Plexiform Lesions in Pulmonary Arterial Hypertension
2011; Elsevier BV; Volume: 179; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.03.040
ISSN1525-2191
AutoresDanny Jonigk, Heiko Golpon, Clemens L. Bockmeyer, Lavinia Maegel, Marius M. Hoeper, Jens Gottlieb, Nils Nickel, Kais Hussein, Ulrich A. Maus, Ulrich Lehmann, Sabina Janciauskiene, Tobias Welte, Axel Haverich, Johanna Rische, Hans Kreipe, Florian Laenger,
Tópico(s)Cardiac Structural Anomalies and Repair
ResumoPulmonary arterial hypertension (PAH) is a debilitating disease with a high mortality rate. A hallmark of PAH is plexiform lesions (PLs), complex vascular formations originating from remodeled pulmonary arteries. The development and significance of these lesions have been debated and are not yet fully understood. Some features of PLs resemble neoplastic disorders, and there is a striking resemblance to glomeruloid-like lesions (GLLs) in glioblastomas. To further elucidate PLs, we used in situ methods, such as (fluorescent) IHC staining, three-dimensional reconstruction, and laser microdissection, followed by mRNA expression analysis. We generated compartment-specific expression patterns in the lungs of 25 patients (11 with PAH associated with systemic shunts, 6 with idiopathic PAH, and 8 controls) and GLLs from 5 glioblastomas. PLs consisted of vascular channels lined by a continuously proliferating endothelium and backed by a uniform myogenic interstitium. They also showed up-regulation of remodeling-associated genes, such as HIF1a, TGF-β1, VEGF-α, VEGFR-1/-2, Ang-1, Tie-2, and THBS1, but also of cKIT and sprouting-associated markers, such as NOTCH and matrix metalloproteinases. The cellular composition and signaling seen in GLLs in neural neoplasms differed significantly from those in PLs. In conclusion, PLs show a distinct cellular composition and microenvironment, which contribute to the plexiform phenotype and set them apart from other processes of vascular remodeling in patients with PAH. Neoplastic models of angiogenesis seem to be of limited use in further study of plexiform vasculopathy. Pulmonary arterial hypertension (PAH) is a debilitating disease with a high mortality rate. A hallmark of PAH is plexiform lesions (PLs), complex vascular formations originating from remodeled pulmonary arteries. The development and significance of these lesions have been debated and are not yet fully understood. Some features of PLs resemble neoplastic disorders, and there is a striking resemblance to glomeruloid-like lesions (GLLs) in glioblastomas. To further elucidate PLs, we used in situ methods, such as (fluorescent) IHC staining, three-dimensional reconstruction, and laser microdissection, followed by mRNA expression analysis. We generated compartment-specific expression patterns in the lungs of 25 patients (11 with PAH associated with systemic shunts, 6 with idiopathic PAH, and 8 controls) and GLLs from 5 glioblastomas. PLs consisted of vascular channels lined by a continuously proliferating endothelium and backed by a uniform myogenic interstitium. They also showed up-regulation of remodeling-associated genes, such as HIF1a, TGF-β1, VEGF-α, VEGFR-1/-2, Ang-1, Tie-2, and THBS1, but also of cKIT and sprouting-associated markers, such as NOTCH and matrix metalloproteinases. The cellular composition and signaling seen in GLLs in neural neoplasms differed significantly from those in PLs. In conclusion, PLs show a distinct cellular composition and microenvironment, which contribute to the plexiform phenotype and set them apart from other processes of vascular remodeling in patients with PAH. Neoplastic models of angiogenesis seem to be of limited use in further study of plexiform vasculopathy. Pulmonary arterial hypertension (PAH) may occur either as a primary disease of unknown cause [idiopathic PAH (IPAH)] or as an associated manifestation of other diseases or malformations [eg, congenital shunts between the systemic and pulmonary circulation; associated PAH (APAH)].1Simonneau G. Galie N. Rubin L.J. Langleben D. Seeger W. Domenighetti G. Gibbs S. Lebrec D. Speich R. Beghetti M. Rich S. Fishman A. Clinical classification of pulmonary hypertension.J Am Coll Cardiol. 