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. 2011 Mar 1;183(5):649-58.
doi: 10.1164/rccm.201005-0746OC. Epub 2010 Oct 1.

Autophagic protein LC3B confers resistance against hypoxia-induced pulmonary hypertension

Seon-Jin Lee  1 , Akaya Smith, Lanping Guo, Tero-Pekka Alastalo, Molong Li, Hirofumi Sawada, Xiaoli Liu, Zhi-Hua Chen, Emeka Ifedigbo, Yang Jin, Carol Feghali-Bostwick, Stefan W Ryter, Hong Pyo Kim, Marlene Rabinovitch, Augustine M K Choi
Affiliations

Autophagic protein LC3B confers resistance against hypoxia-induced pulmonary hypertension

Seon-Jin Lee et al. Am J Respir Crit Care Med. .

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(-/-)), or early growth response-1 (Egr-1(-/-)), 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(-/-) or Egr-1(-/-), but not Beclin 1(+/-), mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α 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(-/-) and Egr-1(-/-) 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.

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Figures

Figure 1.
Figure 1.
Autophagy is increased in the lung and lung vasculature of patients with human pulmonary hypertension (PH), and in the lung and lung vascular tissue of mice exposed to chronic hypoxia. (A) Western blot analysis of light chain-3B (LC3B)-I/-II expression in lung tissue from normal patients (N) and patients with idiopathic pulmonary arterial hypertension (IPAH) or non-IPAH forms of PH (see Table 1 for demographics). β-actin served as the standard (n = 4 normal, n = 5 PAH, and n = 5 patients with PH). (B) The level of LC3B mRNA from normal (n = 5) and patients with PH (n = 10) was measured by reverse transcriptase polymerase chain reaction. 18S rRNA served as the standard. Data are mean ± SD. **P < 0.0001. (C) Immunohistochemical analysis of LC3B protein in large (left) and small (right) human pulmonary vessels from normal or PH lung. Blue arrows indicate staining in the vessel intima and adventitia. Scale bars represent 10–100 μm as indicated. (D) Expression of LC3B in lung tissues from mice exposed to chronic hypoxia (10% O2, 3 wk) or normoxia (N). Total LC3B expression was normalized to β-actin. (E) Representative electron micrograph of mouse lung tissue from chronic hypoxia–treated mice and corresponding normoxic controls. Immature autophagic vacuoles (AVi) and degradative autophagic vacuoles (AVd) are detected in an endothelial cell (EC). AV were scored as described elsewhere (22). A red blood cell (RBC) is observed within the vessel lumen. Scale bar represents 500 nm. Total AV formation per 100 μm2 is quantitated at right. Data represent mean ± SD. n = 15 electron microscope images per condition, #P < 0.01. (F) The expression of LC3B is localized in EC and smooth muscle actin (SMA) in mice lung tissue. Sections of vessels isolated from hypoxia-treated mice were stained for LC3B (rabbit antimouse) using fluorescein isothiocyanate–conjugated secondary antibodies, or stained for α-SMA or platelet endothelial cell adhesion molecule-1 (PECAM) (rat antimouse) as indicated, using Cy3-conjugated IgG secondary antibodies. LC3B localized to the vessel wall. Merged images demonstrate colocalization to the endothelial and smooth muscle cells as indicated by α-SMA and PECAM antibodies. All panels are at ×40 magnification. Scale bars = 20 μm.
Figure 2.
Figure 2.
