Neprilysin Null Mice Develop Exaggerated Pulmonary Vascular Remodeling in Response to Chronic Hypoxia
2009; Elsevier BV; Volume: 174; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2009.080345
ISSN1525-2191
AutoresEdward C. Dempsey, Marilee J. Wick, Vijaya Karoor, Erica Barr, Dustin W. Tallman, Carol A. Wehling, Sandra J. Walchak, Sven Laudi, Mysan Le, Masahiko Oka, Susan M. Majka, Carlyne D. Cool, Karen A. Fagan, Dwight J. Klemm, Louis B. Hersh, Norma P. Gerard, Craig Gérard, York E. Miller,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoNeprilysin is a transmembrane metalloendopeptidase that degrades neuropeptides that are important for both growth and contraction. In addition to promoting carcinogenesis, decreased levels of neprilysin increases inflammation and neuroendocrine cell hyperplasia, which may predispose to vascular remodeling. Early pharmacological studies showed a decrease in chronic hypoxic pulmonary hypertension with neprilysin inhibition. We used a genetic approach to test the alternate hypothesis that neprilysin depletion increases chronic hypoxic pulmonary hypertension. Loss of neprilysin had no effect on baseline airway or alveolar wall architecture, vessel density, cardiac function, hematocrit, or other relevant peptidases. Only lung neuroendocrine cell hyperplasia and a subtle neuropeptide imbalance were found. After chronic hypoxia, neprilysin-null mice exhibited exaggerated pulmonary hypertension and striking increases in muscularization of distal vessels. Subtle thickening of proximal media/adventitia not typically seen in mice was also detected. In contrast, adaptive right ventricular hypertrophy was less than anticipated. Hypoxic wild-type pulmonary vessels displayed close temporal and spatial relationships between decreased neprilysin and increased cell growth. Smooth muscle cells from neprilysin-null pulmonary arteries had increased proliferation compared with controls, which was decreased by neprilysin replacement. These data suggest that neprilysin may be protective against chronic hypoxic pulmonary hypertension in the lung, at least in part by attenuating the growth of smooth muscle cells. Lung-targeted strategies to increase neprilysin levels could have therapeutic benefits in the treatment of this disorder. Neprilysin is a transmembrane metalloendopeptidase that degrades neuropeptides that are important for both growth and contraction. In addition to promoting carcinogenesis, decreased levels of neprilysin increases inflammation and neuroendocrine cell hyperplasia, which may predispose to vascular remodeling. Early pharmacological studies showed a decrease in chronic hypoxic pulmonary hypertension with neprilysin inhibition. We used a genetic approach to test the alternate hypothesis that neprilysin depletion increases chronic hypoxic pulmonary hypertension. Loss of neprilysin had no effect on baseline airway or alveolar wall architecture, vessel density, cardiac function, hematocrit, or other relevant peptidases. Only lung neuroendocrine cell hyperplasia and a subtle neuropeptide imbalance were found. After chronic hypoxia, neprilysin-null mice exhibited exaggerated pulmonary hypertension and striking increases in muscularization of distal vessels. Subtle thickening of proximal media/adventitia not typically seen in mice was also detected. In contrast, adaptive right ventricular hypertrophy was less than anticipated. Hypoxic wild-type pulmonary vessels displayed close temporal and spatial relationships between decreased neprilysin and increased cell growth. Smooth muscle cells from neprilysin-null pulmonary arteries had increased proliferation compared with controls, which was decreased by neprilysin replacement. These data suggest that neprilysin may be protective against chronic hypoxic pulmonary hypertension in the lung, at least in part by attenuating the growth of smooth muscle cells. Lung-targeted strategies to increase neprilysin levels could have therapeutic benefits in the treatment of this disorder. 