Autophagic Protein LC3B Confers Resistance against Hypoxia-induced Pulmonary Hypertension

Abstract

Rationale: Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown. Objectives: To determine the mechanisms by which autophagic proteins regulate tissue responses during PH. Methods: Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B 2/2 ), or early growth response-1 (Egr-1 2/2 ), were evaluated for vascular morphology and hemodynamics. Measurements and Main Results: Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B 2/2 or Egr-1 2/2 , but not Beclin 1 1/2 , mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxiainducible factor-1a stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia. Conclusions:The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B 2/2 and Egr-1 2/2 mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.Keywords: autophagy; hypoxia; hypertension, pulmonary Pulmonary hypertension (PH) is functionally characterized by elevations in mean pulmonary pressure and the development of secondary right ventricular failure. Category I PH, also known as pulmonary arterial hypertension (PAH), includes idiopathic (IPAH), familial, and acquired forms resulting from collagen vascular disease, drugs, HIV infection, portal hypertension, and pulmonary artery shunts (1-5). Categories II-V of PH include those forms secondary to left-heart disease; lung disease or hypoxia; chronic thrombotic or embolic disease; or other diseases rarely associated with PH, such as sarcoid or mediastinitis (1, 2). The molecular and cellular basis for the vascular changes associated with PH remains unclear (6). Impaired production of nitric oxide can result in vasoconstriction and smooth muscle cell proliferation, suggesting a role for endothelial dysfunction in PH (7). In addition, variations in the production, expression, or activity of vascular mediators (i.e., endothelin-1, prostacyclins, voltage-gated K 1 -channels), signaling molecules, and growth factors (e.g., transforming growth factor-b, platelet-derived growth factor [PDGF]) have been implicated in hypoxic remodelin...

Autophagy: A Crucial Moderator of Redox Balance, Inflammation, and Apoptosis in Lung Disease

“…In vascular cells, hypoxia induced the expression of Beclin 1, the conversion of LC3B-I to LC3B-II, and increased autophagosome formation (91). These observations were consistent with increased autophagic flux during hypoxia, as determined by increased LC3B-II levels with the addition of bafilomycin-A1.…”

“…These observations were consistent with increased autophagic flux during hypoxia, as determined by increased LC3B-II levels with the addition of bafilomycin-A1. Similar findings were described after exposure to chronic hypoxia in vivo, with mouse lungs displaying increased LC3B-II formation, and increased autophagosome formation (91). Genetic interference of LC3B using siRNA increased the proliferation of endothelial and smooth muscle cells in vitro and also inhibited smooth muscle cell apoptosis in response to pro-apoptotic agents (91).…”

“…Additionally, autophagy can be modulated during altered states of oxygen tension, including hyperoxia, and hypoxia, corresponding to increased or decreased pO 2 , respectively (91,171). Autophagy may be regarded as a secondary defense to oxidative stress, by virtue of its ability to facilitate the turnover of damaged or modified substrates, such as protein, which may accumulate during this condition.…”

Transcriptional and epigenetic profiling of nutrient-deprived cells to identify novel regulators of autophagy

“…EGR1 is an immediate-early response gene, of which its expression can be induced within minutes after stimulation [30]. Mitogens [31,32], growth factors [33], and stress stimuli, such as cigarette smoke [34–36], hypoxia [37,38], and nutrient deprivation [39] regulate EGR1. For example, in agreement with our data, glucose restriction rapidly increases EGR1 protein levels in multiple cell lines [39].…”

“…The transcription factor EGR1 has been implicated in numerous processes, for example apoptosis, angiogenesis, proliferation, cell differentiation, and migration [40,41]. EGR1 has been linked to cigarette smoke, hypoxia, and irradiation-induced autophagy, by induction of ATG4B [32] and LC3B protein or gene expression [35,38]. Additionally, egr1 −/- mice are more resistant to the pro-autophagic effects of chronic cigarette smoke exposure [35].…”