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• W1574865824 abstract "The mechanisms underlying schistosomiasis-induced pulmonary hypertension (PH), one of the most common causes of PH worldwide, remain unclear. We sought to determine whether Schistosoma mansoni causes experimental PH associated with pulmonary vascular remodeling in an interleukin (IL)-13-dependent manner. IL-13Rα1 is the canonical IL-13 signaling receptor, whereas IL-13Rα2 is a competitive nonsignaling decoy receptor. Wild-type, IL-13Rα1−/−, and IL-13Rα2−/− C57BL/6J mice were percutaneously infected with S. mansoni cercariae, followed by i.v. injection of eggs. We assessed PH with right ventricular catheterization, histological evaluation of pulmonary vascular remodeling, and detection of IL-13 and transforming growth factor-β signaling. Infected mice developed pulmonary peri-egg granulomas and arterial remodeling involving predominantly the vascular media. In addition, gain-of-function IL-13Rα2−/− mice had exacerbated vascular remodeling and PH. Mice with loss of IL-13Rα1 function did not develop PH and had reduced pulmonary vascular remodeling. Moreover, the expression of resistin-like molecule-α, a target of IL-13 signaling, was increased in infected wild-type and IL-13Rα2−/− but not IL-13Rα1−/− mice. Phosphorylated Smad2/3, a target of transforming growth factor-β signaling, was increased in both infected mice and humans with the disease. Our data indicate that experimental schistosomiasis causes PH and potentially relies on up-regulated IL-13 signaling. The mechanisms underlying schistosomiasis-induced pulmonary hypertension (PH), one of the most common causes of PH worldwide, remain unclear. We sought to determine whether Schistosoma mansoni causes experimental PH associated with pulmonary vascular remodeling in an interleukin (IL)-13-dependent manner. IL-13Rα1 is the canonical IL-13 signaling receptor, whereas IL-13Rα2 is a competitive nonsignaling decoy receptor. Wild-type, IL-13Rα1−/−, and IL-13Rα2−/− C57BL/6J mice were percutaneously infected with S. mansoni cercariae, followed by i.v. injection of eggs. We assessed PH with right ventricular catheterization, histological evaluation of pulmonary vascular remodeling, and detection of IL-13 and transforming growth factor-β signaling. Infected mice developed pulmonary peri-egg granulomas and arterial remodeling involving predominantly the vascular media. In addition, gain-of-function IL-13Rα2−/− mice had exacerbated vascular remodeling and PH. Mice with loss of IL-13Rα1 function did not develop PH and had reduced pulmonary vascular remodeling. Moreover, the expression of resistin-like molecule-α, a target of IL-13 signaling, was increased in infected wild-type and IL-13Rα2−/− but not IL-13Rα1−/− mice. Phosphorylated Smad2/3, a target of transforming growth factor-β signaling, was increased in both infected mice and humans with the disease. Our data indicate that experimental schistosomiasis causes PH and potentially relies on up-regulated IL-13 signaling. Pulmonary hypertension (PH) is a frequent and potentially fatal sequel to several chronic lung, collagen vascular, and liver diseases.1Tuder RM Marecki JC Richter A Fijalkowska I Flores S Pathology of pulmonary hypertension.Clin Chest Med. 2007; 28 (vii): 23-42Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar Furthermore, pulmonary arterial hypertension (PAH) occurs in a fraction of patients with HIV infection, and idiopathic PAH has been linked to the human herpesvirus type 8.2Cool CD Rai MD Yeager ME Hernandez-Saavedra D Serls AE Bull TM Geraci MW Brown KK Routes JM Tuder RM Voelkel NF Expression of human herpesvirus 8 in primary pulmonary hypertension.N Engl J Med. 2003; 349: 1113-1122Crossref PubMed Scopus (253) Google Scholar The pathogenesis of PH involves an imbalance of vascular cell proliferation vis-à-vis cell death, driven by excessive growth factor stimulation, including transforming growth factor (TGF)-β family signaling,3Richter A Yeager ME Zaiman A Cool CD Voelkel NF Tuder RM Impaired transforming growth factor-β signaling in idiopathic pulmonary arterial hypertension.Am J Respir Crit Care Med. 2004 Dec 15; 170: 1340-1348Crossref PubMed Scopus (112) Google Scholar platelet derived growth factor,4Schermuly RT Dony E Ghofrani HA Pullamsetti S Savai R Roth M Sydykov A Lai YJ Weissmann N Seeger W Grimminger F Reversal of experimental pulmonary hypertension by PDGF inhibition.J Clin Invest. 