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We tested the hypothesis that NEP protects the lung vasculature from the development of PHTN in response to chronic hypoxia at least in part by suppressing the growth of PA SMCs. Our approach was to first carefully examine important baseline normoxic features of wild-type (NEP+/+) and NEP null (NEP−/−) mouse lung and heart. Then, we tested the effect of NEP deletion on susceptibility to chronic hypoxic PHTN and vascular remodeling. We defined temporal and spatial changes in NEP expression in response to hypoxia in NEP+/+ mice, and compared the pattern of medial/adventitial remodeling and growth of PA SMCs from NEP+/+ and NEP−/− mice. Finally, we tested the effect of NEP replacement strategies (recombinant and lentiviral) on growth of PA SMCs from NEP−/− mice.Materials and MethodsMaterialsRecombinant human NEP (rNEP) was provided by Dr. Catherine Magill, AXYS Pharmaceuticals, Inc (South San Francisco, CA). Other chemicals were from Sigma (St. Louis, MO) unless specified.AnimalsBreeder mice for the C57BL/6 NEP null (NEP−/−) colony were originally provided by Drs. NP and C Gerard.33Lu B Figini M Emanueli C Geppetti P Grady EF Gerard NP Ansell J Payan DG Gerard C Bunnett N The control of microvascular permeability and blood pressure by neutral endopeptidase.Nat Med. 1997; 3: 904-907Crossref PubMed Scopus (139) Google Scholar C57BL/6 NEP+/+ littermate controls and NEP−/− mice were born at Denver altitude. Additional wild-type (NEP+/+) mice were obtained from Jackson Laboratories (Bar Harbor, ME) and Harlan (Indianapolis, IN). At 11 to 14 weeks of age, mice were randomly assigned to normoxic (Nx) and hypoxic (Hx) treatment groups. Each gender was equally represented. Approval of animal protocols was obtained from the University of Colorado and Denver VA Medical Center Institutional Animal Care and Use Committees.GenotypingAmplification of genomic DNA from mouse tail was performed using primer sets specific for NEP and Neomycin (NEP forward: 5′-CCAAACTTAAGCCTATTCTTAC-3′, reverse: 5′-CCATTATGAACCTCCAGGAC-3′ and Neomycin forward: 5′-GATGGATTGCACGCAGGTTCT-3′) according to the following PCR protocol: 94°C for 45 seconds, 55°C for 1 minute, 72°C for 2 minutes plus 10 seconds' autoextension repeated for 30 cycles, and 1 cycle of 72°C for 3 minutes (Figure 1). NEP expression was confirmed in mouse lung by demonstrating the presence or absence of NEP by immunoblot (as shown in Figure 1) and immunostaining. Finally, NEP activity was detected in lung lysate from NEP+/+, but not NEP−/−, mice.Hypoxia ExposureNEP+/+ and NEP−/− mice were exposed to normoxia (5200 ft, Denver altitude) or hypoxia (18,000 ft, hypobaric chamber) for up to 5 weeks with interruptions of <1 hour every 3 to 4 days for animal maintenance. In preliminary studies, C57BL/6 mice were maintained in a simulated sea level chamber (hyperbaric normoxia; pressurized to 1 atmosphere) for 5 weeks and the impact of Denver altitude on RSVP was found to be minimal (1 to 3 mmHg). Simulated sea level was achieved by the use of inline compressed air directed through a chamber fabricated with fixed and variable resistance valves. In parallel with the fixed leak from the chamber was a solenoid valve controlled leak. A Stratham transducer regulated the solenoid valve to maintain pressure in the chamber at 5.8 inches of mercury relative to Denver altitude (equal to sea level). The air in the chamber exchanged at least 10 times per hour.Invasive Hemodynamic MeasurementsAt the end of normoxic or hypoxic exposures, mice were anesthetized with Ketamine-Rompun (100 and 15 mg/kg; Fort Dodge, Madison, NJ and Miles Laboratories, Elkhart, IN), and while spontaneously breathing, underwent closed-chest measurements of right ventricular systolic pressure (RVSP) using a pressure transducer (Statham; n = 3 to 5 measurements/mouse) as previously described.17Littler CM Wehling CA Wick MJ Fagan KA Cool CD Messing RO Dempsey EC Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia.Am J Physiol Lung Cell Mol Physiol. 2005; 289: L1083-L1093Crossref PubMed Scopus (21) Google Scholar, 46Fagan KA Fouty BW Tyler RC Morris Jr, KG Hepler LK Sato K LeCras TD Abman SH Weinberger HD Huang PL McMurtry IF Rodman DM The pulmonary circulation of homozygous or heterozygous eNOS-null mice is hyperresponsive to mild hypoxia.J Clin Invest. 