Hypoxia Inducible-Factor1α Regulates the Metabolic Shift of Pulmonary Hypertensive Endothelial Cells
2010; Elsevier BV; Volume: 176; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2010.090832
ISSN1525-2191
AutoresIwona J. Fijałkowska, Weiling Xu, Suzy Comhair, Allison J. Janocha, Lori Mavrakis, Balaji Krishnamachary, Lijie Zhen, Thianzi Mao, Amy Richter, Serpil C. Erzurum, Rubin M. Tuder,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoSevere pulmonary hypertension is irreversible and often fatal. Abnormal proliferation and resistance to apoptosis of endothelial cells (ECs) and hypertrophy of smooth muscle cells in this disease are linked to decreased mitochondria and preferential energy generation by glycolysis. We hypothesized this metabolic shift of pulmonary hypertensive ECs is due to greater hypoxia inducible-factor1α (HIF-1α) expression caused by low levels of nitric oxide combined with low superoxide dismutase activity. We show that cultured ECs from patients with idiopathic pulmonary arterial hypertension (IPAH-ECs) have greater HIF-1α expression and transcriptional activity than controls under normoxia or hypoxia, and pulmonary arteries from affected patients have increased expression of HIF-1α and its target carbonic anhydrase IX. Decreased expression of manganese superoxide dismutase (MnSOD) in IPAH-ECs paralleled increased HIF-1α levels and small interfering (SI) RNA knockdown of MnSOD, but not of the copper-zinc SOD, increased HIF-1 protein expression and hypoxia response element (HRE)-driven luciferase activity in normoxic ECs. MnSOD siRNA also reduced nitric oxide production in supernatants of IPAH-ECs. Conversely, low levels of a nitric oxide donor reduced HIF-1α expression in normoxic IPAH-ECs. Finally, mitochondria numbers increased in IPAH-ECs with knockdown of HIF-1α. These findings indicate that alterations of nitric oxide and MnSOD contribute to pathological HIF-1α expression and account for lower numbers of mitochondria in IPAH-ECs. Severe pulmonary hypertension is irreversible and often fatal. Abnormal proliferation and resistance to apoptosis of endothelial cells (ECs) and hypertrophy of smooth muscle cells in this disease are linked to decreased mitochondria and preferential energy generation by glycolysis. We hypothesized this metabolic shift of pulmonary hypertensive ECs is due to greater hypoxia inducible-factor1α (HIF-1α) expression caused by low levels of nitric oxide combined with low superoxide dismutase activity. We show that cultured ECs from patients with idiopathic pulmonary arterial hypertension (IPAH-ECs) have greater HIF-1α expression and transcriptional activity than controls under normoxia or hypoxia, and pulmonary arteries from affected patients have increased expression of HIF-1α and its target carbonic anhydrase IX. Decreased expression of manganese superoxide dismutase (MnSOD) in IPAH-ECs paralleled increased HIF-1α levels and small interfering (SI) RNA knockdown of MnSOD, but not of the copper-zinc SOD, increased HIF-1 protein expression and hypoxia response element (HRE)-driven luciferase activity in normoxic ECs. MnSOD siRNA also reduced nitric oxide production in supernatants of IPAH-ECs. Conversely, low levels of a nitric oxide donor reduced HIF-1α expression in normoxic IPAH-ECs. Finally, mitochondria numbers increased in IPAH-ECs with knockdown of HIF-1α. These findings indicate that alterations of nitric oxide and MnSOD contribute to pathological HIF-1α expression and account for lower numbers of mitochondria in IPAH-ECs. Severe pulmonary arterial hypertension is characterized by significant increases in pulmonary artery pressures to levels present in the systemic circulation. Pulmonary hypertension (PH) is a major determinant of morbidity and mortality in several pulmonary and heart diseases. The pathogenesis of severe pulmonary arterial hypertension has revolved around excessive vasoconstriction and/or abnormal pulmonary vascular remodeling. Recent experimental evidence has linked the pulmonary vascular disease in severe pulmonary arterial hypertension to an abnormal proliferative vascular cell phenotype, which is also characterized by resistance to endothelial and/or vascular smooth muscle cell apoptosis.1Lee SD Shroyer KR Markham NE Cool CD Voelkel NF Tuder RM Monoclonal endothelial cell proliferation is present in primary but not secondary pulmonary hypertension.J Clin Invest. 