Article

Dominant negative mutation of the TGF-β eceptor blocks hypoxia-induced pulmonary vascular remodeling

Vascular Biology and Hypertension Program, Division of Cardiovascular Disease, Dept. of Medicine, Univ. of Alabama at Birmingham, UAB Station, Birmingham, AL 35294-0007, USA.
Journal of Applied Physiology (Impact Factor: 3.43). 03/2006; 100(2):564-71. DOI: 10.1152/japplphysiol.00595.2005
Source: PubMed

ABSTRACT The present study utilized a novel transgenic mouse model that expresses an inducible dominant negative mutation of the transforming growth factor (TGF)-beta type II receptor (DnTGFbetaRII mouse) to test the hypothesis that TGF-beta signaling plays an important role in the pathogenesis of chronic hypoxia-induced increases in pulmonary arterial pressure and vascular and alveolar remodeling. Nine- to 10-wk-old male DnTGFbetaRII and control nontransgenic (NTG) mice were exposed to normobaric hypoxia (10% O2) or air for 6 wk. Expression of DnTGFbetaRII was induced by drinking 25 mM ZnSO4 water beginning 1 wk before hypoxic exposure. Hypoxia-induced increases in right ventricular pressure, right ventricular mass, pulmonary arterial remodeling, and muscularization were greatly attenuated in DnTGFbetaRII mice compared with NTG controls. Furthermore, the stimulatory effects of hypoxic exposure on pulmonary arterial and alveolar collagen content, appearance of alpha-smooth muscle actin-positive cells in alveolar parenchyma, and expression of extracellular matrix molecule (including collagen I and III, periostin, and osteopontin) mRNA in whole lung were abrogated in DnTGFbetaRII mice compared with NTG controls. Hypoxic exposure had no effect on systemic arterial pressure or heart rate in either strain. These data support the hypothesis that endogenous TGF-beta plays an important role in pulmonary vascular adaptation to chronic hypoxia and that disruption of TGF-beta signaling attenuates hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, pulmonary arterial hypertrophy and muscularization, alveolar remodeling, and expression of extracellular matrix mRNA in whole lung.