2005; 115: 2811-2821Crossref PubMed Scopus (870) Google Scholar and more recently, Notch receptor signaling.5Li X Zhang X Leathers R Makino A Huang C Parsa P Macias J Yuan JX Jamieson SW Thistlethwaite PA Notch3 signaling promotes the development of pulmonary arterial hypertension.Nat Med. 2009; 15: 1289-1297Crossref PubMed Scopus (261) Google Scholar In idiopathic PAH, dysregulation of TGF-β signaling is central to the pathobiology of the disease, including mutations of bone morphogenetic protein receptor 2.6Zaiman AL Podowski M Medicherla S Gordy K Xu F Zhen L Shimoda LA Neptune E Higgins L Murphy A Chakravarty S Protter A Sehgal PB Champion HC Tuder RM Role of the TGF-β/Alk5 signaling pathway in monocrotaline-induced pulmonary hypertension.Am J Respir Crit Care Med. 2008; 177: 896-905Crossref PubMed Scopus (116) Google Scholar Recent evidence has linked the cellular events underlying pulmonary vascular remodeling to hypoxia-like metabolic alterations, largely driven by hypoxia-inducible factor-1α in both experimental models7Bonnet S Michelakis ED Porter CJ ndrade-Navarro MA Thebaud B Bonnet S Haromy A Harry G Moudgil R McMurtry S Weir EK Archer SL An abnormal mitochondrial-hypoxia inducible factor-1α-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: similarities to human pulmonary arterial hypertension.Circulation. 2006; 113: 2630-2641Crossref PubMed Scopus (467) Google Scholar and in the human disease.8Fijalkowska I Xu W Comhair SA Janocha AJ Mavrakis LA Krishnamachary B Zhen L Mao T Richter A Erzurum SC Tuder RM Hypoxia inducible-factor1alpha regulates the metabolic shift of pulmonary hypertensive endothelial cells.Am J Pathol. 2010; 176: 1130-1138Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar Moreover, both the human disease and experimental models have a variable contribution by inflammatory cells, whose precise pathogenetic role has not been clarified thus far. Schistosomiasis is one of the most common causes of PAH, occurring in 2 to 5% of the over 200 million individuals chronically infected worldwide.9Butrous G Ghofrani HA Grimminger F Pulmonary vascular disease in the developing world.Circulation. 2008 Oct 21; 118: 1758-1766Crossref PubMed Scopus (81) Google Scholar Schistosoma are snail-borne trematodes that percutaneously infect individuals, transiently pass through the lungs, and migrate to the portal venous system where they reproduce and release eggs. Four to 8% of those chronically infected develop hepatosplenic disease, in which the host immunological response and disruption of portal hepatic blood flow to the egg antigens leads to progressive liver fibrosis and portal hypertension.10Andrade ZA Schistosomiasis and liver fibrosis.Parasite Immunol. 2009; 31: 656-663Crossref PubMed Scopus (174) Google Scholar Over time, the increased pressure causes opening of portosystemic shunts, allowing the passage of eggs to the pulmonary arterial circulation.11Lapa M Dias B Jardim C Fernandes CJ Dourado PM Figueiredo M Farias A Tsutsui J Terra-Filho M Humbert M Souza R Cardiopulmonary manifestations of hepatosplenic schistosomiasis.Circulation. 2009; 119: 1518-1523Crossref PubMed Scopus (151) Google Scholar The pulmonary vascular pathology is associated with progressive clinical PH and right heart failure. PH almost exclusively occurs in those infected with the species Schistosoma mansoni rather than the other endemic species Schistosoma japonicum and Schistosoma hematobium. There has been an intense effort to better understand the pathogenesis of liver fibrosis due to schistosomiasis, uncovering a type 2 helper T cell (Th-2) predominant immunological response, highlighted by the involvement of interleukin (IL)-4 and IL-13 as key mediators of granuloma size, and most importantly, of the fibrogenic response with liver injury.12Burke ML Jones MK Gobert GN Li YS Ellis MK McManus DP Immunopathogenesis of human schistosomiasis.Parasite Immunol. 2009; 1931: 163-176Crossref Scopus (296) Google Scholar More recently, Andrade has noted that the portal fibrosis may arise from a destruction of the portal vein, with occlusive lesions.10Andrade ZA Schistosomiasis and liver fibrosis.Parasite Immunol. 