1998; 101: 927-934Crossref PubMed Scopus (346) Google Scholar The identification that there is a clonal expansion of endothelial cells (ECs) in idiopathic pulmonary arterial hypertension (IPAH)1Lee SD Shroyer KR Markham NE Cool CD Voelkel NF Tuder RM Monoclonal endothelial cell proliferation is present in primary but not secondary pulmonary hypertension.J Clin Invest. 1998; 101: 927-934Crossref PubMed Scopus (346) Google Scholar and somatic and germline mutations in the transforming growth factor β superfamily, particularly of bone morphogenetic protein receptor 2,2Deng Z Morse JH Slager SL Cuervo N Moore KJ Venetos G Kalachikov S Cayanis E Fischer SG Barst RJ Hodge SE Knowles JA Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene.Am J Hum Genet. 2000; 67: 737-744Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar, 3The International PPH Consortium Lane KB Machado RD Pauciulo. M.W. Thompson JR Philips III, JA Loyd JE Nichols WC Trembath RC Heterozygous germline mutations in BMPR2 encoding a TGF-B receptor cause familiar pulmonary hypertension.Nat Genet. 2000; 26: 81-84Crossref PubMed Scopus (1132) Google Scholar led to the concept that this abnormal vascular cell proliferation process resembles that seen in neoplastic processes.4Voelkel NF Cool CD Lee SD Wright L Geraci MW Tuder RM Primary pulmonary hypertension between inflammation and cancer.Chest. 1999; 114: 225S-230SCrossref Scopus (169) Google Scholar The hypoxia-driven activation of hypoxia inducible factor (HIF), which contributes to several of the key features present in neoplastic processes,5Rai PR Cool CD King JAC Stevens T Burns N Winn RA Kasper M Voelkel NF The cancer paradigm of severe pulmonary arterial hypertension.Am J Respir Crit Care Med. 2008; 178: 558-564Crossref PubMed Scopus (187) Google Scholar also plays a role in the pathogenesis of experimental pulmonary hypertension.6Yu AY Shimoda LA Iyer NV Huso DL Sun X McWilliams R Beaty T Sham JS Wiener CM Sylvester JT Semenza GL Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1 alpha.J Clin Invest. 1999; 103: 691-696Crossref PubMed Scopus (525) Google Scholar, 7Brusselmans K Compernolle V Tjwa M Wiesener MS Maxwell PH Collen D Carmeliet P Heterozygous deficiency of hypoxia-inducible factor-2α protects mice against pulmonary hypertension and right ventricular dysfunction during prolonged hypoxia.J Clin Invest. 2003; 111: 1519-1527Crossref PubMed Scopus (220) Google Scholar HIF, a heterodimer of the HIF-1α or -2α, and HIF-1β, mediates adaptive molecular responses to low oxygen availability,8Semenza GL Life with oxygen.Science. 2007; 318: 62-64Crossref PubMed Scopus (502) Google Scholar leading to transcriptional activation of genes that regulate energy metabolism, erythropoiesis, vasomotor tone, and angiogenesis.9Semenza GL Targeting HIF-1 for cancer therapy.Nat Rev Cancer. 2003; 3: 721-732Crossref PubMed Scopus (4816) Google Scholar HIF-1α therefore plays a pathological role in tumor angiogenesis, neoplastic invasion,9Semenza GL Targeting HIF-1 for cancer therapy.Nat Rev Cancer. 2003; 3: 721-732Crossref PubMed Scopus (4816) Google Scholar and in the metabolic shift of cancer cells toward glycolysis, which underlies the Warburg phenomena.10Warburg O Posener K Negelein E Ueber den Stoffwechsel der Carcinomzelle.Biochem. 1924; Z152: 319-344Google Scholar HIF-2α coordinates fetal lung development11Compernolle V Brusselmans K Acker T Hoet P Tjwa M Beck H Plaisance S Dor Y Keshet E Lupu F Nemery B Dewerchin M Van Veldhoven P Plate K Moons L Collen D Carmeliet P Loss of HIF-2[alpha] and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice.Nat Med. 2002; 8: 702-710Crossref PubMed Google Scholar and adaptive lung responses to chronic hypoxia, which include the control of expression of genes involved in pulmonary vascular cell proliferation and angiogenesis.12Tuder RM Flook BE Voelkel NF Increased gene expression for VEGF and the VEGF receptors KDR/Flk and Flt in lungs exposed to acute or to chronic hypoxia. Modulation of gene expression by nitric oxide.J Clin Invest. 