Modulation of Intersectin-1s Lung Expression Induces Obliterative Remodeling and Severe Plexiform Arteriopathy in the Murine Pulmonary Vascular Bed
2017; Elsevier BV; Volume: 187; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2016.11.012
ISSN1525-2191
AutoresMonal Patel, Dan Predescu, Cristina Bardita, Jiwang Chen, Niranjan Jeganathan, Melanie Pritchard, Salvatore DiBartolo, Roberto F. Machado, Sanda Predescu,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoMurine models of pulmonary arterial hypertension (PAH) that recapitulate the plexiform and obliterative arteriopathy seen in PAH patients and help in defining the molecular mechanisms involved are missing. Herein, we investigated whether intersectin-1s (ITSN) deficiency and prolonged lung expression of an ITSN fragment with endothelial cell (EC) proliferative potential (EHITSN), present in the lungs of PAH animal models and human patients, induce formation of plexiform/obliterative lesions and defined the molecular mechanisms involved. ITSN-deficient mice (knockout/heterozygous and knockdown) were subjected to targeted lung delivery of EHITSN via liposomes for 20 days. Immunohistochemistry and histological and morphometric analyses revealed a twofold increase in proliferative ECs and a 1.35-fold increase in proliferative α-smooth muscle actin–positive cells in the lungs of ITSN-deficient mice, transduced with the EHITSN relative to wild-type littermates. Treated mice developed severe medial wall hypertrophy, intima proliferation, and various forms of obliterative and plexiform-like lesions in pulmonary arteries, similar to PAH patients. Hemodynamic measurements indicated modest increases in the right ventricular systolic pressure and right ventricle hypertrophy. Transcriptional and protein assays of lung tissue indicated p38MAPK-dependent activation of Elk-1 transcription factor and increased expression of c-Fos gene. This unique murine model of PAH-like plexiform/obliterative arteriopathy induced via a two-hit pathophysiological mechanism without hypoxia provides novel druggable targets to ameliorate and, perhaps, reverse the EC plexiform phenotype in severe human PAH. Murine models of pulmonary arterial hypertension (PAH) that recapitulate the plexiform and obliterative arteriopathy seen in PAH patients and help in defining the molecular mechanisms involved are missing. Herein, we investigated whether intersectin-1s (ITSN) deficiency and prolonged lung expression of an ITSN fragment with endothelial cell (EC) proliferative potential (EHITSN), present in the lungs of PAH animal models and human patients, induce formation of plexiform/obliterative lesions and defined the molecular mechanisms involved. ITSN-deficient mice (knockout/heterozygous and knockdown) were subjected to targeted lung delivery of EHITSN via liposomes for 20 days. Immunohistochemistry and histological and morphometric analyses revealed a twofold increase in proliferative ECs and a 1.35-fold increase in proliferative α-smooth muscle actin–positive cells in the lungs of ITSN-deficient mice, transduced with the EHITSN relative to wild-type littermates. Treated mice developed severe medial wall hypertrophy, intima proliferation, and various forms of obliterative and plexiform-like lesions in pulmonary arteries, similar to PAH patients. Hemodynamic measurements indicated modest increases in the right ventricular systolic pressure and right ventricle hypertrophy. Transcriptional and protein assays of lung tissue indicated p38MAPK-dependent activation of Elk-1 transcription factor and increased expression of c-Fos gene. This unique murine model of PAH-like plexiform/obliterative arteriopathy induced via a two-hit pathophysiological mechanism without hypoxia provides novel druggable targets to ameliorate and, perhaps, reverse the EC plexiform phenotype in severe human PAH. Pulmonary arterial hypertension (PAH) is a severe human disease characterized by narrowing of the small pulmonary arteries, leading to a progressive increase in pulmonary vascular resistance, which frequently leads to right-sided heart failure and death.1Tuder R.M. Cool C.D. Yeager M. Taraseviciene-Stewart L. Bull T.M. Voelkel N.F. The pathobiology of pulmonary hypertension: endothelium.Clin Chest Med. 2001; 22: 405-418Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 2Tuder R.M. Marecki J.C. 