2004; 43: 5S-12SAbstract Full Text Full Text PDF PubMed Scopus (1547) Google Scholar, 2Firth A.L. Mandel J. Yuan J.X. Idiopathic pulmonary arterial hypertension.Dis Model Mech. 2010; 3: 268-273Crossref PubMed Scopus (48) Google Scholar Characteristic histologic findings of PAH include remodeling of small pulmonary arteries and arterioles with varying degrees of endothelial cell proliferation, muscular hypertrophy, and intimal fibrosis, ultimately leading to an obliteration of precapillary vessels. Morphologic hallmarks of severe PAH are the so-called plexiform lesions (PLs): complex, glomeruloid-like vascular structures originating from the pulmonary arteries.3Sakao S. Tatsumi K. Voelkel N.F. Endothelial cells and pulmonary arterial hypertension: apoptosis, proliferation, interaction and transdifferentiation.Respir Res. 2009; 10: 95Crossref PubMed Scopus (163) Google Scholar, 4Pietra G.G. Edwards W.D. Kay J.M. Rich S. Kernis J. Schloo B. Ayres S.M. Bergofsky E.H. Brundage B.H. Detre K.M. Histopathology of primary pulmonary hypertension: a qualitative and quantitative study of pulmonary blood vessels from 58 patients in the National Heart, Lung, and Blood Institute, Primary Pulmonary Hypertension Registry.Circulation. 1989; 80: 1198-1206Crossref PubMed Scopus (408) Google Scholar Whether PLs represent just a morphologic “indicator lesion” or play a role in the pathogenesis and/or progression of PAH has not yet been clarified. On a more basal level, even the actual cellular composition of PLs has not been conclusively determined: the high plasticity of the (mesenchymal) cells involved, ie, their ability to change their phenotype depending on the current local microenvironment, have hampered analysis.5Stevens T. Molecular and cellular determinants of lung endothelial cell heterogeneity.Chest. 2005; 128: 558S-564SCrossref PubMed Scopus (48) Google Scholar, 6Owens G.K. Kumar M.S. Wamhoff B.R. Molecular regulation of vascular smooth muscle cell differentiation in development and disease.Physiol Rev. 2004; 84: 767-801Crossref PubMed Scopus (2582) Google Scholar The favored consensus describes the PL as a proliferating network of endothelial-lined vascular channels supported by a core of specialized and apoptosis-resistant myofibroblasts, smooth muscle cells, or even undifferentiated mesenchymal cells.7Cool C.D. Stewart J.S. Werahera P. Miller G.J. Williams R.L. Voelkel N.F. Tuder R.M. Three-dimensional reconstruction of pulmonary arteries in plexiform pulmonary hypertension using cell-specific markers: evidence for a dynamic and heterogeneous process of pulmonary endothelial cell growth.Am J Pathol. 1999; 155: 411-419Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar Animal models using chronic hypoxia combined with the application of SU5416 (semaxinib), a vascular endothelial growth factor receptor (VEGFR) blocker, have produced glomeruloid lesions that mimic the formation of PLs to a certain extent.8Abe K. Toba M. Alzoubi A. Ito M. Fagan K.A. Cool C.D. Voelkel N.F. McMurtry I.F. Oka M. Formation of plexiform lesions in experimental severe pulmonary arterial hypertension.Circulation. 