1995; 95: 1798-1807Crossref PubMed Scopus (509) Google Scholar Identification of vascular endothelial growth factor, HIF-1α, and HIF1β expression within endothelial plexiform lesions13Tuder RM Chacon M Alger LA Wang J Taraseviciene-Stewart L Kasahara Y Cool CD Bishop AE Geraci MW Semenza GL Yacoub M Polak JM Voelkel NF Expression of angiogenesis-related molecules in plexiform lesions in severe pulmonary hypertension: evidence for a process of disordered angiogenesis.J Pathol. 2001; 195: 367-374Crossref PubMed Scopus (367) Google Scholar and in pulmonary artery medial smooth muscle cells14Bonnet 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-1 alpha-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 (421) Google Scholar suggests that HIF-dependent signaling may contribute to the proliferative vasculopathy of IPAH. The potential pathogenic role of HIF-1α in PH was supported by the findings that HIF-1α6Yu AY Shimoda LA Iyer NV Huso DL Sun X McWilliams R Beaty T Sham JS Wiener CM Sylvester JT Semenza GL Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1 alpha.J Clin Invest. 1999; 103: 691-696Crossref PubMed Scopus (525) Google Scholar or HIF-2α7Brusselmans K Compernolle V Tjwa M Wiesener MS Maxwell PH Collen D Carmeliet P Heterozygous deficiency of hypoxia-inducible factor-2α protects mice against pulmonary hypertension and right ventricular dysfunction during prolonged hypoxia.J Clin Invest. 2003; 111: 1519-1527Crossref PubMed Scopus (220) Google Scholar heterozygous mice have decreased hypoxic PH, when compared with littermates, and abnormalities in HIF-1α underlie the mitochondria pathology and PH in the Fawn-Hooded rat model of spontaneous disease.14Bonnet 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-1 alpha-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 (421) Google Scholar The role of abnormal HIF-dependent signaling in human IPAH has not been addressed. Our prior work indicated that cultured ECs derived from human IPAH lungs (IPAH-ECs) exhibit an abnormal metabolic phenotype that is characterized by low numbers of mitochondria and decreased oxygen consumption, significantly higher glycolytic rate,15Xu W Koeck T Lara AR Neumann D DiFilippo FP Koo M Janocha AJ Masri FA Arroliga AC Jennings C Dweik RA Tuder RM Stuehr DJ Erzurum SC Alterations of cellular bioenergetics in pulmonary artery endothelial cells.Proc Natl Acad Sci USA. 2007; 104: 1342-1347Crossref PubMed Scopus (255) Google Scholar apoptosis-resistance, and increased cell proliferation.16Masri FA Xu W Comhair SA Asosingh K Koo M Vasanji A Drazba J nand-Apte B Erzurum SC Hyperproliferative apoptosis-resistant endothelial cells in idiopathic pulmonary arterial hypertension.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L548-L554Crossref PubMed Scopus (245) Google Scholar Moreover, pulmonary and total body nitric oxide (NO) are lower in IPAH patients, as compared with healthy controls.17Girgis RE Champion HC Diette GB Johns RA Permutt S Sylvester JT Decreased exhaled nitric oxide in pulmonary arterial hypertension: response to bosentan therapy.Am J Respir Crit Care Med. 2005; 172: 352-357Crossref PubMed Scopus (100) Google Scholar, 18Kaneko FT Arroliga AC Dweik RA Comhair SA Laskowski D Oppedisano R Thomassen MJ Erzurum SC Biochemical reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension.Am J Respir Crit Care Med. 1998; 158: 917-923Crossref PubMed Scopus (173) Google Scholar, 19Machado RF Londhe Nerkar MV Dweik RA Hammel J Janocha A Pyle J Laskowski D Jennings C Arroliga AC Erzurum SC Nitric oxide and pulmonary arterial pressures in pulmonary hypertension.Free Radic Biol Med. 2004; 37: 1010-1017Crossref PubMed Scopus (59) Google Scholar, 20Ozkan M Dweik RA Laskowski D Arroliga AC Erzurum SC High levels of nitric oxide in individuals with pulmonary hypertension receiving