Chronic Hypoxia-Enhanced Murine Pulmonary Vasoconstriction

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system under normoxic conditions: its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 1997; 272:22642–22647 Tanimoto K, Makino Y, Pereira T, et al. Mechanism of regulation of the hypoxia-inducible factor-1␣ by the von Hippel-Lindau tumor suppressor protein. EMBO J 2000; 19:4298 – 4309 Hirsila M, Koivunen P, Gunzler V, et al. Characterization of the human prolyl 4-hydroxylases that modify the hypoxiainducible factor. J Biol Chem 2003; 278:30772–30780 Dames SA, Martinez-Yamout M, De Guzman RN, et al. Structural basis for HIF-1␣/CBP recognition in the cellular hypoxic response. Proc Natl Acad Sci U S A 2002; 99:5271– 5276 Freedman SJ, Sun ZY, Poy F, et al. Structural basis for recruitment of CBP/p300 by hypoxia-inducible factor-1␣. Proc Natl Acad Sci U S A 2002; 99:5367–5372 Lando D, Peet DJ, Gorman JJ, et al. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes Dev 2002; 16:1466 –1471 Lando D, Peet DJ, Whelan DA, et al. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science 2002; 295:858 – 861 Mahon PC, Hirota K, Semenza GL. FIH-1: a novel protein that interacts with HIF-1␣ and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev 2001; 15:2675– 2686 Yu AY, Frid MG, Shimoda LA, et al. Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible factor-1 in the lung. Am J Physiol 1998; 275:L818 –L826 Feldser D, Agani F, Iyer NV, et al. Reciprocal positive regulation of hypoxia-inducible factor 1␣ and insulin-like growth factor 2. Cancer Res 1999; 59:3915–3918 Hellwig-Burgel T, Stiehl DP, Jelkmann W. Hypoxia-inducible factor 1: more than a hypoxia-inducible transcription factor. In: Lahiri S, Semenza GL, Prabhakar NR, eds. Oxygen sensing: responses and adaptation to hypoxia. New York, NY: Marcel Dekker, 2003; 95–108 Fukuda R, Hirota K, Fan F, et al. Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem 2002; 277:38205–38211 Laughner E, Taghavi P, Chiles K, et al. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1␣ (HIF-1␣) synthesis: a novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol 2001; 21:3995– 4004 Treins C, Giorgetti-Peraldi S, Murdaca J, et al. Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway. J Biol Chem 2002; 277:27975–29781 Iyer NV, Kotch LE, Agani F, et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1␣. Genes Dev 1998; 12:149 –162 Ryan HE, Lo J, Johnson RS. HIF-1␣ is required for solid tumor formation and embryonic vascularization. EMBO J 1998; 17:3005–3015 Yu AY, Shimoda LA, Iyer NV, et al. Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1␣. J Clin Invest 1999; 103:691– 696 Shimoda LA, Manalo DJ, Sham JS, et al. Partial HIF-1␣ deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia. Am J Physiol 2001; 281:L202– L208 Wang J, Juhaszova M, Rubin LJ, et al. Hypoxia inhibits gene expression of voltage-gated K⫹ channel alpha subunits in

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pulmonary artery smooth muscle cells. J Clin Invest 1997; 100:2347–2353 Hu CJ, Wang LY, Chodosh LA, et al. Differential roles of hypoxia-inducible factor 1␣ (HIF-1␣) and HIF-2␣ in hypoxic gene regulation. Mol Cell Biol 2003; 23:9361–9374 Scortegagna M, Ding K, Oktay Y, et al. Multiple organ pathology, metabolic abnormalities and impaired homeostasis of reactive oxygen species in Epas1-/- mice. Nat Genet 2003; 35:331–340 Sowter HM, Raval R, Moore J, et al. Predominant role of hypoxia-inducible transcription factor (HIF)-1␣ versus HIF-2␣ in regulation of the transcriptional response to hypoxia. Cancer Res 2003; 63:6130 – 6134 Brusselmans K, Compernolle V, Tjwa M, et al. Heterozygous deficiency of hypoxia-inducible factor-2␣ protects mice against pulmonary hypertension and right ventricular dysfunction during prolonged hypoxia. J Clin Invest 2003; 111: 1519 –1527 Tuder RM, Chacon M, Alger L, et al. Expression of angiogenesis-related molecules in plexiform lesions in severe pulmonary hypertension: evidence for a process of disordered angiogenesis. J Pathol 2001; 195:367–374