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 (267) Google Scholar, 3Voelkel N.F. Cool C. Pathology of pulmonary hypertension.Cardiol Clin. 2004; 22 (v): 343-351Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar A common histological finding in patients with severe PAH is the presence of plexiform lesions that obliterate the small to mid-sized pulmonary arterioles.4Tuder R.M. Abman S.H. Braun T. Capron F. Stevens T. Thistlethwaite P.A. Haworth S.G. Development and pathology of pulmonary hypertension.J Am Coll Cardiol. 2009; 54: S3-S9Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 5Rabinovitch M. Molecular pathogenesis of pulmonary arterial hypertension.J Clin Invest. 2008; 118: 2372-2379Crossref PubMed Scopus (311) Google Scholar The plexiform pulmonary vascular lesions found at branching points in the small pulmonary arterioles are lumen-obliterating, glomeruloid-like vascular structures, predominantly composed of actively dividing and phenotypically abnormal apoptosis-resistant endothelial cells (ECs).6Tuder 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, 7Sakao S. Tatsumi K. Voelkel N.F. Endothelial cells and pulmonary arterial hypertension: apoptosis, proliferation, interaction and transdifferentiation.Respir Res. 2009; 10: 95Crossref PubMed Scopus (164) Google Scholar The cellular and molecular mechanisms responsible for the development of plexiform lesions are poorly understood. Recent evidence suggests the involvement of inflammatory mechanisms in the development of PAH.8Savai R. Pullamsetti S.S. Kolbe J. Bieniek E. Voswinckel R. Fink L. Scheed A. Ritter C. Dahal B.K. Vater A. Klussmann S. Ghofrani H.A. Weissmann N. Klepetko W. Banat G.A. Seeger W. Grimminger F. Schermuly R.T. Immune and inflammatory cell involvement in the pathology of idiopathic pulmonary arterial hypertension.Am J Respir Crit Care Med. 2012; 186: 897-908Crossref PubMed Scopus (248) Google Scholar Studies have indicated that inflammation associated with human PAH attracts CD8+ T lymphocytes, which release the cytotoxic protease granzyme B.8Savai R. Pullamsetti S.S. Kolbe J. Bieniek E. Voswinckel R. Fink L. Scheed A. Ritter C. Dahal B.K. Vater A. Klussmann S. Ghofrani H.A. Weissmann N. Klepetko W. Banat G.A. Seeger W. Grimminger F. Schermuly R.T. Immune and inflammatory cell involvement in the pathology of idiopathic pulmonary arterial hypertension.Am J Respir Crit Care Med. 2012; 186: 897-908Crossref PubMed Scopus (248) Google Scholar, 9Buzza M.S. Hirst C.E. Bird C.H. Hosking P. McKendrick J. Bird P.I. The granzyme B inhibitor, PI-9, is present in endothelial and mesothelial cells, suggesting that it protects bystander cells during immune responses.Cell Immunol. 2001; 210: 21-29Crossref PubMed Scopus (76) Google Scholar, 10Austin E.D. Rock M.T. Mosse C.A. Vnencak-Jones C.L. Yoder S.M. Robbins I.M. Loyd J.E. Meyrick B.O. T lymphocyte subset abnormalities in the blood and lung in pulmonary arterial hypertension.Respir Med. 2010; 104: 454-462Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar Recently, we reported that granzyme B is closely associated with ECs in the small pulmonary arterioles and plexiform lesions in human PAH specimens; we have also shown that granzyme B cleaves intersectin-1s (ITSN), a prosurvival protein of lung ECs, and generates an NH2-terminal fragment (EHITSN) with EC proliferative potential, which is mediated via sustained phosphorylation of p38MAPK and Elk-1 transcription factor.11Patel M. Predescu D. Tandon R. Bardita C. Pogoriler J. Bhorade S. Wang M. Comhair S. Hemnes A.R. Chen J. Machado R. Husain A. Erzurum S. Predescu S. A novel p38 mitogen-activated protein kinase/Elk-1 transcription factor-dependent molecular mechanism underlying abnormal endothelial cell proliferation in plexogenic pulmonary arterial hypertension.J Biol Chem. 2013; 288: 25701-25716Crossref PubMed Scopus (27) Google Scholar Moreover, lung tissue of PAH animal models as well as human PAH lung tissue and pulmonary artery ECs isolated from PAH subjects are