2009; 31: 656-663Crossref PubMed Scopus (174) Google Scholar Of note, experimental PH is also associated with early endothelial cell apoptosis,6Zaiman AL Podowski M Medicherla S Gordy K Xu F Zhen L Shimoda LA Neptune E Higgins L Murphy A Chakravarty S Protter A Sehgal PB Champion HC Tuder RM Role of the TGF-β/Alk5 signaling pathway in monocrotaline-induced pulmonary hypertension.Am J Respir Crit Care Med. 2008; 177: 896-905Crossref PubMed Scopus (116) Google Scholar which might lead to abnormal endothelial proliferation, genetic instability, and occlusive lesions.13Marecki JC Cool CD Parr JE Beckey VE Luciw PA Tarantal AF Carville A Shannon RP Cota-Gomez A Tuder RM Voelkel NF Flores SC HIV-1 Nef is associated with complex pulmonary vascular lesions in SHIV-nef-infected macaques.Am J Respir Crit Care Med. 2006; 174: 437-445Crossref PubMed Scopus (155) Google Scholar Despite the focus on the immunological and hepatic alterations caused by the parasite in humans and the growing realization of the epidemiological importance of schistosomiasis-associated PAH, little is known regarding the pathogenesis of this complication related to the parasite infection. In a subset of patients with the hepatosplenic form of schistosomiasis, a concurrent pulmonary arterial vasculopathy develops with medial and intimal lesions, including plexiform lesions reminiscent of the pathology of idiopathic PAH.14Tuder RM Pathology of pulmonary arterial hypertension.Semin Respir Crit Care Med. 2009; 1930: 376-385Crossref Scopus (77) Google Scholar This remodeling involves an excessive number of smooth muscle cells in the intima and media and proliferation of endothelial cells.14Tuder RM Pathology of pulmonary arterial hypertension.Semin Respir Crit Care Med. 2009; 1930: 376-385Crossref Scopus (77) Google Scholar Mice chronically infected with schistosomes can develop pulmonary peri-egg granulomas and pulmonary vascular remodeling, but have not been previously demonstrated to develop PH.15Crosby A Jones FM Southwood M Stewart S Schermuly R Butrous G Dunne DW Morrell NW Pulmonary vascular remodeling correlates with lung eggs and cytokines in murine schistosomiasis.Am J Respir Crit Care Med. 2010; 181: 279-288Crossref PubMed Scopus (93) Google Scholar The acute infection in mice causes granulomas reliant on a Th-1 response, while chronic infection transitions to a Th-2 response with increased IL-13 and larger granulomas often with fibrosis.16Mentink-Kane MM Cheever AW Thompson RW Hari DM Kabatereine NB Vennervald BJ Ouma JH Mwatha JK Jones FM Donaldson DD Grusby MJ Dunne DW Wynn TA IL-13 receptor α2 down-modulates granulomatous inflammation and prolongs host survival in schistosomiasis.Proc Natl Acad Sci USA. 2004; 101: 586-590Crossref PubMed Scopus (119) Google Scholar, 17Ramalingam TR Pesce JT Sheikh F Cheever AW Mentink-Kane MM Wilson MS Stevens S Valenzuela DM Murphy AJ Yancopoulos GD Urban Jr, JF Donnelly RP Wynn TA Unique functions of the type II interleukin 4 receptor identified in mice lacking the interleukin 13 receptor α1 chain.Nat Immunol. 2008; 9: 25-33Crossref PubMed Scopus (144) Google Scholar A downstream product of the IL-4 and IL-13 signaling pathways, resistin-like molecule (RELM)-α (also called found in inflammatory zone-1, hypoxia-induced mitogenic factor, and Retnla), was recently identified as an important regulator of the host inflammatory response in both the liver and the lungs, as mice lacking RELM-α have larger granulomas.18Pesce JT Ramalingam TR Wilson MS Mentink-Kane MM Thompson RW Cheever AW Urban JF Wynn TA Retnla (relmα/fizz1) suppresses helminth-induced Th2-type immunity.PLoS Pathog. 