Chronic Hypoxia-Enhanced Murine Pulmonary Vasoconstriction* Role of Superoxide and gp91phox John Q. Liu, MD; Efua M. Erbynn, BS; and Rodney J. Folz, PhD

Chronic hypoxia (CH) is a common cause of pulmonary hypertension (PH). Accumulating evidence suggests that changes in the activity of endothelin (ET)-1 receptors may play an important role in CH-induced PH. After 3 weeks of CH (10% O2) exposure, we found that the isolated intra-pulmonary artery (PA) constrictor response to ET-1 was significantly increased in wild-type (wt) mice. The administration of Cu/Zn superoxide dismutase (SOD) markedly reduced the CH-enhanced maximal PA constrictor response to ET-1, demonstrating the contribution of superoxide to CH-enhanced PA constrictor responses. Using mice that are completely deficient in gp91phox (a subunit protein of the superoxide producing nicotinamide adenine dinucleotide phosphate [NADPH] oxidase), we found that CH-enhanced PA constriction to *From the Departments of Medicine and Cell Biology, Division of Pulmonary, Allergy, and Critical Care Medicine, Duke University Medical Center, Durham, NC. This study was supported, in part, by American Heart Association (Mid-Atlantic Affiliates) Beginning Grant-in-Aid to J.Q. Liu and by NIH grant R01 (HL-64894) to R.J. Folz. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: John Q. Liu, MD, Room 341 MSRB, Division of Pulmonary, Allergy, and Critical Care Medicine, Duke University Medical Center, Durham, NC 27710; e-mail: john.liu@ duke.edu

47th Annual Thomas L. Petty Lung Conference: Cellular and Molecular Pathobiology of Pulmonary Hypertension

ET-1 was completely blocked (decreases in mean [ⴞ SE] maximal isometric tension from 5.43 ⴞ 0.35 to 3.33 ⴞ 0.19 mN; n ⴝ 7; p < 0.01). Using a lucigenin-enhanced chemiluminescence technique to measure superoxide, we found that the 3 weeks of CH significantly increased superoxide levels in PA isolated from wt mice. The addition of ET-1 further increased superoxide production. To demonstrate that the increased chemiluminescence is due to superoxide generation, we added Cu/Zn SOD, which markedly decreased chemiluminescence, demonstrating the specificity of this assay. When gp91phox knockout mice were exposed to CH, they had significantly reduced levels of superoxide compared to CH-treated wt mice. Our results demonstrate that the CH-enhanced PA constrictor response to ET-1 is mediated by NADPH oxidase (gp91phox)-derived superoxide overproduction that may contribute to the pathogenesis of CH-induced PH. (CHEST 2005; 128:594S–596S)

PA Ring Contractility Studies PA rings (internal diameter, 100 to 150 ␮m) were isolated from the mouse intra-PA and were placed in a small-vessel wire myograph chamber. Changes in PA isometric tension were recorded.7 Measurement of Superoxide The measurement of superoxide levels in isolated murine PAs were performed using a lucigenin-enhanced chemiluminescence technique.2,7,8

Results CH exposure enhanced PA constriction to endothelin (ET)-1 in wt mice (increase in mean [⫾ SE] maximal isometric tension from 3.58 ⫾ 0.23 to 5.43 ⫾ 0.35 mN; n ⫽ 6; p ⬍ 0.01) [Fig 1, top, A]. This was reduced by the