deficient of ITSN compared to controls and express the EHITSN product.11Patel M. Predescu D. Tandon R. Bardita C. Pogoriler J. Bhorade S. Wang M. Comhair S. Hemnes A.R. Chen J. Machado R. Husain A. Erzurum S. Predescu S. A novel p38 mitogen-activated protein kinase/Elk-1 transcription factor-dependent molecular mechanism underlying abnormal endothelial cell proliferation in plexogenic pulmonary arterial hypertension.J Biol Chem. 2013; 288: 25701-25716Crossref PubMed Scopus (27) Google Scholar Given these findings, we hypothesized that the EHITSN may be responsible for angioproliferation in advanced human PAH. Thus, in the present study, we addressed the in vivo effects of EHITSN expression on lung vascular remodeling using two murine models of ITSN-1 deficiency, the ITSN knockdown mouse (KDITSN)12Bardita C. Predescu D. Justice M.J. Petrache I. Predescu S. In vivo knockdown of intersectin-1s alters endothelial cell phenotype and causes microvascular remodeling in the mouse lungs.Apoptosis. 2013; 18: 57-76Crossref PubMed Scopus (17) Google Scholar and the ITSN knockout/heterozygous mouse (K0ITSN+/−),13Yu Y. Chu P.Y. Bowser D.N. Keating D.J. Dubach D. Harper I. Tkalcevic J. Finkelstein D.I. Pritchard M.A. Mice deficient for the chromosome 21 ortholog Itsn1 exhibit vesicle-trafficking abnormalities.Hum Mol Genet. 2008; 17: 3281-3290Crossref PubMed Scopus (71) Google Scholar both transduced with a myc-tagged EHITSN plasmid. Herein, we show that this unique murine model, induced via a two-hit pathophysiological mechanism (ITSN deficiency and EHITSN expression) with no hypoxia and no exposure to any chemical or synthetic compound, develops pulmonary obliterative vascular remodeling and severe plexiform arteriopathy, as seen in human PAH patients, via activation of p38/Elk-1/c-Fos signaling. All animal studies were performed in accordance with the guidelines of the Rush University Institutional Animal Care and Use Committee. K0ITSN+/− mice, strain 129SV/J genetic background, were kindly provided by Dr. Melanie Pritchard (Monash University, Clayton, VIC, Australia). Breeding colonies were maintained in the university animal facility. Mice were genotyped by tail snipping standard procedure. All mice, 6 to 8 weeks old, 20 to 30 g weight, were kept under standardized housing and feeding conditions. Long-term siRNA-induced KDITSN mice were generated by repeated delivery (every 72 hours, for 18 days) of a specific ITSN-1 siRNA sequence (Dharmacon ON-TARGETplus siRNA; 100 μg siRNA per mouse; Dharmacon, Lafayette, CO) using cationic liposomes, by retro-orbital injection into mouse lungs (CD1 females; Jackson Labs, Bar Harbor, ME), as described by us.12Bardita C. Predescu D. Justice M.J. Petrache I. Predescu S. In vivo knockdown of intersectin-1s alters endothelial cell phenotype and causes microvascular remodeling in the mouse lungs.Apoptosis. 2013; 18: 57-76Crossref PubMed Scopus (17) Google Scholar, 14Predescu D.N. Neamu R. Bardita C. Wang M. Predescu S.A. Impaired caveolae function and upregulation of alternative endocytic pathways induced by experimental modulation of intersectin-1s expression in mouse lung endothelium.Biochem Res Int. 2012; 2012: 672705Crossref PubMed Scopus (20) Google Scholar Mice were sacrificed at 2, 6, 12, and 18 days; three mice [control wild-type (wt), vehicle and nonspecific (scrambled) siRNA treated, as well as ITSN siRNA treated] were used per experimental condition and per experiment, with experiments repeated at least three times. Equal doses of the scrambled siRNA and ITSN siRNA were used. No changes in pulmonary cell viability, as indicated by caspase-3 cleavage and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, were recorded between days 1 and 18 of this experimental approach. No mouse mortality occurred. Pulmonary inflammatory infiltrates or vascular enlargements were not detected in KDITSN mouse lungs. In addition, the Dharmacon ON-TARGETplus siRNA reagents and the siRNA concentration used allowed us to minimize the off-target effects and provide a highly specific on-target gene knockdown, while reducing not only the off-target gene modulation but also the extent of ITSN knockdown in other organs.12Bardita C. Predescu D. Justice M.J. Petrache I. Predescu S. In vivo knockdown of intersectin-1s alters endothelial cell phenotype and causes microvascular remodeling in the mouse lungs.Apoptosis. 