2010; 121: 2747-2754Crossref PubMed Scopus (381) Google Scholar These lesions develop early after treatment with SU5416 combined with hypoxia, a situation that does not fully mirror the situation in humans, where there is evidence that severe pulmonary arterial pressure over a longer period is needed to induce PLs.9Abe M. Kimura T. Morimoto T. Taniguchi T. Yamanaka F. Nakao K. Yagi N. Kokubu N. Kasahara Y. Kataoka Y. Otsuka Y. Kawamura A. Miyazaki S. Horiuchi K. Ito A. Hoshizaki H. Kawaguchi R. Setoguchi M. Inada T. Kishi K. Sakamoto H. Morioka N. Imai M. Shiomi H. Nonogi H. Mitsudo K. Sirolimus-eluting stent versus balloon angioplasty for sirolimus-eluting stent restenosis: insights from the j-Cypher Registry.Circulation. 2010; 122: 42-51Crossref PubMed Scopus (37) Google Scholar Moreover, neoangiogenesis in peritumoral tissue, such as the so-called glomeruloid-like lesions (GLLs) in high-grade neural tumors, have also been discussed as a putative model for PLs.10Tuder R.M. Voelkel N.F. Plexiform lesion in severe pulmonary hypertension: association with glomeruloid lesion.Am J Pathol. 2001; 159: 382-383Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 11Skuli N. Liu L. Runge A. Wang T. Yuan L. Patel S. Iruela-Arispe L. Simon M.C. Keith B. Endothelial deletion of hypoxia-inducible factor-2α (HIF-2α) alters vascular function and tumor angiogenesis.Blood. 2009; 114: 469-477Crossref PubMed Scopus (190) Google Scholar, 12Sakao S. Tatsumi K. The effects of antiangiogenic compound SU5416 in a rat model of pulmonary arterial hypertension.Respiration. 2011; 81: 253-261Crossref PubMed Scopus (52) Google Scholar, 13Tuder R.M. Groves B. Badesch D.B. Voelkel N.F. Exuberant endothelial cell growth and elements of inflammation are present in plexiform lesions of pulmonary hypertension.Am J Pathol. 1994; 144: 275-285PubMed Google Scholar This experimental approach is compatible with the concept of the PL, or rather the adjacent artery from which it arises, as a circumscript angiogenic reservoir or niche that accommodates endothelial cells with a “quasi neoplastic” behavior, which contributes to remodeling of the pulmonary vasculature.14Rai P.R. Cool C.D. King J.A. Stevens T. Burns N. Winn R.A. Kasper M. Voelkel N.F. The cancer paradigm of severe pulmonary arterial hypertension.Am J Respir Crit Care Med. 2008; 178: 558-564Crossref PubMed Scopus (206) Google Scholar Thus, although extensive analyses have been performed in animal experiments or alternative, tumor-associated vascular models that show a degree of morphologic similarity, actual PLs in humans have not yet been studied in more detail. In this work, we studied the cellular composition, architecture, and local signaling of PLs in human PAH lungs. We compared the microenvironment in PLs with that in tumor-associated GLLs to examine the relevance of neoplastic models as a research platform for PLs. Furthermore, we aimed to explore whether PLs represent an epiphenomenon in severe PAH and whether the “angiogenic niche” plays a major role in vascular remodeling. We chose to focus on vascular remodeling in APAH associated with congenital heart disease. To this end, we selected 11 bilateral lung explants from patients with Eisenmenger's syndrome (New York Heart Association class IV). Of these patients, 7 showed prominent PLs (age at transplantation: arithmetic mean ± SD, 37.4 ± 9.9 years; median, 38 years) and 4 did not (age at transplantation: arithmetic mean ± SD, 34.5 ± 19.3 years; median, 28.8 years). Unagitated, nonremodeled pulmonary arteries taken from downsized lung tissue of donor lungs (n = 8) resected immediately before transplantation served as a reference for mRNA and protein expression analyses (Table 1).Table 1Characteristics of the 11 Study Patients and 8 ControlsNo.SexAPAH with PLsAPAH without PLsHeath-Edwards gradeControlsAge at Tx (years)Underlying conditionNYHA class