1999; 103: 291-299Crossref PubMed Scopus (265) Google Scholar, 47Littler CM Morris Jr, KG Fagan KA McMurtry IF Messing RO Dempsey EC Protein kinase C-epsilon-null mice have decreased hypoxic pulmonary vasoconstriction.Am J Physiol Heart Circ Physiol. 2003; 284: H1321-H1331PubMed Google Scholar In preliminary studies, this transthoracic approach yielded the same RVSP results as an intravascular Millar catheter technique with more consistency and less time expended. The transthoracic approach was therefore used routinely here.Baseline Echocardiographic AssessmentTransthoracic echocardiography was performed using 10 and 13 MHz ultrasound probes with a Vivid Five System (General Electrics Vingmed Ultrasound, Horton, Norway). Data were analyzed with EchoPac 6.3.6 software.17Littler CM Wehling CA Wick MJ Fagan KA Cool CD Messing RO Dempsey EC Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia.Am J Physiol Lung Cell Mol Physiol. 2005; 289: L1083-L1093Crossref PubMed Scopus (21) Google Scholar The mice were anesthetized with Ketamine-Rompun. Left heart dimensions were obtained in short-axis view. Intraluminal diameter of PA and flow in the PA was obtained in the parasternal longitudinal axis. Cardiac output and index were calculated using standardized methods.48Kawahara Y Tanonaka K Daicho T Nawa M Oikawa R Nasa Y Takeo S Preferable anesthetic conditions for echocardiographic determination of murine cardiac function.J Pharmacol Sci. 2005; 99: 95-104Crossref PubMed Scopus (83) Google Scholar Echocardiographic measurements were only made as part of the baseline assessment.Blood and Tissue HarvestingAfter hemodynamic measurements were completed, blood was collected from the RV via percutaneous stick, for measurement of hematocrit and preparation of serum. Then the chest was opened and 100 units of heparin were injected into the RV. After gentle perfusion of the pulmonary circulation with PBS (pressure = 40 cmH2O), the left lung was removed and quick-frozen in liquid nitrogen. The right lung was inflated to an airway pressure of 30 cmH20 with a mixture of 1% prewarmed agarose (GIBCO, Grand Island, NY) and 4% paraformaldehyde in PBS before preparation of sections for staining.17Littler CM Wehling CA Wick MJ Fagan KA Cool CD Messing RO Dempsey EC Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia.Am J Physiol Lung Cell Mol Physiol. 2005; 289: L1083-L1093Crossref PubMed Scopus (21) Google ScholarMeasurement of RV HypertrophyHeart ventricles were dissected, dried for 7 days, and weighed.17Littler CM Wehling CA Wick MJ Fagan KA Cool CD Messing RO Dempsey EC Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia.Am J Physiol Lung Cell Mol Physiol. 2005; 289: L1083-L1093Crossref PubMed Scopus (21) Google Scholar Hearts not dissected were fixed with paraformaldehyde and either processed for histological analysis with H&E to assess cardiac structure or dissected and examined for valvular and PA outflow tract anomalies.Quantitation of Neuropeptide Levels by RIA/Enzyme ImmunoassayLung lysates and serum from NEP+/+ and NEP−/− mice exposed to normoxia and 3 and 7 days hypoxia were prepared for quantitation of selected neuropeptides. BLP levels were measured by RIA; ET-1 and sub P levels were measured by enzyme immunoassay (all from Phoenix Pharmaceuticals, St. Joseph, MO).Tissue and ImmunostainingParaformaldehyde-fixed, paraffin-embedded sections (4 μm) of mouse lung and heart tissues were prepared and routinely stained with H&E or for increased definition of distal airway structure in selected cases immunostained for pan-cytokeratin (AE1/AE3, Ventana, Tucson, AZ).17Littler CM Wehling CA Wick MJ Fagan KA Cool CD Messing RO Dempsey EC Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia.Am J Physiol Lung Cell Mol Physiol. 2005; 289: L1083-L1093Crossref PubMed Scopus (21) Google Scholar For immunostaining of lung sections, antigen retrieval with citrate buffer was performed and tissue was blocked with 1% H2O2, avidin, biotin, and/or mouse-on-mouse blocking reagent (Vector Lab, Burlingame, CA), as appropriate. Sections were incubated with dilu
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