2013; 18: 57-76Crossref PubMed Scopus (17) Google Scholar Myc-EHITSN DNA (amino acids 1 to 271), cloned into the pReceiver/myc-M43 vector, as in the study by Patel et al,11Patel M. Predescu D. Tandon R. Bardita C. Pogoriler J. Bhorade S. Wang M. Comhair S. Hemnes A.R. Chen J. Machado R. Husain A. Erzurum S. Predescu S. A novel p38 mitogen-activated protein kinase/Elk-1 transcription factor-dependent molecular mechanism underlying abnormal endothelial cell proliferation in plexogenic pulmonary arterial hypertension.J Biol Chem. 2013; 288: 25701-25716Crossref PubMed Scopus (27) Google Scholar was delivered to mouse lungs similar to siRNA. DNA-liposome complexes were prepared at a ratio of 8 nmoles liposomes/1 μg myc-EHITSN DNA, a ratio found in pilot studies to induce efficient protein expression without pulmonary toxicity. Long-term myc-EHITSN protein expression was achieved by repeated myc-EHITSN DNA-liposome delivery, every 48 hours, for 18 days. A mutant EHITSN-W263A fragment in which the W263 was substituted with Ala [(A), a substitution that reduces NPF (Asn-Pro-Phe); main target of the EH domains, binding beyond detection15de Beer T. Carter R.E. Lobel-Rice K.E. Sorkin A. Overduin M. Structure and Asn-Pro-Phe binding pocket of the Eps15 homology domain.Science. 1998; 281: 1357-1360Crossref PubMed Scopus (110) Google Scholar] was cloned into the same vector and used as control. At the end of the treatment period, mice were anesthetized by i.p. delivery of 1 mL/kg body weight of ketamine hydrochloride/xylazine hydrochloride solution (Sigma-Aldrich, St. Louis, MO). Hemodynamic measurements or tissue harvesting for histology, immunohistochemistry, and morphometric and biochemical analyses were performed. Mice were divided in five groups: group 1, EHITSN-transduced KDITSN; group 2, EHITSN-transduced K0ITSN+/−; group 3, untreated KDITSN; group 4, untreated K0ITSN+/−; and group 5, wt mice. A number of 70 K0ITSN+/− mice, strain 129SV/J genetic background, and 110 CD1 mice were used. Age and body weight matched mice were used. K0ITSN+/− mice were genotyped by tail snipping standard procedure. Briefly, mouse tails were chopped and digested in 50 μL digestion buffer (1× modified gitschier buffer, 0.5% Triton, and 1% 2-β-mercaptoethanol), for 3 minutes at 93°C, followed by addition of proteinase K at a final concentration of 1 mg/mL and incubation at 60°C for 2 hours (shaking every 15 minutes). After the 2-hour incubation, proteinase K was denatured at 95°C for 5 minutes. The tubes were then centrifuged at 9,500 × g for 10 minutes, and 1 μL of each extracted DNA sample was used for conventional PCR, along with the ITSN primers, as in the study by Patel et al.11Patel M. Predescu D. Tandon R. Bardita C. Pogoriler J. Bhorade S. Wang M. Comhair S. Hemnes A.R. Chen J. Machado R. Husain A. Erzurum S. Predescu S. A novel p38 mitogen-activated protein kinase/Elk-1 transcription factor-dependent molecular mechanism underlying abnormal endothelial cell proliferation in plexogenic pulmonary arterial hypertension.J Biol Chem. 2013; 288: 25701-25716Crossref PubMed Scopus (27) Google Scholar The PCR products were resolved onto a 1.2% agarose gel and visualized using ethidium bromide. Mouse lung tissue was homogenized in buffer A (20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, and protease and phosphatase inhibitors), using a Brinkmann Polytron homogenizer (Brinkmann, Oxford, CT). Total lysates were prepared by adding SDS and Triton X-100 to a final concentration of 0.3% and 1%, respectively, for 2 hours, at 4°C. The resulting lysates were clarified by centrifugation in a Beckman Optima Max-XP ultracentrifuge with a TLA-55 rotor (Beckman, Indianapolis, IN) at 191,500 × g for 45 minutes at 4°C. Protein concentration was determined using the bicinchoninic acid (Pierce, Rockford, IL) method with a bovine serum albumin standard. Protein samples normalized for total protein were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Strips of nitrocellulose