at TxMean PAP at Tx (mmHg)1FX451Atrial septal defectIV652MX442Atrial septal defectIV703FX445Ventricular septal defectIV794FX431Ventricular septal defectIV725MX438Complex cardiac malformationIV656FX434Patent ductus arteriosusIV627FX421Univentricular heartIV688MX323Complex cardiac malformationIV659MX358Complex cardiac malformationIV6810FX342Complex cardiac malformationIV7011MX315Alström's syndromeIV8012FX66NANANA13FX56NANANA14FX46NANANA15FX42NANANA16MX49NANANA17MX43NANANA18MX43NANANA19MX20NANANAF, female; M, male; NA, not applicable; NYHA, New York Heart Association; PAP, pulmonary arterial pressure; Tx, treatment. Open table in a new tab F, female; M, male; NA, not applicable; NYHA, New York Heart Association; PAP, pulmonary arterial pressure; Tx, treatment. In addition, we selected six bilateral lung explants from patients with IPAH (New York Heart Association class IV), all of them exhibiting prominent PLs (age at transplantation: arithmetic mean ± SD, 32.7 ± 17.1 years; median, 34 years). One of these patients carried a mutation of the BMP receptor 2 (BMPR2) (see Supplemental Table S1 at http://ajp.amjpathol.org). All the specimens were inflated with formalin by the main bronchi and were formalin-fixed overnight before being extensively sampled and paraffin-embedded (FFPE). Subsequently, they were histologically evaluated, graded according to the Heath-Edwards classification, and correlated with clinical data to confirm the (histopathologic) diagnosis.15Heath D. Edwards J.E. The pathology of hypertensive pulmonary vascular disease: a description of six grades of structural changes in the pulmonary arteries with special reference to congenital cardiac septal defects.Circulation. 1958; 18: 533-547Crossref PubMed Google Scholar In addition, five high-grade glial neoplasms (glioblastomas multiforme) with prominent peritumoral neoangiogenesis forming so-called GLLs were examined. The FFPE samples were retrieved from the archives of the Institute of Pathology of Hannover Medical School and were handled anonymously, following the requirements of the local ethics committee. Serially cut slides were IHC stained for different markers of endothelial and smooth muscle differentiation, vascular remodeling, fibrosis, and inflammation-associated markers using monoclonal antibodies and following a standard ABC protocol [angiopoietin (Ang)-1, Ang-2, CD3, CD20, CD31, CD34, CD68 (KP1), CD117 (c-KIT), CD141 (thrombomodulin), desmin Ki-67, mast cell tryptase, myocardin, β-type platelet-derived growth factor receptor, podoplanin, SMA, smooth muscle myosin heavy chain (smmhc), transforming growth factor (TGF)-β1, thrombospondin (THBS)-1, and VEGF-α; Table 2]. The staining intensity in different compartments was assessed in at least two locations in both lobes of the lung specimens as no reactivity, barely visible reactivity at high magnification (“weak”), well-recognizable reactivity at medium magnification (“intermediate”), and high protein expression levels visible even at low magnification (“strong”).16Ruschoff J. Dietel M. Baretton G. Arbogast S. Walch A. Monges G. Chenard M.P. Penault-Llorca F. Nagelmeier I. Schlake W. Hofler H. Kreipe H.H. HER2 diagnostics in gastric cancer-guideline validation and development of standardized immunohistochemical testing.Virchows Arch. 2010; 457: 299-307Crossref PubMed Scopus (398) Google Scholar The range of positive cells in the different compartments was scored semiquantitatively as no apparent reaction (score 0), positivity in <30% (score 1), positivity in ≥30% and <60% (score 2), and positivity in ≥60% (score 3) of cells.17Jonigk D. Merk M. Hussein K. Maegel L. Theophile K. Muth M. Lehmann U. Bockmeyer C.L. Mengel M. Gottlieb J. Welte T. Haverich A. Golpon H. Kreipe H. Laenger F. Obliterative airway remodeling molecular evidence for shared pathways in transplanted and native lungs.Am J Pathol. 