2009; 2005: e1000393Crossref Scopus (185) Google Scholar In multiple secondary forms of severe PH, including connective tissue disease and schistosomiasis-associated PH, inflammation likely underlies the pathogenesis.19Dorfmuller P Perros F Balabanian K Humbert M Inflammation in pulmonary arterial hypertension.Eur Respir J. 2003; 1922: 358-363Crossref Scopus (496) Google Scholar Whereas Schistosoma eggs are usually found within granulomas, their direct role or connection with ensuing inflammation in the pathogenesis of schistosomiasis-associated PH remains unclear. Although most of the experimental models of PH involve challenge with chronic hypoxia or endothelial cell toxicity with the alkaloid monocrotaline pyrrole, schistosomiasis-associated PH might involve the direct interplay of inflammatory cells and cytokines with well-known aforementioned signaling pathways that control pulmonary vascular reactivity and/or remodeling. The interactions between cell signaling induced by the peri-egg granulomatous response and the altered cellular and molecular framework underlying pulmonary vasculopathy, including the role of altered TGF-β signaling and imbalance between vascular cell death versus proliferation, have not been studied. The mouse model of schistosomiasis-induced PH offers a unique system to interrogate the interplay of growth factors that control pulmonary vascular cell function and Th-2 signaling. In the present study, we postulated that schistosome eggs and the ensuing Th-2 inflammatory response they induce are key drivers in experimental PH caused by S. mansoni. To address this hypothesis, we developed a model system to test whether mice infected with S. mansoni develop PH and pulmonary vascular remodeling; furthermore, we interrogated the potential participation of Th-2-dependent cytokines and growth factors that may have led to the pulmonary vascular remodeling, including RELM-α and TGF-β. We used wild-type C57Bl6/J and two genetically modified mice: IL-13Rα1−/−, lacking the canonical IL-13 receptor resulting in a loss-of-function of IL-13 signaling, and mice lacking the soluble and membrane-bound “decoy” IL-13 receptor (IL-13Rα2−/−), which results in a gain-of-function for IL-13 signaling. We also analyzed human pulmonary autopsy tissue from patients with schistosomiasis-associated PH to compare signaling pathways between the mouse model and human disease. Breeding pairs of C57BL/6J IL-13Rα1−/− mice (N10) were provided by Regeneron Pharmaceuticals (Tarrytown, NY), and C57BL/6J IL-13Rα2−/− mice (N10) were provided by Wyeth Research (Cambridge, MA). The phenotypes of these knockout models have been described previously.17Ramalingam TR Pesce JT Sheikh F Cheever AW Mentink-Kane MM Wilson MS Stevens S Valenzuela DM Murphy AJ Yancopoulos GD Urban Jr, JF Donnelly RP Wynn TA Unique functions of the type II interleukin 4 receptor identified in mice lacking the interleukin 13 receptor α1 chain.Nat Immunol. 2008; 9: 25-33Crossref PubMed Scopus (144) Google Scholar, 20Wood N Whitters MJ Jacobson BA Witek J Sypek JP Kasaian M Eppihimer MJ Unger M Tanaka T Goldman SJ Collins M Donaldson DD Grusby MJ Enhanced interleukin (IL)-13 responses in mice lacking IL-13 receptor α2.J Exp Med. 2003; 197: 703-709Crossref PubMed Scopus (177) Google Scholar The wild-type control mice were also of the C57BL/6J background and were purchased from Taconic. All mice were bred and housed under specific pathogen-free conditions in an American Association for the Accreditation of Laboratory Animal Care-approved facility. All experimental procedures in rodents were approved by the Animal Care and Use Committees at the National Institutes of Health, Johns Hopkins University, and the University of Colorado. All experiments were performed in a coded format, with the investigators lacking knowledge of the specific experimental group identifiers before final data reporting. S. mansoni cercariae and eggs were obtained from the Biomedical Research Institute (Rockville, MD), and the mice were percutaneously infected with cercariae and intravenously challenged with S. mansoni eggs as described previously.21Boros DL The role of cytokines in the formation of the schistosome egg granuloma.Immunobiology. 1994; 191: 441-450Crossref PubMed Scopus (60) Google Scholar Briefly, percutaneous infection was performed by placing the mouse tail in a vial containing 30 to 35 cercariae for 30 minutes. Fifty-five days later, mice were challenged intravenously by injecting 5000 viable eggs suspended in 0.5 ml of sterile saline into the tail vein. The intravenous challenge with eggs mimics the deposition of eggs in the lung by collateral shunts, which normally form in chronically infected mice. Sixty-two days after cercarial infection and 7 days after i.v. egg administration, measurement of the right ventricular pressure was performed as described previously.22Hemnes AR Zaiman A Champion HC PDE5A inhibition attenuates bleomycin-induced pulmonary fibrosis and pulmonary hypertension through inhibition of ROS generation and RhoA/Rho kinase activation.Am J Physiol Lung Cell Mol Physiol. 