membranes were incubated with the primary and reporter antibodies (Abs) and processed, as in the study by Patel et al.11Patel M. Predescu D. Tandon R. Bardita C. Pogoriler J. Bhorade S. Wang M. Comhair S. Hemnes A.R. Chen J. Machado R. Husain A. Erzurum S. Predescu S. A novel p38 mitogen-activated protein kinase/Elk-1 transcription factor-dependent molecular mechanism underlying abnormal endothelial cell proliferation in plexogenic pulmonary arterial hypertension.J Biol Chem. 2013; 288: 25701-25716Crossref PubMed Scopus (27) Google Scholar The reaction was visualized using the enhanced chemiluminescence kit (Pierce) and HyBlot CL films (Denville Scientific Inc., South Plainfield, NJ). Specific Abs were obtained from the following sources: p38 polyclonal Ab, phospho-p38 monoclonal Ab, c-Fos polyclonal Ab, and myc polyclonal Ab from Cell Signaling Technology (Beverly, MA); ITSN-1 monoclonal Ab from BD Biosciences (San Jose, CA); actin monoclonal Ab from Sigma-Aldrich; and Elk1 polyclonal Ab from Santa Cruz Biotechnology (Dallas, TX). The primary Abs were diluted 1:500 (Elk-1), 1:1000 (p38, phospho-p38, myc, ITSN, and c-Fos), and 1:2000 (actin) using 5% Blotto (Rockland, Limerick, PA) in tris-buffered saline. Horseradish peroxidase–conjugated reporter Abs were from Cappel, Organon Teknika (Durham, NC) and used at 1:1000 dilution; X-ray films will be subjected to densitometry using the ImageJ software version 1.51b (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Nuclear extracts of controls and myc-EHITSN transduced mice were prepared using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce), as previously described.11Patel M. Predescu D. Tandon R. Bardita C. Pogoriler J. Bhorade S. Wang M. Comhair S. Hemnes A.R. Chen J. Machado R. Husain A. Erzurum S. Predescu S. A novel p38 mitogen-activated protein kinase/Elk-1 transcription factor-dependent molecular mechanism underlying abnormal endothelial cell proliferation in plexogenic pulmonary arterial hypertension.J Biol Chem. 2013; 288: 25701-25716Crossref PubMed Scopus (27) Google Scholar The nuclear extracts were then analyzed by enzyme-linked immunosorbent assay (TransAM Kit; Active Motif, Carlsbad, CA), with colorimetric readout quantifiable by spectrophotometry, in a 96-well plate containing the immobilized Elk-1 consensus site oligonucleotide. Activated Elk-1 was detected via an Ab against phosphorylated Elk-1, followed by a horseradish peroxidase–conjugated reporter Ab. The plates were read at 450 nm using a Dynex plate reader (Chantilly, VA). Data from triplicate wells in three different experiments were expressed as means ± SEM. Mouse lungs were inflated with 1% low-melting point agarose in 10% formalin at a constant pressure of 25 cm H2O, allowing for homogeneous expansion of lung parenchyma, and then fixed in 4% paraformaldehyde for 48 hours and paraffin embedded.12Bardita C. Predescu D. Justice M.J. Petrache I. Predescu S. In vivo knockdown of intersectin-1s alters endothelial cell phenotype and causes microvascular remodeling in the mouse lungs.Apoptosis. 2013; 18: 57-76Crossref PubMed Scopus (17) Google Scholar Thin sections (4 to 5 μm thick), cut longitudinally, were stained with hematoxylin/eosin. Images were acquired with a ×20 lens using a Zeiss AxioImagerM1 microscope (Zeiss, Thornwood, NY) equipped with a color digital camera. Bromodeoxyuridine (BrdU) solution (Roche, Indianapolis, IN) was injected i.p., every 24 hours, for 2 consecutive days. Sections of paraffin-embedded mouse lung tissue were subjected to double immunohistochemistry using BrdU Ab (Santa Cruz Biotechnology), with either CD31 Ab (Abbiotec, San Diego, CA) or α-smooth muscle actin (α-SMA) Ab (Abcam, Cambridge, MA), followed by the appropriate Alexa Fluor 488 or Alexa Fluor 594 conjugated reporters (Molecular Probes, Eugene, OR), as in the study by Patel et al.11Patel M. Predescu D. Tandon R. Bardita C. Pogoriler J. Bhorade S. Wang M. Comhair S. Hemnes A.R. Chen J. Machado R. Husain A. Erzurum S. Predescu S. A novel p38 mitogen-activated protein kinase/Elk-1 transcription factor-dependent molecular mechanism underlying abnormal endothelial cell proliferation in plexogenic pulmonary arterial