Abbreviations: CH ⫽ chronic hypoxia; ET ⫽ endothelin; KO ⫽ knockout; NADPH ⫽nicotinamide adenine dinucleotide phosphate; PA ⫽ pulmonary artery; PH ⫽ pulmonary hypertension; RLU ⫽ relative light units; SOD ⫽ superoxide dismitase; wt ⫽ wild-type

hypoxia (CH) is a major cause of pulmonary C hronic hypertension (PH) and right ventricular hypertrophy

in patients with COPD.6 Some studies2– 4 have reported that CH-induced PH is due to increased vasoconstrictor activities and/or depressed endothelium-dependent vasodilatation along with pulmonary vascular remodeling. Other studies1,4,5,10,11 have suggested that changes in the production of reactive oxygen species, such as superoxide, may mediate these changes. Nonphagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, such as vascular NADPH oxidases, are the major source of reactive oxygen species in the cardiovascular system.7 Recent studies3 have suggested that gp91phox is an important subunit protein that is required for NADPH oxidase activity. However, no studies have examined the role of NADPH oxidase (gp91phox) on CH-increased pulmonary vasoconstrictor activities. We hypothesized that exposure to CH leads to enhanced gp91phox-dependent superoxide production, which in turn enhances PA constrictor responses. In this study, we used a previously described small-vessel bioassay7 to demonstrate the important role that gp91phox plays in CH-enhanced murine pulmonary artery (PA) constrictor responsiveness.

Experimental Procedures Mice and CH Exposures Wild-type (wt) C57BL/6 mice and hemizygous NADPH gp91phox knockout (KO) mice (⫺/Y) [C57BL/6] (The Jackson Laboratory; Bar Harbor, ME) were used for this study. All mice were 10 to 20 weeks of age, and weighed between 22 and 30 g. Mice were housed for 3 weeks in a flow chamber gassed with 10% O2. www.chestjournal.org

Figure 1. Top, A: effects of ET-1 in pulmonary arteries isolated from wt and gp91phox KO mice following 3 weeks of CH. Cu/Zn SOD (150 U/mL). Changes of contraction tension are expressed as the mean ⫾ SE. Bottom, B: murine PA superoxide production as measured by lucigenin-enhanced chemiluminescence. PAs were isolated from wt and gp91phox KO mice under the conditions indicated. Changes in chemiluminescence signal are expressed as the mean ⫾ SE. CHEST / 128 / 6 / DECEMBER, 2005 SUPPLEMENT

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administration of a superoxide scavenger, Cu/Zn superoxide dismutase (SOD) (150 U/mL) [decrease in maximal isometric tension from 5.43 ⫾ 0.35 to 3.74 ⫾ 0.44 mN; n ⫽ 5; p ⬍ 0.05]. The CH-enhanced PA constrictor response to ET-1 was completely blocked in PAs isolated from gp91phox KO mice (wt ⫹ CH mice, 5.43 ⫾ 0.35 mN; gp91phox KO ⫹ CH mice, 3.33 ⫾ 0.19 mN; n ⫽ 7; p ⬍ 0.01). CH increased PA superoxide production (increase in mean chemiluminescence from 12.0 ⫾ 0.40 to 24.8 ⫾ 1.74 relative light units [RLU]/s; n ⫽ 6; p ⬍ 0.05) [Fig 1, bottom, B] in wt mice that was further increased in the presence of 10 nmol/L ET-1 (37.5 ⫾ 1.8 RLU/s; n ⫽ 6; p ⬍ 0.01). However, this CH-increased PA chemiluminescence was significantly reduced either by treatment with Cu/Zn SOD (150 U/mL; 18.7 ⫾ 1.17 RLU/s; n ⫽ 6; p ⬍ 0.01) or in PA isolated from gp91phox KO mice (14.4 ⫾ 0.66 RLU/s; n ⫽ 6; p ⬍ 0.01).

Discussion This study supports a model of CH-enhanced superoxide formation via a gp91phox-dependent NADPH oxidase pathway. Activation of this pathway leads to enhanced PA vasoconstrictor responses. Enhanced vasoconstrictor response to ET-1, via ETA or ETB receptors, has been documented in various models of CH-associated PH.9 Prior studies have established that CH-induced PH is associated with increased PA vasoconstrictor activity, but the mechanisms by which these PA constrictor responses are modulated remain unclear. In CH-induced PH in rats, ETA or ETB receptor-mediated PA vasoconstriction was increased.9 We show that the exogenous addition of SOD can significantly reduce CH-enhanced vasoconstrictor responses to ET-1 (Fig 1, top, A). This strongly suggests that CH-enhanced vasoconstrictor responses are mediated, at least in part, by an overproduction of superoxide radicals. In PA isolated from gp91phox KO mice, the CH-enhanced vasoconstrictor responses to ET-1 were completely blocked, and the CH-induced superoxide overproduction was markedly reduced (Fig 1, bottom, B), demonstrating that NADPH oxidase (gp91phox) is the major superoxide generator in this model. In conclusion, our studies demonstrate that superoxide overproduction, via NADPH oxidase (gp91phox) following CH, plays a central role in enhancing ET-1-mediated PA vasoconstriction.