epoprostenol therapy.Lung. 2001; 179: 233-243Crossref PubMed Scopus (55) Google Scholar, 21Xu W Kaneko FT Zheng S Comhair SA Janocha AJ Goggans T Thunnissen FB Farver C Hazen SL Jennings C Dweik RA Arroliga AC Erzurum SC Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension.FASEB J. 2004; 18: 1746-1748PubMed Google Scholar Here, we hypothesize that IPAH-ECs have altered hypoxia sensing, with increased expression of HIF-1α, which accounts for the decreased mitochondria in IPAH-ECs.15Xu W Koeck T Lara AR Neumann D DiFilippo FP Koo M Janocha AJ Masri FA Arroliga AC Jennings C Dweik RA Tuder RM Stuehr DJ Erzurum SC Alterations of cellular bioenergetics in pulmonary artery endothelial cells.Proc Natl Acad Sci USA. 2007; 104: 1342-1347Crossref PubMed Scopus (255) Google Scholar Moreover, we postulate that the oxidative stress seen in IPAH lungs,22Bowers R Cool C Murphy RC Tuder RM Hopken MW Flores SC Voelkel NF Oxidative stress in severe pulmonary hypertension.Am J Respir Crit Care Med. 2004; 169: 764-769Crossref PubMed Google Scholar, 23Grobe AC Wells SM Benavidez E Oishi P Azakie A Fineman JR Black SM Increased oxidative stress in lambs with increased pulmonary blood flow and pulmonary hypertension: role of NADPH oxidase and endothelial NO synthase.Am J Physiol−Lung Cell MPH. 2006; 290: L1069-L1077Crossref PubMed Scopus (123) Google Scholar, 24Liu JQ Zelko IN Erbynn EM Sham JS Folz RJ Hypoxic pulmonary hypertension: role of superoxide and NADPH oxidase (gp91phox).Am J Physiol Lung Cell Mol Physiol. 2006; 290: L2-L10Crossref PubMed Scopus (230) Google Scholar, 25Wedgwood S Black SM Role of reactive oxygen species in vascular remodeling associated with pulmonary hypertension.Antioxid Redox Signal. 2003; 5: 759-769Crossref PubMed Scopus (68) Google Scholar caused by decreased expression of manganese superoxide dismutase and nitric oxide, accounts for the up-regulation of HIF-1α, and ultimately, for the decreased mitochondria numbers in IPAH-ECs. Our findings using disease-relevant cells provide the rationale for the development of therapies targeting the energetic shift and HIF-1 activation in pulmonary arterial hypertension. IPAH patients were identified by the clinical classification of pulmonary hypertension,26Simonneau G Galie N Rubin LJ 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 (1467) Google Scholar recently updated in the fourth World Symposium on Pulmonary Hypertension.27Simonneau G Robbins IM Beghetti M Channick RN Delcroix M Denton CP Elliott CG Gaine SP Gladwin MT Jing ZC Krowka MJ Langleben D Nakanishi N Souza R Updated clinical classification of pulmonary hypertension.J Am Coll Cardiol. 2009; 54: S43-S54Abstract Full Text Full Text PDF PubMed Scopus (1793) Google Scholar Altogether, five IPAH and three control subjects were studied. Clinical characteristics among subjects were similar (age in years, IPAH 43 ± 6, control 29 ± 12; sex [female/male], IPAH 4/1, control 2/1; race [Caucasian/African American/Hispanic], IPAH 5/0/0, control 3/0/0). Pulmonary hypertension was diagnosed by right heart catheterization performed for clinical care (pulmonary artery pressures [mm Hg], IPAH, systolic 88 ± 3, diastolic 37 ± 4, mean 59 ± 3). The study was approved by the Cleveland Clinic Institutional Review Board. The immunohistochemical studies were performed in histological sections of IPAH (n = 5) or normal lungs (n = 5) (cases P1 to P5, see table in28Richter 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; 170: 1340-1348Crossref PubMed Scopus (103) Google Scholar). These five patients were treated with intravenous prostacyclin before lung transplant. The study was approved by the Western Institutional Review Board (in lieu of the Johns Hopkins Institutional Review Board). Human pulmonary artery ECs were dissociated and cultured as described before16Masri FA Xu W Comhair SA Asosingh K Koo M Vasanji A Drazba J nand-Apte B Erzurum SC Hyperproliferative apoptosis-resistant endothelial cells in idiopathic pulmonary arterial hypertension.