hypertension.J Biol Chem. 2013; 288: 25701-25716Crossref PubMed Scopus (27) Google Scholar The Abs were diluted in phosphate-buffered saline containing 1% bovine serum albumin as follows: 1:100 (BrdU), 1:250 (CD31), and 1:500 (α-SMA). All reporters were used at 1:500 dilution in 1% bovine serum albumin in phosphate-buffered saline. The Prolong Antifade reagent with DAPI (Molecular Probes, Eugene, OR) was used for mounting. Micrographs were acquired using a Zeiss AxioImagerM1 microscope equipped with a color digital camera. For the hemodynamic measurements in mice, we used the Millar system, which includes a microtip catheter transducer (model PVR-1030), a pressure volume system, and Power Lab Data acquisition system using Lab Chart Pro software version 7.0 (AD Instruments Inc., Colorado Spring, CO). The catheter was inserted into the right ventricle (RV) via the external right jugular vein. The Fulton index (the ratio of RV weight to left ventricle + septum weight) or RV weight relative to the animal's body weight was determined as a measurement for RV hypertrophy. Quantification of BrdU+ nuclei was performed on small- and medium-sized (20 μm ≥ diameter ≤ 100 μm) blood vessels, as in the study by Bardita et al.12Bardita C. Predescu D. Justice M.J. Petrache I. Predescu S. In vivo knockdown of intersectin-1s alters endothelial cell phenotype and causes microvascular remodeling in the mouse lungs.Apoptosis. 2013; 18: 57-76Crossref PubMed Scopus (17) Google Scholar For counting the number of BrdU+ cells, a minimum of 25 vessels per section were used (three sections per mouse, three to five mice per experimental condition). All experiments were performed at least three times, with reproducible results. For assessment of the extent of pulmonary vascular remodeling, we performed a stereological assessment of the (intima + media) thickness and mean linear intercept values on 30 slides for every lung. The stereological software Stepanizer version beta 2.28 (http://www.stepanizer.com)16Tschanz S.A. Burri P.H. Weibel E.R. A simple tool for stereological assessment of digital images: the STEPanizer.J Microsc. 2011; 243: 47-59Crossref PubMed Scopus (204) Google Scholar was used, with a 256-point grid (for assessment of pulmonary artery area) and subsampling with a 16-point (course) grid for assessment of alveolar septa, as in the study by Stacher et al.17Stacher E. Graham B.B. Hunt J.M. Gandjeva A. Groshong S.D. McLaughlin V.V. Jessup M. Grizzle W.E. Aldred M.A. Cool C.D. Tuder R.M. Modern age pathology of pulmonary arterial hypertension.Am J Respir Crit Care Med. 2012; 186: 261-272Crossref PubMed Scopus (440) Google Scholar The thickness of pulmonary arteries was determined on the basis of sum of point hits in pulmonary artery (intima + media) per 50 histological fields. The mean linear intercept was calculated as described in the study by Bardita et al.12Bardita C. Predescu D. Justice M.J. Petrache I. Predescu S. In vivo knockdown of intersectin-1s alters endothelial cell phenotype and causes microvascular remodeling in the mouse lungs.Apoptosis. 2013; 18: 57-76Crossref PubMed Scopus (17) Google Scholar The number of profiles of plexiform lesions per slide was determined by the identification of lesion profiles, with the characteristics described in human PAH patients2Tuder R.M. Marecki J.C. 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 (267) Google Scholar, 6Tuder 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 and SUGEN 5416 rat model of PAH.18Abe 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 (384) Google Scholar Uncertain plexiform lesions were not considered for final count. We also quantified the percentage of affected vessels by sorting the pulmonary in three groups: occluded (either slit-like or no lumen), muscularized (irregular or complete layer of muscle), and nonmuscular. Quantification of affected vessels was performed on small- and medium-sized blood vessels (20 μm ≥ diameter ≤ 100 μm), as above, using three sections per mouse, with three mice in the control group and five mice in the experimental group, with the experiments performed at least three times with reproducible results. This number of mice