2008; 294: L24-L33Crossref PubMed Scopus (127) Google Scholar Briefly, mice were anesthetized with isofluorane and ventilated through a transtracheal catheter. Before cardiac catheterization a right jugular catheter was placed and 0.2 ml of 5% albumin instilled. The abdominal and thoracic cavities were opened, and a four-electrode pressure-volume catheter (Scisense model FTS-1212B-4518, London, Ontario, Canada) was placed through the right ventricle apex to transduce the pressure. The blood was flushed out of the lungs, the right bronchus was sutured, and 2% agarose instilled into the left lung through the transtracheal catheter. The left lung was removed, formalin-fixed, and processed for paraffin embedding. The right lung was removed and frozen. The right ventricle free wall was dissected off of the heart, weighed relative to the septum and left ventricle, and then formalin-fixed and paraffin-embedded. The number of S. mansoni eggs present in the mouse lung tissue was determined as previously described.23Cheever AW Conditions affecting the accuracy of potassium hydroxide digestion techniques for counting Schistosoma mansoni eggs in tissues.Bull World Health Organ. 1968; 39: 328-331PubMed Google Scholar Briefly, 20 to 30 mg of frozen right lung tissue was digested in 4% potassium hydroxide for 18 hours at 33°C, and the number of eggs present in aliquots of the digest was counted. A sample of the right lung frozen tissue was macerated and sonicated in PBS containing anti-proteases, and 20 μg of protein from each sample was used to detect specific proteins by Western blot using the reagents listed in the Table 1. Double-antibody sandwich enzyme-linked immunosorbent assays were used to quantify cytokine levels in the tissue lysates using the following kits: IL-13 (M1300CB; R&D Systems, Minneapolis, MN), TGF-β1 (MB100B; R&D Systems), and tumor necrosis factor (TNF)-α (DY410; R&D Systems), and following the manufacturer's recommended protocols. Immunohistochemistry, immunofluorescence, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining was performed using the reagents listed in the Table 2. Images were acquired with a Nikon Eclipse E800 microscope with filter wheels, a color charge-coupled device camera (Nikon, Melville, NY) and a cooled black and white charge-coupled device camera (Photometrics, Tucson, AZ). Images selected for presentation and analysis were representative of the findings seen in each model, and granulomas were selected with only a single egg nidus visible.Table 1Reagents for ImmunoblotsImmunoblotBlockPrimary antibody*All primary antibodies incubated overnight at 4°C unless otherwise stated.Secondary antibodyTertiary reagentRELM-αStartingBlock 2 hours at room temperature (Thermo Scientific 37542)1/50,000 Abcam 39626Horseradish peroxidase-bound goat anti-rabbit 1/5000 1 hour at room temperature (Vector Laboratories PI-1000)Enhanced chemiluminescence detection 5 minutes at room temperature (GE Healthcare, RPN2106)p-Smad2/31/2,000 Cell Signaling Technology 3101Smad2/31/2,000 Cell Signaling Technology 3102p-ERK1/21/2,000 Cell Signaling Technology 9101ERK1/21/2,000 Cell Signaling Technology 9102TGF-β1/2,000 Cell Signaling Technology 3711β-Actin1/20,000 Cell Signaling Technology 4967 (also used 1/5,000 for 1 hour at room temperature)* All primary antibodies incubated overnight at 4°C unless otherwise stated. Open table in a new tab Table 2Reagents for Immunostains on Mouse TissueImmunostainAntigen retrievalBlockPrimary antibodySecondary antibodyTertiary reagentThrombomodulin (CD141)Citrate buffer 30 minutes in steamer (Vector H-3300)10% Horse serum in 1/1 5% BSA: Superblock (ScyTek AAA5000) 1 hour at room temperature1/1000 1 hour at room temperature (R&D Systems AF3894)1/200 AF594 Donkey anti-goat (Invitrogen A11058) 1 hour at room temperatureNoneProliferating cell nuclear AgCitrate buffer 30 minutes in steamer (Vector H-3300)10% Horse serum in 1/1 5% BSA: Superblock (ScyTek AAA5000) 1 hour at room temperature1/50 1 hour at room temperature (Santa Cruz Biotechnology 7907)1/200 