References 1 Adnot S, Raffestin B, Eddahibi S, et al. Loss of endotheliumdependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia. J Clin Invest 1991; 87:155–162 2 Brandes RP, Barton M, Philippens KM, et al. Endothelialderived superoxide anions in pig coronary arteries: evidence from lucigenin chemiluminescence and histochemical techniques. J Physiol 1997; 500:331–342 3 Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res 2000; 86:494 –501 4 Griffith SL, Rhoades RA, Packer CS. Pulmonary arterial smooth muscle contractility in hypoxia-induced pulmonary hypertension. J Appl Physiol 1994; 77:406 – 414 5 Herget J, Wilhelm J, Novotna J, et al. A possible role of the 596S

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oxidant tissue injury in the development of hypoxic pulmonary hypertension. Physiol Res 2000; 49:493–501 Kennedy TP, Michael JR, Summer W. Calcium channel blockers in hypoxic pulmonary hypertension. Am J Med 1985; 78:18 –26 Liu JQ, Folz RJ. Extracellular superoxide enhances 5-HTinduced murine pulmonary artery vasoconstriction. Am J Physiol Lung Cell Mol Physiol 2004; 287:L111–L118 Liu JQ, Zelko IN, Folz RJ. Reoxygenation-induced constriction in murine coronary arteries: the role of endothelial NADPH oxidase (gp91phox) and intracellular superoxide. J Biol Chem 2004; 279:24493–24497 MacLean MR, McCulloch KM, Baird M. Effects of pulmonary hypertension on vasoconstrictor responses to endothelin-1 and sarafotoxin S6C and on inherent tone in rat pulmonary arteries. J Cardiovasc Pharmacol 1995; 26:822– 830 Morrell NW, Atochina EN, Morris KG, et al. Angiotensin converting enzyme expression is increased in small pulmonary arteries of rats with hypoxia-induced pulmonary hypertension. J Clin Invest 1995; 96:1823–1833 Weissmann N, Tadic A, Hanze J, et al. Hypoxic vasoconstriction in intact lungs: a role for NADPH oxidase-derived H(2)O(2)? Am J Physiol Lung Cell Mol Physiol 2000; 279: L683–L690

Phenotypic Diversity of the Lung Vasculature in Experimental Models of Metastases* Yun Oh; Imran Mohiuddin; Yan Sun; Joseph B. Putnam, Jr; Waun Ki Hong; Wadih Arap, MD, PhD; and Renata Pasqualini, PhD

In vivo phage display is a screening method in which peptides homing to specific vascular beds are selected after IV administration of a random peptide library. This strategy has revealed a vascular address system that allows tissue-specific targeting of normal blood vessels and angiogenesis-related targeting of tumor blood vessels by selected peptides. Many vascular receptors or “addresses” targeted by homing peptides have been identified. One such vascular *From the Departments of Thoracic/Head & Neck Medical Oncology (Drs. Oh and Hong), Thoracic Surgery (Drs. Mohiuddin and Putnam), and Genitourinary Medical Oncology (Drs. Sun, Arap, and Pasqualini), The University of Texas M.D. Anderson Cancer Center, Houston, TX. This work was funded in part by an award from the GillsonLongenbaugh Foundation (Drs. Arap and Pasqualini) and the Biology, Education, Screening, Chemoprevention and Treatment (BESCT) Lung Cancer Program (contract No. DAMD17– 01-1– 0689; Dr. Oh). Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Wadih Arap, MD, PhD, or Renata Pasqualini, PhD, M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: [email protected] or [email protected]

47th Annual Thomas L. Petty Lung Conference: Cellular and Molecular Pathobiology of Pulmonary Hypertension