was considered sufficient to detect relevant differences when significance is set at P < 0.05. For comparison of two groups, t-tests (two-tailed) were performed; for multiple groups (three or more groups), one-way analysis of variance and post hoc statistical comparisons with the Bonferroni adjustment were conducted. The data are shown as means ± SEM, if not otherwise specified. A value of P < 0.05 was considered significant. To address the proliferative potential of myc-EHITSN in vivo, mice were transduced with the myc-EHITSN/lipoplexes, as described in Materials and Methods. Briefly, myc-EHITSN lipoplexes were delivered repeatedly, every 48 hours, by retro-orbital injection to wt mice, to long-term siRNA-induced KDITSN mice, and to K0ITSN+/− mice. Compelling published work, including our studies, demonstrates the efficient and highly specific uptake of the i.v. delivered DNA via cationic liposomes by lung endothelium, mainly proliferative ECs, with no cytotoxicity, as lung endothelium is the first vascular bed encountered by the DNA-lipoplexes delivered retro-orbitally; the positive charge of the cationic liposomes mediates their interaction/fusion with the negatively charged cell membrane, followed by endocytic internalization.19McLean J.W. Fox E.A. Baluk P. Bolton P.B. Haskell A. Pearlman R. Thurston G. Umemoto E.Y. McDonald D.M. Organ-specific endothelial cell uptake of cationic liposome-DNA complexes in mice.Am J Physiol. 1997; 273: H387-H404PubMed Google Scholar, 20Thurston G. McLean J.W. Rizen M. Baluk P. Haskell A. Murphy T.J. Hanahan D. McDonald D.M. Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice.J Clin Invest. 1998; 101: 1401-1413Crossref PubMed Google Scholar, 21Miyawaki-Shimizu K. Predescu D. Shimizu J. Broman M. Predescu S. Malik A.B. siRNA-induced caveolin-1 knockdown in mice increases lung vascular permeability via the junctional pathway.Am J Physiol Lung Cell Mol Physiol. 2006; 290: L405-L413Crossref PubMed Scopus (117) Google Scholar, 22Sakurai F. Inoue R. Nishino Y. Okuda A. Matsumoto O. Taga T. Yamashita F. Takakura Y. Hashida M. Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression.J Control Release. 2000; 66: 255-269Crossref PubMed Scopus (147) Google Scholar Also, the liposomes/DNA ratio and liposomes' formulation are critical for endosomal escape, nuclear transport, and transfection efficiency.23Zuhorn I.S. Bakowsky U. Polushkin E. Visser W.H. Stuart M.C. Engberts J.B. Hoekstra D. Nonbilayer phase of lipoplex-membrane mixture determines endosomal escape of genetic cargo and transfection efficiency.Mol Ther. 2005; 11: 801-810Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 24Zuhorn I.S. Kalicharan R. Hoekstra D. Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis.J Biol Chem. 2002; 277: 18021-18028Crossref PubMed Scopus (285) Google Scholar, 25Zhu L. Mahato R.I. Lipid and polymeric carrier-mediated nucleic acid delivery.Expert Opin Drug Deliv. 2010; 7: 1209-1226Crossref PubMed Scopus (115) Google Scholar The ratio of 1 μg EHITSN DNA/8 nmoles liposomes and the liposomes containing the commonly used lipid imethyldioctadecyl ammonium bromide as cationic head and cholesterol, as hydrophobic group, proved critical for efficient EHITSN expression. Our previous studies indicated that this treatment induces efficient and highly specific ITSN-1 knockdown with <40% down-regulation of ITSN in three other murine organs surveyed (ie, heart, liver, and kidney).12Bardita C. Predescu D. Justice M.J. Petrache I. Predescu S. In vivo knockdown of intersectin-1s alters endothelial cell phenotype and causes microvascular remodeling in the mouse lungs.Apoptosis. 2013; 18: 57-76Crossref PubMed Scopus (17) Google Scholar Although K0ITSN+/− mice require less intervention for experimental manipulation of ITSN expression, the turning down ITSN gene by a certain amount using the siRNA approach more closely resembles the PAH state, allowing us to observe in real-time the changes in the ECs and other cells' phenotype, and to fashion a useful and refined model of the disease. Western blot (WB) analyses of long-term KDITSN mouse lung lysates using ITSN Ab follo
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