2011; 178: 599-608Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 18Jonigk D. Theophile K. Hussein K. Bock O. Lehmann U. Bockmeyer C.L. Gottlieb J. Fischer S. Simon A. Welte T. Maegel L. Kreipe H. Laenger F. Obliterative airway remodelling in transplanted and non-transplanted lungs.Virchows Arch. 2010; 457: 369-380Crossref PubMed Scopus (23) Google ScholarTable 2Antibodies Used in the StudyProteinCompanyDilutionAng-1R&D Systems, Minneapolis, MN1:15Ang-2R&D Systems1:50CD3Dako, Carpinteria, CA1:200CD20Dako1:50CD31Dako1:75CD34Leica Microsystems GmbH, Wetzlar, GermanyReady to useCD68 (KP1)Dako1:100CD117 (c-KIT)Zytomed Systems GmbH, Berlin, Germany1:50CD141 (thrombomodulin)AbD Serotec, Raleigh, NC1:20DesminDako1:75Ki-67Thermo Fisher Scientific, Waltham, MA1:100Mast cell tryptaseLeica Microsystems GmbH1:50MyocardinCovalab S.A.S., Villeurbanne France1:200β-Type platelet-derived growth factor receptorAcris Antibodies Inc., San Diego, CA1:50PodoplaninAcris Antibodies Inc.1:25SMADako1:25SmmhcUS Biological, Swampscott, MA1:100TGF-β1Acris Antibodies Inc.1:500THBS1Lifespan, Providence, RI1:20VEGF-αSanta Cruz Biotechnology, Santa Cruz, CA1:50The detection system was the DAB Zytomed HRP kit (Zytomed Systems GmbH) for all. Open table in a new tab The detection system was the DAB Zytomed HRP kit (Zytomed Systems GmbH) for all. We examined PLs and the adjacent arteries from which they sprout: remodeled arteries ≤750 μm from the corresponding PL, with varying degrees of media hypertrophy and (concentric) intimal proliferation. In PAH lungs without PLs, we analyzed remodeled arteries, which showed concentric laminar intimal proliferation and fibrosis (“concentric lesions” with a diameter of ≤500 μm). Unagitated arterial vessels from downsized lung tissue of donor lungs served as controls. Inflammatory cells in PLs and their adjacent vessels and in controls were quantitated by counting the number of positively marked cells per square millimeter (positivity for CD3, CD20, CD45, CD68, and mast cell tryptase; Table 2). Staining intensity, where applicable, was graded as weak, intermediate, or strong.17Jonigk D. Merk M. Hussein K. Maegel L. Theophile K. Muth M. Lehmann U. Bockmeyer C.L. Mengel M. Gottlieb J. Welte T. Haverich A. Golpon H. Kreipe H. Laenger F. Obliterative airway remodeling molecular evidence for shared pathways in transplanted and native lungs.Am J Pathol. 2011; 178: 599-608Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 18Jonigk D. Theophile K. Hussein K. Bock O. Lehmann U. Bockmeyer C.L. Gottlieb J. Fischer S. Simon A. Welte T. Maegel L. Kreipe H. Laenger F. Obliterative airway remodelling in transplanted and non-transplanted lungs.Virchows Arch. 2010; 457: 369-380Crossref PubMed Scopus (23) Google Scholar For negative controls, the primary antibody was replaced by bovine serum albumin. Serial sections (5-μm–thick FFPE tissue) were mounted on a poly-l-lysine–coated membrane fixed onto a metal frame. After deparaffinization and routine (hemalum) staining, the CellCut Plus system (MMI Molecular Machines & Industries AG, Glattbrugg, Switzerland) was used for laser-assisted microdissection. Pathologic and anatomic structures (PLs, adjacent arteries, concentric lesions in patients with APAH without PLs, and controls) were sampled from at least two locations in both lobes of the explants using a no-touch technique, as described elsewhere19Jonigk D. Lehmann U. Stuht S. Wilhelmi M. Haverich A. Kreipe H. Mengel M. Recipient-derived neoangiogenesis of arterioles and lymphatics in quilty lesions of cardiac allografts.Transplantation. 