Hypoxia increased the expression and activation of light chain-3B (LC3B) in pulmonary vascular cells in vitro. (A) pulmonary artery endothelial cells (PAEC) and pulmonary artery vascular smooth muscle cells (PASMC) were exposed to hypoxia for 0–24 hours and evaluated for expression of LC3B-I and LC3B-II by Western analysis. β-actin served as the standard. (B) PAEC were exposed to hypoxia for 24 hours in the absence or presence of bafilomycin A1 (100 nM), and evaluated for the expression of LC3B-I and LC3B-II by Western analysis. β-actin served as the standard. (C) PAEC transfected with green fluorescence protein–LC3 were exposed to hypoxia or normoxia for 24 hours. Cells were visualized by confocal microscopy and the percentage of cells exhibiting punctuated green fluorescence protein–LC3 fluorescence was calculated relative to all green fluorescence protein–positive cells. Data represent mean ± SD. #P < 0.01. (D) Electron microscopic analysis of PAEC and PASMC after 24 hours of hypoxia. Red arrows indicate the presence of immature (AVi) or degradative (AVd) autophagic vacuoles. AV were scored as described in reference (22). Scale bar = 500 nm. Graphs represent quantification of autophagosomes (AVi + AVd) per cell in PAEC and PASMC exposed to hypoxia or normoxia. Data represent mean ± SD. n = 15 electron microscope images per cell type and condition, #P < 0.01.
Figure 3.
Figure 3.
Light chain-3B (LC3B−/−) mice display enhanced indices of pulmonary hypertension after chronic hypoxia. (A) Right ventricular systolic pressure (RVSP) was measured in LC3B+/+ (open square) and LC3B−/− (black square) mice after hypoxia (n = 15) and normoxia (n = 15) for 4 weeks. (B) Right ventricle weight (RVW) (milligrams) normalized for body weight (BW) (grams) in LC3B+/+ (open square) and LC3B−/− (black square) mice. (C) Fulton index of right ventricular hypertrophy (RVH), measured as ratio of the RVW to that of the left ventricle plus septum (RV/LV+S). (A–C) Data represent mean ± SD (n = 5). #P < 0.01; *P < 0.05. (D) Graphic shows quantitation of percent wall thickness of pulmonary arterioles in the lungs of LC3B+/+ (open square) and LC3B−/− (black square) mice after normoxia (n = 10 per group) and hypoxia (n = 10 per group). Data are expressed as mean ± SE (*P < 0.05; #P < 0.01) for hypoxic LC3B−/− mice versus hypoxic LC3B+/+ mice and corresponding normoxic controls.
Figure 4.
Figure 4.
Light chain-3B (LC3B) modulates pulmonary vascular cell proliferation. (A and B) Pulmonary artery endothelial cells (PAEC) and pulmonary artery vascular smooth muscle cells (PASMC) were transfected with control or LC3B siRNA then endothelin (ET)-1 (40 nM) or platelet-derived growth factor (PDGF)-BB (20 ng ml−1) were added to PAEC (A) and PASMC (B) for 48 hours, respectively. Cultures were then exposed to hypoxia or normoxia for an additional 48 hours. Proliferation was assessed by MTT assay. Data represent mean ± SD (n = 3). *P < 0.05; #P < 0.01. (C and D) PAEC and PASMC were transfected with control or pCMV-LC3 expression vector and then ET-1 (40 nM) or PDGF-BB (20 ng ml−1) were added to PAEC (C) and PASMC (D) for 48 hours, respectively. Cells were exposed to hypoxia or normoxia for an additional 48 hours. Proliferation was assessed by MTT assay. Data represent mean ± SD (n = 3). *P < 0.05; #P < 0.01. (A–D) Western analysis was used to validate targeted changes in LC3B expression in PAEC and PASMC with β-actin as the standard (inserts). (E and F) LC3B modulates pulmonary vascular cell apoptosis (E) PAEC and (F) PASMC were transfected with control or LC3B siRNA and then exposed to hypoxia or normoxia for 0–48 hours in the presence of staurosporine (1 μM). Cell death was assessed by crystal violet staining. Data represent mean ± SD (n = 3). *P < 0.05. Western blot analysis was used to assay caspase-3 cleavage in PASMC with β-actin as the standard (insert).
Figure 5.
Figure 5.