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    ABSTRACT: The pulmonary vasculature has the unique ability to undergo vasoconstriction in response to acute hypoxia, a physiological mechanism known as hypoxic pulmonary vasoconstriction (HPV). Sustained or chronic hypoxia, however, leads to proliferation of vascular smooth muscle cells of pulmonary arterioles, which causes a permanent increase in pulmonary vascular resistance, and may lead to right heart dysfunction. The underlying mechanisms of vascular proliferation under chronic hypoxia have not been fully defined. The NADPH oxidases are one family of recently discovered molecules which generate reactive oxygen species (ROS) and have been suggested to be important for cellular signaling under physiological conditions. However, NADPH oxidase generated oxidative stress can also lead to inflammation, vascular smooth muscle cell proliferation and endothelial damage under pathological conditions. Many homologs of NADPH oxidases exist, the classical homolog is gp91phox or NOX2, and the recently discovered homologs include NOX1, NOX3, NOX4, NOX5, DUOX1 and DUOX2. Superoxide production by classical gp91phox is induced by assembly of the cytosolic subunits such as p40phox, p47phox and p67phox with membrane-bound gp91phox complex. A previous report from our laboratory has shown that the knockout mice of p47phox subunit exhibit reduced acute HPV as compared to wild type mice suggesting an essential role of NADPH oxidases in regulation of vascular tone in acute hypoxia. Against this background, the current thesis aimed to elucidate the role of NADPH oxidases in vascular remodeling in chronic hypoxia, and its possible downstream mediators. Screening of NADPH oxidase expression revealed that all subunits were expressed in the lung homogenate and that NOX4 was prominently up-regulated under chronic hypoxia. The NOX4 mRNA was also up-regulated in the microdissected vessels of mice exposed up to three weeks of chronic hypoxia. In addition, a functional interference with NOX4 using NOX4 siRNA resulted in reduced ROS production and reduced proliferation of pulmonary arterial smooth muscle cells (PASMC) revealing an important contribution of NOX4 in PASMC proliferation and particularly in hypoxia induced pulmonary hypertension. Intriguingly, a similar reflection was found in lungs of patients with idiopathic pulmonary hypertension that underwent lung transplantation. Further experiments demonstrated that NOX4 inhibited voltage-gated delayed rectifier K+ channels (KDR) under hypoxia. Pharmacological inhibition with apocynin and genetic ablation with NOX4siRNA resulted in increased KDR current under hypoxia. In addition, the current study demonstrated that NOX4 is essential for ET-1 mediated calcium influx in PASMC as NOX4 knockdown using NOX4 siRNA abolished the ET-1 mediated calcium influx under chronic hypoxia. Thus, the NOX4-ROS-KDR-[Ca2+] pathway may contribute to the development of pulmonary hypertension. Das pulmonale Gefäßsystem besitzt die besondere Eigenschaft, auf akute Hypoxie mit einer Vasokonstriktion zu reagieren. Dies ist ein physiologischer Mechanismus, der als hypoxische pulmonale Vasokonstriktion (HPV) bezeichnet wird. Anhaltende oder chronische Hypoxie führt darüberhinaus zu einer Proliferation der Media der Lungenarteriolen, das einen permanenten Anstieg des pulmonalen Gefäßwiderstandes auslöst, der im weiteren zu einem Cor pulmonale führen kann. Die der HPV zu Grunde liegenden Mechanismen sind bisher noch nicht umfassend geklärt worden. NADPH-Oxidasen sind eine vor kurzem entdeckte Proteinfamilie, die reaktive Sauerstoffspezies (ROS) generieren können und unter physiologischen Bedingungen für die zelluläre Signaltransduktion wichtig sind. Andererseits können die von NADPH-Oxidasen stammenden ROS unter pathologischen Bedingungen zu Entzündung und Proliferation von vaskulären glatten Muskelzellen und zur Schädigung des Endothels führen. In der Literatur sind mehrere Homologe von NADPH-Oxidasen beschrieben, wobei das klassische Homolog als gp91phox oder NOX2 bekannt ist. Weitere, erst kürzlich entdeckte Homologe umfassen NOX1, NOX3, NOX4, NOX5, DUOX1 und DUOX2. Die Superoxidproduktion der klassischen gp91phox wird durch die Anlagerung der zytosolischen Untereinheiten p40phox, p47phox und p67phox an gp91phox induziert. Unsere bisherigen Daten zeigen, dass Mäuse mit einer p47phox-Defizienz im Vergleich zu Wildtyp-Mäusen eine geringere akute HPV aufweisen, das auf eine essentielle Rolle der NADPH-Oxidasen für die Regulation des vaskulären Tonus unter akuter Hypoxie hinweist. Ziel der vorliegenden Arbeit war, die Rolle der NADPH-Oxidasen für den vaskulären Umbauprozess unter chronischer Hypoxie und deren Signaltransduktionsmechanismen aufzuklären. Untersuchungen der NADPH-Oxidasen Expression zeigten, dass alle Untereinheiten im Lungenhomogenat exprimiert waren und die Expression von NOX4 unter chronischer Hypoxie stark hochreguliert war. Darüberhinaus war die NOX4-mRNA auch in den durch Mikrodissektion gewonnenen Gefäßen der Mäuse, die 3 Wochen unter chronischer Hypoxie gehalten wurden, hochreguliert. Zusätzlich bewirkte eine funktionelle Interferenz von NOX4 mit NOX4-siRNA eine reduzierte ROS-Produktion und eine verringerte Proliferation der pulmonalarteriellen glatten Muskelzellen (PASMC). Dies weist auf eine bedeutungsvolle Rolle von NOX4 für die Proliferation von PASMC, insbesondere bei Hypoxia-induzierter pulmonaler Hypertonie, hin. Interessanterweise wurden ähnliche Ergebnisse in Lungen von Patienten mit idiopatischer pulmonaler Hypertonie nach Transplantation der Lunge gewonnen. Weitere Experimente zeigten, dass NOX4 unter chronischer Hypoxie die voltage gated delayed rectifier K+-Kanäle (KDR) hemmt. Eine pharmakologische Inhibition von NOX4 mittels Apocynin oder eine genetische Ablation durch siRNA erhöhten den KDR-Strom unter chronischer Hypoxie. Zusätzlich ist in der vorliegenden Arbeit gezeigt, dass NOX4 essentiell für den ET-1 vermittelten Kalziumeinstrom ist, da durch Reduzierung von NOX4 mittels NOX4 siRNA der ET-1 vermittelte Kalziumeinstrom unter chronischer Hypoxie nicht mehr vorhanden war. Zusammengefasst gesagt, könnte der NOX4-ROS-KDR-[Ca2+]-Signalweg somit zur Entstehung der pulmonalen Hypertonie beitragen.
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    ABSTRACT: Clinical fibrotic syndromes are usually driven by excessive transforming growth factor-beta (TGF-beta) activity. Heme oxygenase-1 (HO-1), which is induced by TGF-beta in many tissues, has antifibrotic effects which are at least partially attributable to down-regulation of TGF-beta signal transduction. This down-regulation is mediated by bilirubin - a physiological inhibitor of NADPH oxidase that suppresses the oxidant stress required for optimal TGF-beta activity - and by carbon monoxide, which acts via guanylate cyclase, cGMP, and protein kinase G to inhibit the nuclear translocation of phosphorylated Smads while also impeding TGF-beta activation by blocking thrombospondin-1 transcription. It may be feasible to mimic these protective effects of HO-1 in clinical fibrotic syndromes by administering bilins (biliverdin or phycocyanobilin) and high-dose biotin - the former can function as clinical NADPH oxidase inhibitors, whereas the latter stimulates guanylate cyclase activity. This strategy may prove useful in the prevention and management of clinical fibrotic syndromes in which TGF-beta activity plays a central mediating role - likely including hepatic cirrhosis, glomerulosclerosis, interstitial fibrosis, ventricular hypertrophy, atrial fibrosis with fibrillation, pulmonary hypertension, cystic fibrosis, idiopathic pulmonary fibrosis, asthma, pancreatic fibrosis, scleroderma, and fibrostenosis in Crohn's disease.
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