2007; 84: 1335-1342Crossref PubMed Scopus (22) Google Scholar (Figure 1). These microdissected areas included the adventitia directly adjacent to the vascular structures (Figure 1). Approximately 1500 cells were harvested from each compartment. The microdissected material was subsequently suspended in a proteinase K digestion buffer by placing it directly in the adhesive cap. After overnight digestion, RNA was isolated using phenol-chloroform extraction and precipitation following the established modus operandi.20Theophile K. Jonigk D. Kreipe H. Bock O. Amplification of mRNA from laser-microdissected single or clustered cells in formalin-fixed and paraffin-embedded tissues for application in quantitative real-time PCR.Diagn Mol Pathol. 2008; 17: 101-106Crossref PubMed Scopus (37) Google Scholar cDNA was synthesized using the High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) and following the manufacturer's protocol. cDNA was preamplified in 14 PCR cycles with target gene–specific PCR primers, thus increasing the sensitivity of the subsequent real-time PCR analysis several thousand-fold (decrease of CT: 14 cycles; TaqMan PreAmp master mix kit, Applied Biosystems), as previously reported.18Jonigk D. Theophile K. Hussein K. Bock O. Lehmann U. Bockmeyer C.L. Gottlieb J. Fischer S. Simon A. Welte T. Maegel L. Kreipe H. Laenger F. Obliterative airway remodelling in transplanted and non-transplanted lungs.Virchows Arch. 2010; 457: 369-380Crossref PubMed Scopus (23) Google Scholar, 20Theophile K. Jonigk D. Kreipe H. Bock O. Amplification of mRNA from laser-microdissected single or clustered cells in formalin-fixed and paraffin-embedded tissues for application in quantitative real-time PCR.Diagn Mol Pathol. 2008; 17: 101-106Crossref PubMed Scopus (37) Google Scholar The preamplified cDNA from laser-microdissected samples was evaluated by real-time PCR (TaqMan 7500 real-time PCR system, Applied Biosystems, Carlsbad, CA). Quantification was performed in reactions containing preamplified cDNA, TaqMan Gene Expression Master Mix, and the individual TaqMan Gene Expression Assay (both from Applied Biosystems). A compilation of proliferation-, apoptosis-, fibrosis-, and inflammation-associated genes was selected and analyzed following the manufacturer's protocol (Table 3). For negative controls, the cDNA was replaced by water. CT values were calculated by normalization to the mean expression of three endogenous controls (POLR2A, β-GUS, and GAPDH) and were converted into 2−ΔCT values using Excel 8.0 (Microsoft Corp., Redmond, WA), which were then statistically analyzed using the Mann-Whitney U-test and GraphPad Prism, version 5.0 (GraphPad Software Inc., San Diego, CA).21Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔ CT method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123392) Google Scholar We considered P ≤ 0.05 to be statistically significant. Expression graphics were created using GraphPad Prism, version 5.0.Table 3Target and Reference GenesNo.Target genesSynonym/full name1Ang-1Angiopoietin-12Ang-2Angiopoietin-23BMP4Bone morphogenetic protein-44BMPR2Bone morphogenetic protein receptor type 25CASP9Caspase-96c-KITCD1177desDesmin8eNOSNitric oxide synthase 3, endothelial cell9FGF-2Fibroblast growth factor-210HIF1aHypoxia-inducible factor 111IL 1bInterleukin-1b12IL 6Interleukin-613MMP9Matrix metalloproteinase 914MMP14Matrix metalloproteinase 1415MYH11Smooth muscle myosin heavy chain 1116MYOCDMyocardin17NOTCH4Neurogenic locus notch homolog protein 418PECAM-1CD3119PDGFRbβ-Type platelet-derived growth factor receptor20SMASmooth muscle actin21TGF-β1Transforming growth factor-beta 122THBS1Thrombospondin-123VEGF-αVascular endothelial growth factor alpha24VEGFR1Vascular endothelial growth factor