Smoking exposure induces human lung endothelial cell adaptation to apoptotic stress

Abstract

Prolonged exposure to cigarette smoking is the main risk factor for emphysema, a component of chronic obstructive pulmonary diseases (COPDs) characterized by destruction of alveolar walls. Moreover, smoking is associated with pulmonary artery remodeling and pulmonary hypertension, even in the absence of COPD, through as yet unexplained mechanisms. In murine models, elevations of intra- and paracellular ceramides in response to smoking have been implicated in the induction of lung endothelial cell apoptosis, but the role of ceramides in human cell counterparts is yet unknown. We modeled paracrine increases (outside-in) of palmitoyl ceramide (Cer16) in primary human lung microvascular cells. In naive cells, isolated from nonsmokers, Cer16 significantly reduced cellular proliferation and induced caspase-independent apoptosis via mitochondrial membrane depolarization, apoptosis-inducing factor translocation, and poly(ADP-ribose) polymerase cleavage. In these cells, caspase-3 was inhibited by ceramide-induced Akt phosphorylation, and by the induction of autophagic microtubule-associated protein-1 light-chain 3 lipidation. In contrast, cells isolated from smokers exhibited increased baseline proliferative features associated with lack of p16(INK4a) expression and Akt hyperphosphorylation. These cells were resistant to Cer16-induced apoptosis, despite presence of both endoplasmic reticulum stress response and mitochondrial membrane depolarization. In cells from smokers, the prominent up-regulation of Akt pathways inhibited ceramide-triggered apoptosis, and was associated with elevated sphingosine and high-mobility group box 1, skewing the cell's response toward autophagy and survival. In conclusion, the cell responses to ceramide are modulated by an intricate cross-talk between Akt signaling and sphingolipid metabolites, and profoundly modified by previous cigarette smoke exposure, which selects for an apoptosis-resistant phenotype.

Figures

Figure 1.

Inhibitory effects of palmitoyl ceramide (ceramide 16:0 [Cer16]) on the metabolic/proliferative activity of human lung microvascular endothelial cells. Metabolic activity measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in cells from nonsmoker (gray bars) and smoker (black bars) in the following conditions: (a) untreated, 16 hours, mean + SEM (n = 3; *P < 0.05); (b) following vehicle (veh; polyethylene glycol 2,000) or Cer16 treatment at the indicated concentrations (6 h), fold change versus veh, mean + SEM (n = 3; *P < 0.05, #P < 0.005 vs. veh); or (c) following veh, or Cer16 treatment for the indicated time in hours (10 μM), mean + SEM (n = 3; *P < 0.0001 and #P < 0.01 vs. vehicle control; ##P < 0.05).

Figure 2.

Apoptosis and survival responses of human lung microvascular endothelial cells to Cer16. (a and b) Apoptosis measured by annexin V/propidium iodide staining and expressed as fold increase in positive cells versus control vehicle in human lung microvascular endothelial cells (a) compared with human small airway epithelial cells (b), both isolated from either nonsmokers (gray bars) or smokers (black bars). Cells were treated with Cer16 or Cer6 (10 μM; 6 h), or with vehicle (PEG 2,000); mean + SEM (n = 4; *P < 0.05, #P = 0.006, and $P < 0.001). (c) Caspase-3 activity of human lung microvascular endothelial cells treated Cer16 (10 μM) or vehicle (control [Ctl]) for the indicated time. Mean + SEM. (n = 9; *P < 0.05, #P = 0.006, and $P = 0.05. (d) Activated and total extracellular signal–regulated kinase (ERK) 1/2 measured by Western blot in lung microvascular endothelial cells (LMVECs) lysates from nonsmoker and smoker donors after treatment with Cer16 (10 μM) or vehicle (veh) for 6 hours (upper panel, Western blot) and 16 hours (lower panel, densitometry). Mean + SEM; n = 4. (e) Activated and total Akt in LMVEC lysates treated with either Cer16 (10 μM) or vehicle for 6 hours (upper panel showing Western blot and lower panel showing densitometry). Mean + SEM (n = 4; two-way ANOVA showing P < 0.05 for both Cer treatment and smoking status). (f) Effect of ERK1/2 or Akt inhibition on LMVEC apoptosis measured by annexin V/PI staining in cells from nonsmoker donors. Cells were pretreated (1 h) with either PD98059 (10 μM) or LY294002 (30 μM) followed by Cer16 (10 μM, 2 h). Results are expressed as fold change versus Cer16. Mean + SEM (n = 3; *P = 0.01, #P < 0.0001). P-Akt, phosphorilated serine/threonine-specific protein kinase; P-ERK, phosphorilated extracellular signal–regulated kinase; Ser, serine; Thr, threonine.