AF488 Donkey anti-Rabbit (Invitrogen A21206) 1 hour at room temperatureNoneMacrophage-3Borg buffer 30 minutes in steamer (Biocare BD1000G1)10% Goat Serum in 1:1 5% BSA: Superblock (ScyTek AAA5000) 1 hour at room temperature1/50 1 hour at room temperature (BD Pharmingen 550292)1/300 AF488 Goat anti-Rat (Invitrogen A11006) 1 hour at room temperaturePro-surfactant protein C (Pro-SPC)Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300), then avidin 10 minutes, then Biotin 10 minutes5% Horse Serum in PBS 1 hour at room temperature1/200 1 Hour at room temperature (Santa Cruz Biotechnology 7705)1/100 Biotinylated donkey anti-Goat (Abcam 6578) 1 hour at room temperatureNoneα-Smooth muscle actinMouse on Mouse (MOM) kit blocking solution (Vector Laboratories BMK-2202) 1 hour at room temperature1/100 30 minutes at room temperature (DakoCytomation M0851)MOM Biotinylated anti-Mouse Reagent (Vector Laboratories BMK-2202) 10 minutes at room temperatureTexas Red-Strepavidin 1/2000 (Invitrogen S872)NoneMajor basic proteinCitrate buffer 30 minutes in steamer (Vector Laboratories H-3300), then Pepsin 10 minutes at room temperature (Invitrogen 00-3009), then avidin 10 minutes, then Biotin 10 minutes1.5% Rabbit Serum in PBS 1 hour at room temperature1/500 1 hour at room temperature (antibody courtesy of Lee Lab, Mayo Clinic)1/100 Biotin-Bound Rabbit anti-Rat (DakoCytomation E0468) 1 hour at room temperatureStrepavidin-horseradish peroxidase (Vector Laboratories SA-5704), then DAB 5 minutes, then hematoxylinCD45Citrate buffer 30 minutes in steamer (Vector H-3300)10% Horse Serum in PBS 1 hour at room temperature1/400 1 Hour at room temperature (R&D Biosystems AF114)1/200 AF594 Donkey anti-Goat (Invitrogen A11058) 1 hour at room temperature or 1/200 AF488 Donkey anti-Goat (Invitrogen A11055) 1 hour at room temperatureNoneRELM-αCitrate buffer 30 minutes in steamer (Vector H-3300)5% Horse Serum in PBS 1 hour at room temperature1/200 overnight at 4°C (Abcam 39626)1/100 AF488 Donkey anti-Rabbit (Invitrogen A21206) 1 hour at room temperatureNonep-Smad2/3Citrate buffer 30 minutes in steamer (Vector H-3300)5% Horse Serum in PBS 1 hour at room temperature1/100 overnight at 4degC (Cell Signaling Technology 3101)1/100 AF488 Donkey anti-Rabbit (Invitrogen A21206) 1 hour at room temperatureNoneLectin from Bandeiraea Simplicifolia (tetramethylrhodamine isothiocyanate-bound)Citrate buffer 30 minutes in steamer (Vector H-3300)None1/100 overnight at 4degC (Sigma-Aldrich L5264)NoneNoneTerminal deoxynucleotidyl transferase dUTP nick end labelingDeadEnd Fluorometric TUNEL System (Promega G3250), per manufacturer's protocol Open table in a new tab The granuloma volumes were measured using the rotator sterologic method.24Tandrup T Gundersen HJ Jensen EB The optical rotator.J Microsc. 1997; 186: 108-120Crossref PubMed Scopus (104) Google Scholar Briefly, paraffin-embedded tissue was stained with H&E, and 10 to 12 images of granulomas were acquired for each sample. The rotator method for object volume estimation was then applied using the egg spine as the central reference point. Masked paraffin-embedded samples were immunofluorescence stained for α-smooth muscle actin and thrombomodulin as described above to identify the smooth muscle cells and endothelial cells demarcating the media and intima, respectively. Twelve to 15 images of vessels at ×40 magnification were randomly acquired from each sample. Image processing software (Image Pro Plus version 4.5.1; Media Cybernetics, Bethesda, MD) was used to identify the cross-sectional areas contained by the external perimeter of the media, the internal perimeter of the media, and the internal perimeter of the intima. A length:breadth ratio of less than 2 was required to ensure tranverse sections through the vessels. The radius ri for each of the three vessel layers i enclosing an area Ai was then calculated using the equationri=Ai/π. The thicknesses of the media and intima were calculated as the differences between the respective radii, and expressed as a fraction of the external media radius. Formalin-fixed and paraffin-embedded histological sections of mouse right ventricle tissue were stained as described above with tetramethylrhodamine isothiocyanate-labeled lectin from Bandeiraea simplicifolia to identify