Light chain-3B (LC3B) knockdown increases intracellular reactive oxygen species production and stabilizes hypoxia-inducible factor (HIF)-1α. Pulmonary artery endothelial cells (PAEC) were infected with LC3B-siRNA or control siRNA. Infected PAECs were treated with hypoxia (1%) exposure for 30 minutes and incubated with 10 μM H2DCF-DA for another 30 minutes at 37°C. After the excess probe was removed, cells were incubated for an additional 20 minutes before fluorescence confocal microscopy. (A) Representative fluorescence images are shown. (B) The relative fluorescence in the treatment groups is quantitated (n = 10). *P < 0.5. (C) Cell lysates were analyzed for HIF-1α stabilization by Western immunoblot analysis. β-actin was used as the standard.
Figure 6.
Figure 6.
Early growth response (Egr)-1 regulates hypoxia-induced light chain-3B (LC3B) expression in vitro and in vivo, and protects against pulmonary hypertension during chronic hypoxia. (A) Pulmonary artery vascular smooth muscle cells (PASMC) were treated with hypoxia (1% O2) for 8 hours. Chromatin samples were immunoprecipitated with anti–Egr-1 and evaluated for factor binding to the LC3B promoter region. (B) Egr-1 expression relative to β-actin was evaluated in lung homogenate from normal patients (N) and patients with pulmonary hypertension (PH) by quantification of Western blots. Data represent mean ± SD (n = 5 normal, n = 10 PH). #P < 0.01. (C) The protein levels of Egr-1 in mouse lung tissues harvested from animals exposed to hypoxia and normoxia were evaluated by Western analysis. Data represent mean ± SD (n = 6). #P < 0.01. (D) Pulmonary artery endothelial cells (PAEC) were infected with Egr-1–specific siRNA or control siRNA, then treated with hypoxia (1% O2) for 0–24 hours. Expression of LC3B and Egr-1 was determined by immunoblotting with β-actin as the standard.
Figure 7.
Figure 7.
Early growth response (Egr-1)−/− mice display enhanced indices of pulmonary hypertension after chronic hypoxia. (A) Egr-1+/+ and Egr-1−/− were exposed to chronic hypoxia. Lung tissue was analyzed for light chain-3B (LC3B) by Western blotting. β-actin served as the standard. *P < 0.05; #P < 0.01. (B) Right ventricular systolic pressure (RVSP) was measured in Egr-1+/+ (open square) and Egr-1−/− (black square) mice after exposure to hypoxia and normoxia for 4 weeks. Data represent mean ± SD (n = 3). (C) Right ventricular weight (RVW, milligrams) normalized for body weight (BW, grams) in Egr-1+/+ (open square) and Egr-1−/− (black square) mice. (D) Fulton index of right ventricle hypertrophy measured as ratio of the weight of the RV to that of the left ventricle (LV) plus septum (RV/LV+S). (C and D) Data represent mean ± SD (n = 3) *P < 0.05; #P < 0.01.
Figure 8.
Figure 8.
Light chain-3B (LC3B) and early growth response (Egr)-1 localize to the lipid rafts and form complexes with caveolin (Cav)-1, which are displaced by hypoxia. (A) Human pulmonary artery endothelial cells (PAEC) and pulmonary artery vascular smooth muscle cells (PASMC) were exposed to hypoxia or normoxia for 24 hours. Cell lysates were subjected to sucrose density gradient ultracentrifugation, to isolate 12 fractions (see Figure E5A). Fractions (–5) corresponding to lipid rafts were immunoblotted for Egr-1, LC3B, or Cav-1. PAEC (B and C) or PASMC (D and E) were exposed to hypoxia for 0–8 hours. Cell lysates were subjected to sucrose density gradient fractionation. Fractions (–5) were subjected to immunoprecipitation with anti–Cav-1 followed by immunoblotting with anti-LC3B (B and D) or anti–Egr-1 (C and E). Reciprocal co-IP was performed for each experiment. IgG served as the standard.
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