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L548-L554Crossref PubMed Scopus (245) Google Scholar in endothelial cell growth medium (EGM-2, Cambrex, Walkersville, MD) on plates precoated with fibronectin 1 μg/cm2 at 37°C for 1 hour. Cells were passaged at 70% to 80% confluence by dissociation from plates with 0.25% trypsin-EDTA (Invitrogen Corporation, Carlsbad, CA). Primary cultures of passages 5 to 8 were used in experiments. HeLa cells were maintained in Dulbecco's Modified Eagle Medium (Invitrogen Corporation) with 10% heat-inactivated fetal calf serum, and 1% penicillin/streptomycin. Human umbilical vein ECs (HUVECs) (Lonza, Walkersville, MD) were cultured in endothelial cell growth medium (EGM-2, Cambrex). For nitric oxide (NO) donor treatment, cells were incubated with DETA NONOate (detaNO) (AXXORA LLC, San Diego, CA) as the time indicated. For hypoxia treatment, cells were placed directly in a 5% CO2 and 95% air incubator (21% O2) or exposed to hypoxia in a sealed chamber or a hypoxic incubator, and cultured at 37°C. Cytosolic and nuclear extracts of pulmonary artery ECs were prepared according to manufacturer's protocol (Panomics Inc, Fremont, CA). Whole cell lysates were prepared as previously described21Xu W Kaneko FT Zheng S Comhair SA Janocha AJ Goggans T Thunnissen FB Farver C Hazen SL Jennings C Dweik RA Arroliga AC Erzurum SC Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension.FASEB J. 2004; 18: 1746-1748PubMed Google Scholar or in radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology, Santa Cruz, CA). Protein was separated by electrophoresis on a 4% to 15% Tris-HCl precast gel (Bio-Rad Lab, Hercules, CA), and then transferred onto polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA). Alternatively, XT Precast Gels (Bio-Rad) and 3-(N-morpholino) propanesulfonic acid buffer were used for SDS polyacrylamide gel electrophoresis. Antibodies used for Western analyses included mouse monoclonal anti-HIF-1α antibody (Ab) (BD Biosciences, Mississauga, ON, Canada), anti-catalase Ab (Sigma-Aldrich, St. Louis, MO), anti-Complex III-2 Ab (Molecular Probes, Inc., Eugene, OR), anti-cytochrome c Ab (Santa Cruz Biotechnology) rabbit polyclonal anti-manganese superoxide dismutase (MnSOD) Ab, anti-CuZnSOD Ab (Santa Cruz Biotechnology), anti-von Hippel-Lindau (VHL) Ab, anti-prolyl hydroxylase 2 Ab (Novus Biologicals, Littleton, CO), or anti-EC nitric oxide synthase (eNOS) Ab (Affinity Bioreagents, Golden, CO), followed by a secondary anti-rabbit or anti-mouse Ab (Amersham, Arlington Heights, IL, or Cell Signaling Technology, Beverly, MA). Goat polyclonal anti-lamin B Ab, rabbit polyclonal anti-enolase Ab, or mouse monoclonal anti-α-tubulin Ab (Santa Cruz), anti-glyceraldehyde phosphate dehydrogenase Ab (Research Diagnostics, Inc., Concord MA), and anti-tubulin Ab (Santa Cruz Biotechnology), were used for protein loading. DNA was isolated and purified from IPAH-ECs and IPAH whole lung tissue specimens. The DNA was then bisulfite modified with Methyl Detector Bisulfite Modification Kit (Active Motif, Carlsbad CA). The positive control consisted of p16Kip1 methylation assessed by nested PCR, using primers included in the kit. Methylation status of the VHL promoter was assessed using the following primers29Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5058) Google Scholar: unmethylated: forward (−185), 5′-GTTGGAGGATTTTTTTGTGTATGT-3′, reverse (−20), 5′-CCCAAACCAAACACCACAAA-3′, with an expected amplicon of 165 bp; and methylated: forward (−183), 5′-TGGAGGATTTTTTTGCGTACGC-3′, reverse (−25), 5′-GAACCGAACGCCGCGAA-3′, with expected amplicon of 158 bp. Methylated DNA and unmethylated DNA from Chemicon were used as positive and negative controls, respectively, in reactions run in parallel with sample DNA. The PCR protocol were as follows: 95°C × 5 minutes, (95°C × 45 seconds, 59°C × 45 seconds, 72°C × 60 seconds) × 35, 72°C × 10 minutes, followed by maintenance at 4°C. Wild-type hypoxia response element (HRE)-luciferase reporter construct and mutant HRE-luciferase reporter construct were generous gifts from Dr. M.C. Simon.30Arsham AM Plas DR Thompson CB Simon MC Phosphatidylinositol 3-kinase/Akt signaling