vascular structures and 4′,6′-diamidino-2-phenylindole. Twelve to 15 images at ×40 magnification were randomly captured of each sample. The component colors of the images were individually thresholded (Metamorph version 6.3; MDS Analytical Technologies, Sunnyvale, CA) to demarcate structures of interest (vessels and nuclei, respectively). Stereologic analysis was performed by projecting the thresholded images onto a grid of 216 points and counting the number of points intersecting with each tissue component. Immunohistochemistry and immunofluorescence staining on formalin-fixed and paraffin embedded samples of lung tissue obtained at autopsy from two patients who died of schistosomiasis-associated PAH in Brazil was performed using the reagents listed in the Table 3.Table 3Reagents for Immunostains on Human TissueImmunostainAntigen retrievalBlockPrimary antibodySecondary antibodyTertiary reagentp-Smad2/3Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300)5% Horse serum in PBS 1 hour at room temperature1/2000 overnight at 4°C (Cell Signaling Technology 3101)1/100 AF488 Donkey anti-Rabbit (Invitrogen A21206) 1 hour at room temperatureNoneα-Smooth muscle actinCitrate buffer 30 minutes in steamer (Vector Laboratories H-3300)5% Goat serum in PBS 1 hour at room temperature1/200 1 hour at room temperature (DakoCytomation M0851)1/100 AF594 Goat anti-mouse (Invitrogen A11005) 1 hour at room temperatureNoneCD31Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300)1.5% Rabbit serum in PBS 1 hour at room temperature1:500 1 hour at room temperature (antibody courtesy of Lee Lab, Mayo Clinic)1/100 Biotin-bound rabbit anti-rat (DakoCytomation E0468) 1 hour at room temperatureStrepavidin-horseradish peroxidase (Vector Laboratories SA-5704), then DAB 5 minutes, then hematoxylin Open table in a new tab Analysis of variance was used to compare multiple groups (Kruskal-Wallis one-way analysis of variance on ranks for nonnormally distributed data), and the posthoc Tukey test (for normally distributed data) or the posthoc pairwise multiple comparison by Dunn's method (for nonnormally distributed data) was used to identify individual statistical differences within the groups. Graphs are presented as mean ± SE, and statistical significance was taken as P ≤ 0.05. Initially, we addressed whether PH can be experimentally induced by infecting mice with S. mansoni to reproduce the natural infection cycle in susceptible vertebrates, or through infusion of eggs directly into the venous circulation leading to embolization to the lungs. Although percutaneous cercarial infection, the normal route of infection, causes extensive portal and prehepatic disease, it leads to inconsistent shunting of eggs to the lungs. We found that percutaneous infection with cercariae alone or intravenous injection of schistosome eggs alone did not cause an increase in right ventricular pressure, suggesting each mode of infection appears incapable to incite PH in the mouse,15Crosby A Jones FM Southwood M Stewart S Schermuly R Butrous G Dunne DW Morrell NW Pulmonary vascular remodeling correlates with lung eggs and cytokines in murine schistosomiasis.Am J Respir Crit Care Med. 2010; 181: 279-288Crossref PubMed Scopus (93) Google Scholar but both sequential cercarial infection followed by intravenous egg challenge and i.p. sensitization with eggs followed by intravenous egg challenge resulted in an elevation in right ventricular pressure as compared with uninfected and unchallenged mice (Supplemental Figure 1, see http://ajp.amjpathol.org). We chose to further investigate the sequential cercariae and egg infection model as this disease process combines the normal route of infection with a reproducible egg burden in the lung. Histological examination of wild-type, IL-13Rα1−/−, and IL-13Rα2−/− left lungs revealed no significant pulmonary vascular remodeling in uninfected and unchallenged mice, while infected wild-type mice developed a widespread arterial vasculopathy, often seen" @default.
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• W1574865824 date "2010-09-01" @default.
• W1574865824 modified "2023-10-18" @default.
• W1574865824 title "Schistosomiasis-Induced Experimental Pulmonary Hypertension" @default.
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