receptor 125VEGFR2Vascular endothelial growth factor receptor 2Reference genes26POLR2ARNA-polymerase 2 subunit A27GUSBβ-glucuronidase28GAPDHGlyceraldehyde-3-phosphate dehydrogenase Open table in a new tab To confirm the findings of conventional IHC and compartment-specific mRNA expression analyses regarding endothelial and/or smooth muscle differentiation of PLs, we performed immunofluorescence double staining for CD31 and SMA, CD31 and Ki-67, and SMA and Ki-67. Serially cut 5-μm–thick FFPE slides were deparaffinized, and antigens were retrieved following the established procedure. Target structures were marked using monoclonal primary- and fluorescence-labeled secondary antibodies [Cy-conjugated AffiniPure F(ab')2 fragment antibodies (H+L); Jackson ImmunoResearch Laboratories Inc., West Grove, PA]. The nuclear structures were counterstained using DAPI (40 μg/mL; Carl Roth GmbH, Karlsruhe, Germany) before the slides were mounted.19Jonigk D. Lehmann U. Stuht S. Wilhelmi M. Haverich A. Kreipe H. Mengel M. Recipient-derived neoangiogenesis of arterioles and lymphatics in quilty lesions of cardiac allografts.Transplantation. 2007; 84: 1335-1342Crossref PubMed Scopus (22) Google Scholar Circumscribed peritumoral vascular formations, so-called GLLs in glioblastomas multiforme (n = 5), were identified by light microscopy. Vital nonnecrotic GLLs were stained by fluorescence IHC, followed by laser-microdissection and processing for RNA expression analysis as described previously herein for PLs. To further assess the structural composition of PLs, we used 30-μm-thick slides and (fluorescence) double stained them for CD31 and SMA as described previously herein. To generate three-dimensional (3-D) images of PLs, we used a microscope with an automated drive (Micro Focus, Rockville, MD) and special acquisition software (BX51 microscope and Cell P Software 3.3, Olympus Europa GmbH, Hamburg, Germany) and systematically scanned the whole width of the slides. The resulting 2-D images were merged into 3-D shapes. Unspecific, out-of-focus fluorescence was removed from the resulting 3-D images using deconvolution software (advanced maximum likelihood estimation). Images were displayed using the cell* Voxel Viewer (Olympus Europa GmbH). To analyze the role of apoptosis in the remodeling process in PLs beyond caspase mRNA expression (see previously herein), we assessed the content of fragmented DNA in PLs, adjacent vessels, and controls using an apoptosis detection kit (ApopTag plus peroxidase in situ; Millipore, Temecula, CA) on 5-μm FFPE samples following the manufacturer's protocol. PLs showed significant mRNA up-regulation of the endothelial markers CD31 and endothelial nitric oxide synthase and an increase in SMA expression compared with the adjacent arteries (Figure 2). Because PLs are mainly composed of “vascular channels,” (over)expression of markers of endothelial differentiation is not surprising, especially considering the comparable numbers of microdissected cells from the different compartments. The complementary IHC staining concurred with the results of mRNA expression analysis: CD31 showed strong laminar positivity in luminal cells of vascular channels in PLs and their adjacent arteries. The staining patterns of the endothelial markers CD34 and CD141 were comparable. SMA stained strongly in the slender, nonluminal interstitium, composed of a homogenous population of tightly layered mesenchymal/myogenic cells (Figure 3 and Table 3). These staining patterns in regular IHC were confirmed using immunofluorescence double staining: the CD31-positive endothelium of the vascular channels was continuously backed by SMA-positive cells, arranged in a uniform stratum less than or equal to four cell layers thick. 