is neither required for hypoxic stabilization of HIF-1 alpha nor sufficient for HIF-1-dependent target gene transcription.J Biol Chem. 2002; 277: 15162-15170Crossref PubMed Scopus (156) Google Scholar Both plasmids have three copies of HRE in tandem and cloned upstream of the Firefly luciferase gene in the pGL2 vector. According to the manufacturer protocol of Lipofectamine 2000 Reagent (Invitrogen Corp), IPAH-ECs, or control human pulmonary artery ECs were transiently transfected with wild-type or mutant constructs for 5 hours and cotransfected with Renilla luciferase construct for normalization of transfection efficiency. Alternatively, cells were transfected using Fugene6 (Roche Applied Science, Indianapolis, IN) or Targeting Systems (El Cajon, CA) instead of lipofectamine. In parallel experiments, enhanced green fluorescence protein was used to determine percentage of cells transfected. After transfection, cells were exposed to hypoxia with 2.5% O2, treated with 125 μmol/L CoCl2, or left untreated under normoxia (21% O2); 48-hour after transfection, cells were assayed for Western blot, luciferase activity using a Dual-Luciferase Reporter Assay System (Promega Corp, Madison, WI), and for protein content by Bradford method (BioRad Laboratories, Inc., Hercules, CA). Wild-type HRE-luciferase activity was determined by values of Firefly luciferase normalized by the Renilla luciferase activity, protein content, and mutant HRE-luciferase reporter construct activity. Fold-induction of HRE binding activation was the ratio of normalized HRE-luciferase activity under treatment with CoCl2 or hypoxia over the relative activity in cells exposed to normoxia. Immunohistochemical detection of HIF-1α was performed with the mouse anti-human HIF-1α Ab (Novus Biol.) at 1:8000 dilution for 30 minutes at 37C using the Dako Cytomation CSA system for mouse antibodies (Dako). Histological sections of formalin-fixed, paraffin-embedded lung tissues were dehydrated followed by antigen retrieval with a preheated citrate buffer (Dako). The sections were then blocked with avidin-biotin and then treated with 3% H2O2 for 7 minutes. Following incubation with primary Ab or negative control (mouse IgG), the slides were incubated with the link Ab for 15 minutes, then with streptavidin-biotin for 15 minutes, and the biotinylated tyramide amplification reagent diluted 1:10 in 0.10 M Tris.HCl, 0.15 M NaCl, 0.5% BMP blocking buffer. The secondary streptavidin-horseradish peroxidase was incubated for 15 minutes followed by development with diaminobenzidine. Carbonic anhydrase IX (CAIX) expression was detected with a rabbit anti-human CAIX (Novus Biol) at 1:500 dilution for 1 hour at room temperature. Histological sections of formalin-fixed, paraffin-embedded lung tissues were dehydrated and blocked with serum block for 30 minutes, followed by incubation with primary Ab or rabbit serum. The slides were then developed by the avidin-biotin system with diaminobenzidine as chromogen (Vector). MnSOD small interfering (si)RNA was synthesized by Ambion (Austin, TX). The sense and antisense MnSOD siRNA were 5′-GGAACAACAGGCCUUAUUCtt-3′ (sense) and 5′-GAAUAAGGCCUGUUGUUCCtt-3′ (antisense). SilencerTM Negative control #1 siRNA (siRNA control) (Ambion, Austin TX) was used as a negative control.31Comhair SAA Xu W Ghosh S Thunnissen FBJM Almasan A Calhoun WJ Janocha AJ Zheng L Hazen SL Erzurum SC Superoxide dismutase inactivation in pathophysiology of asthmatic airway remodeling and reactivity.Am J Pathol. 