptor 3 (EPHA3), high-mobility group nucleosome-binding domain 1 (HMGN1), microtubule affinity regulating kinase 1 (MARK1), microtubule-associated serine/threonine kinases 2 and 4 (MAST2/4), nuclear factor of activated T cells 3 (NFATC3), protein kinase AMP-activated catalytic subunit α 1 (PRKAA1), serine/arginine-rich splicing factor protein kinase 2 (SRPK2), protein kinase-like protein sugen kinase 196 (SGK196), transforming growth factor-β regulator 4 (TBRG4), Tau tubulin kinase 2 (TTBK2), vaccinia virus B1R-related kinase 2 (VRK2), and several mitogen-activated protein kinases (MAPK), whereas decreased expression was found for G-protein–coupled receptor kinase 6 (GRK6) and IL-10 (IL10). C-X-C motif chemokine receptor 4 (CXCR4) showed decreased expression in ACD and increased expression in PAH. Apart from these equally regulated genes, the study focused on genes that are known to be involved in lung development, vasculogenesis, angiogenesis, and the development or progression of pulmonary hypertension. In this respect, there was increased expression of transforming growth factor-β3 (TGFB3), activin A receptor type 2A (ACVR2A), budding uninhibited by benzimidazoles 1 mitotic checkpoint serine/threonine kinase (BUB1), calcium/calmodulin-dependent protein kinase II delta (CAMK2D), cyclin-dependent kinases 1, 6, and 20 (CDK1/6/20), death domain–associated protein (DAXX), glycogen synthase kinase 3β (GSK3B), homeodomain-interacting protein kinase 3 (HIPK3), integrin-linked kinase (ILK), protein tyrosine kinase 7 (PTK7), receptor tyrosine kinase-like orphan receptor 2 (ROR2), transcription factor 4 (TCF4), nuclear factor of activated T cells 3 (NFATC3), and thymic stromal lymphopoietin (TSLP), as well as a decreased expression of arachidonate 5-lipoxygenase (ALOX5), CD163 (CD163), cyclin-dependent kinase 5 (CDK5), colony-stimulating factor 3 (CSF3), CXCL2/9, C-X-C motif chemokine receptor 1 (CXCR1), feline Gardner-Rasheed sarcoma viral oncogene homolog proto-oncogene, Src family tyrosine kinase (FGR), IL-6 (IL6), IL-18 receptor accessory protein (IL18RAP), matrix metalloproteinase 9 (MMP9), prostaglandin E receptor 1 and 4 (PTGER1/4), ras homolog family member A (RHOA), as well as toll-like receptors 2 and 4 (TLR2/4).Figure 2Venn diagram of differentially regulated genes in alveolar capillary dysplasia (ACD) and pulmonary arterial hypertension (PAH). Arrowheads indicate increased (black arrowheads) or decreased (red arrowheads) activity of the respective genes in each group; in the overlapping area (middle), left and right arrowheads indicate the expression in ACD and PAH, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Biological PathwaysBiological pathway analysis revealed several activated or inhibited molecular pathways in ACD and PAH, as shown in Supplemental Table S2. In comparison to healthy controls, increased activity was observed for angiopoietin, CXCR4 signaling, hypoxia-inducible factor 1α (HIF1A) signaling, transforming growth factor-β (TGFB) signaling, bone morphogenetic protein (BMP) signaling, vascular endothelial growth factor (VEGF) signaling, STAT3 signaling, NF-κB signaling, mechanistic target of rapamycin kinase signaling, and cardiac hypertrophy signaling pathways in both diseases. TLR signaling, acute-phase response signaling, and IL-6 signaling showed lower activity in ACD but higher activity in PAH.Protein ExpressionOn the basis of the disease-specific mRNA expression, the results of the biological pathway analysis and the morphologic findings of an increased prevalence of intussusceptive angiogenesis, we selected TEK receptor tyrosine kinase (TIE2), CXCL12, HIF1A, bone morphogenetic protein receptor 2 (BMPR2), Wnt family member 10b (Wnt10b), STAT3, and VEGF receptor 1 (VEGFR1) as target molecules. The results of the immunohistochemical staining are depicted in Figures 3 and 4.Figure 3Immunostaining with established markers of intussusceptive angiogenesis in alveolar capillary dysplasia (ACD) with misalignment of the pulmonary veins, idiopathic pulmonary arterial hypertension (PAH), and pediatric healthy controls. A: Large clusters of macrophages within the alveolar spaces as well as migrating macrophages within the alveolar septa (arrowhead) were observed in ACD samples. All macrophages showed a consistent positivity for TEK receptor tyrosine kinase (TIE2). B and C: In contrast, in PAH lungs (B), all macrophages appeared TIE2– (arrowheads); and in controls (C), only few scattered TIE2+ macrophages were found between predominantly TIE2– macrophages (arrowheads). D and G: CXCL12 (D) and hypoxia-inducible factor 1α (HIF1A; G) showed a cytoplasmic enrichment in endothelial cells (arrowhead), respiratory epithelial cells, and septa, as well as in bronchus-associated lymphoid tissue (BALT; asterisk) in ACD lungs. E, F, H, and I: In PAH lungs, CXCL12 (E) was negative, whereas HIF1A (H) was enhanced,
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