3-D reconstruction of PLs validated this cellular composition (Figure 4).Figure 4The-3-D reconstruction of a PL. A: A fluorescent double-stained PL: the luminal vascular channels stain positive for CD31 (red) and the adjacent interstitium shows homogenous positivity for SMA (green). B: Systematically scanned images of the whole width of the PL were merged into 3-D shapes. C: Images were skeletonized into a voxel model for increased clarity. Note the distinct separation of the endothelial layer and the interstitium in the PL. D: GLLs in high-grade neural tumors show a rather disorganized composition without an equally clear separation of endothelial layer and interstitium. Original magnification: ×630 (A–C); ×1000 (D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The adjacent remodeled arteries with prominent media hypertrophy and intimal thickening showed significantly increased mRNA levels of desmin, myocardin, and smmhc compared with PLs (Figure 2), which was largely anticipated given the ratio of smooth muscle to endothelial cells exceeding 9:10 in these vessels. The corresponding IHC staining confirmed these expression patterns, with strong positivity in the prominent media layer for all three targets in pulmonary arteries but not in PLs. Whereas PLs, the adjacent arteries, concentric lesions in APAH without PLs, and controls did not stain for podoplanin, there was a marked accumulation of podoplanin-positive lymphatic vessels around PLs. Aside from the presence of PLs themselves, there were no reproducible morphologic differences between APAH specimens with and without PLs (Figure 3 and Table 3, Table 4).Table 4IHC Staining Results in Vascular CompartmentsProteinPLsAdjacent arteriesConcentric lesions in APAH without PLsControlsGLLsAng-1 Semiquantitative score11111 Staining intensityWeakWeakWeakWeakIntermediateAng-2 Semiquantitative score22322 Staining intensityStrongIntermediateIntermediateWeakStrongc-KIT (CD117) Semiquantitative score11110 Staining intensityIntermediateWeakWeakWeakAbsentCD141 Semiquantitative score21112 Staining intensityStrongStrongStrongStrongStrongCD31 Semiquantitative score21112 Staining intensityStrongStrongStrongStrongStrongCD34 Semiquantitative score21112 Staining intensityStrongIntermediateStrongStrongStrongDesmin Semiquantitative score23330 Staining intensityIntermediateStrongStrongStrongAbsentMyocardin Semiquantitative score22321 Staining intensityWeakIntermediateIntermediateIntermediateWeakPDGFRb Semiquantitative score22221 Staining intensityWeakIntermediateIntermediateIntermediateWeakPodoplanin Semiquantitative score00000 Staining intensityAbsentAbsentAbsentAbsentAbsentSMA Semiquantitative score33333 Staining intensityStrongStrongStrongIntermediateStrongSmmhc Semiquantitative score33332 Staining intensityIntermediateStrongStrongIntermediateIntermediateTGF-β1 Semiquantitative score21112 Staining intensityStrongWeakWeakWeakStrongTHBS1 Semiquantitative score21222 Staining intensityWeakWeakIntermediateIntermediateIntermediateVEGF-A Semiquantitative score22222 Staining intensityStrongWeakWeakIntermediateStrongSemiquantitative scores indicate the range of positive cells (0, no apparent reaction; 1, positivity in <30%; 2, positivity in ≥30% and <60%; and 3, positivity in ≥60%). Staining intensity is indicated as absent, weak, intermediate, or strong.PDGFRb, β-type platelet-derived growth factor receptor. Open table in a new tab Semiquantitative scores indicate the range of positive cells (0, no apparent reaction; 1, positivity in <30%; 2, positivity in ≥30% and <60%; and 3, positivity in ≥60%). Staining intensity is indicated as absent, weak, intermediate, or strong. PDGFRb, β
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