2005; 166: 663-674Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar SOD activity determined by the rate of reduction of cytochrome c with one unit (U) of SOD activity is defined as the amount of SOD required to inhibit the rate of cytochrome c reduction by 50%. HIF-1α micro (mi)RNA (AF304431) served as the target sequence. BLOCK-iT RNAi Designer from Invitrogen was used to design two single-stranded DNA oligonucleotides encoding the target pre-miRNA. The top and bottom strands were annealed to generate a double-stranded oligonucleotide suitable for cloning into BLOCK-iT Pol II miR RNAi Expression Vectors (pcDNA6.2-GW/EmGFP-miR). This vector contains enhanced green fluorescent protein coding sequence that allows visual delivery assessment. Overall 12 different constructs with double-stranded oligos were tested and pcDNA6.2-GW/EmGFP-miR-neg control plasmid (Invitrogen) which is predicted not to target any known vertebrate gene was used as a negative control. The most effective blocking sequence was selected by Dual-Luciferase Reporter Assay (Promega), based on the inhibition of HRE-luciferase activity. Percent inhibition was calculated according to the formula: (A − B)/A × 100%, where A = relative (fold) HRE-luciferase activity induced by CoCl2 or hypoxia in control vector transfected cells, B = relative (fold) HRE-luciferase activity induced by CoCl2 or hypoxia in HIF-1α miRNA-transfected cells. The most potent silencing sequence used for further experiments targeted the sequence AGGATCAGACACCTAGTCCT (1483 to 1503) within HIF-1α mRNA, which had 54% to 75% inhibition of HRE-luciferase activity as determined for each cell line separately. IPAH-ECs and control cells in chamber slides were transfected with HIF-1α miRNA or control miRNA. Seventy-two hours following transfection, mitochondria were stained with Mitotracker Red 580 (Invitrogen). The slides were mounted with Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (Vector Labs, Burlingame, CA), sealed, and analyzed by confocal laser-scanning microscopy (TCS-40; Leica Microsystems, Cambridge, UK). As previously described,15Xu W Koeck T Lara AR Neumann D DiFilippo FP Koo M Janocha AJ Masri FA Arroliga AC Jennings C Dweik RA Tuder RM Stuehr DJ Erzurum SC Alterations of cellular bioenergetics in pulmonary artery endothelial cells.Proc Natl Acad Sci USA. 2007; 104: 1342-1347Crossref PubMed Scopus (255) Google Scholar total DNA extracted from IPAH-EC was digested with restriction enzyme PvuII, electrophoresed through a 0.8% agarose gel, and transferred to Duralon-UV membranes (Stratagene, Cedar Creek, TX). The filters were hybridized with the 32P-labeled PCR generated mitochondrial DNA (mtDNA) probe and visualized with autoradiography. Data were shown as mean ± SE. All statistical comparisons were performed using the Student's t-test, paired t-test, analysis of variance, or Wilcoxon nonparametric analyses as appropriate. The level of significance set at or below 0.05. All data were analyzed by the JMP 7 software (SAS Institute, Cary, NC). As HIF-1α contributes to proliferative properties of neoplastic cells,5Rai PR Cool CD King JAC Stevens T Burns N Winn RA Kasper M Voelkel NF The cancer paradigm of severe pulmonary arterial hypertension.Am J Respir Crit Care Med. 2008; 178: 558-564Crossref PubMed Scopus (187) Google Scholar is expressed in plexiform lesions in IPAH,13Tuder RM Chacon M Alger LA Wang J Taraseviciene-Stewart L Kasahara Y Cool CD Bishop AE Geraci MW Semenza GL Yacoub M Polak JM Voelkel NF Expression of angiogenesis-related molecules in plexiform lesions in severe pulmonary hypertension: evidence for a process of disordered angiogenesis.J Pathol. 2001; 195: 367-374Crossref PubMed Scopus (367) Google Scholar and has been recently implicated in experimental pulmonary hypertension,14Bonnet 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-1 alpha-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 (421) Google Scholar we evaluated HIF-1α expression in the highly proliferative IPAH-ECs. HIF-1α was expressed in cultured IPAH-ECs (n = 3) but not control ECs (n = 3) under normoxia (control: 1.00 ± 0.14 vs. IPAH: 1.49 ± 0.27, normalized relative expression levels, n = 10 replicate experiments, Student's t-test, P < 0.05). IPAH-ECs had approximately twofold higher HIF-1α expression under hypoxia as well (Figure 1, A–B). Hypoxia dose response showed that HIF-1α in IPAH-ECs expression was greater than controls at any oxygen tension (paired t-test, P = 0.02). Consistent with greater HIF-1α expression, IPAH-ECs (n = 3) had greater HRE-luciferase reporter activation than control cells (n = 3) under normo
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