Figure 3.

Mitochondria changes in human lung microvascular endothelial cells treated with Cer16. (a) Mitochondria depolarization in cells treated with Cer16 (10 μM, 6 h) or vehicle (Veh) and effect of Akt inhibitor LY294002 (30 μM, 1 h pretreatment). Mean + SEM (n = 3; *P < 0.05, #P < 0.001, $P = 0.05). (b and c) Apoptosis-inducing factor (AIF) translocation from mitochondria to nucleus detected by Western blot (b) in respective cellular subfractions of cells treated with Cer16 (10 μM; for the indicated time in hours) or its Veh (PEG 2000; Ctl1) compared with staurosporine (Stauro; 0.2 μM, 2 h) or its Veh (Ctl2). Loading controls for each subcellular fraction, voltage-dependent anion channel (VDAC1) and TATA-binding protein (TBP), were detected in the lower lanes. Densitometry of AIF expression normalized by loading control is indicated numerically in between lanes. (c) Representative fluorescence micrographs of cells immunostained for AIF (FITC-labeled). Mitochondria are stained in red (MitoTracker Red) and nuclei in blue with 4′,6-diamidino-2-phenylindole (DAPI) after treatment with Cer16 (10 μM) or Veh; scale bar, 50 μm. (d) Detection of cleaved poly(ADP-ribose) polymerase (PARP) in total protein lysates of cells: untreated, grown in regular full serum–containing media (FM); or treated with Veh or Cer16 (10 μM, 6 h), and either Akt inhibitor (30 μM LY294002; 1 h pretreatment) or autophagy inhibitor, 3-methyladenine (3-MA; 5 mM, 1 h pretreatment). Vinculin expression was used as loading control.

Figure 4.

Autophagy and endoplasmic reticulum (ER) stress in human lung microvascular endothelial cells treated with Cer16. (a) Western blot of microtubule-associated protein-1 light-chain 3 (LC3) -I and LC3-II and vinculin (as loading control) in cells treated with Cer16 (10 μM, 16 h) or Veh, and effect of general caspase inhibitor, known commercially as ZVAD-fmk (0.1 mM, 1 h pretreatment) or autophagy inhibitor 3-MA (5 mM, 1 h pretreatment). (b) Representative electron microscopy images of cells after treatment with Cer16 or Veh for 6 or 16 hours. Noted are: nuclei (n), ER swelling (arrowheads, lower panel), autophagosomes (magnified in lower panel inset, arrows), and autophagosome–lysosome fusion (magnified in upper panel inset, arrow). (c) Western blot of phospho- and total eukaryotic translation initiation factor 2a (eIF2α) in cells similarly treated as in (a). Cells were treated with Cer16 or Veh control (PEG 2,000; 10 μM, 16 h) and pretreated with ZVAD-fmk (0.1 mM, 1 h) or 3-MA (5 mM, 1 h). β-tubulin was used as loading control.

Figure 5.

Autophagy and apoptosis interactions in human lung microvascular endothelial cells treated with Cer16. (a and b) Apoptosis measured by caspase-3 activity (a) or mitochondrial depolarization (b) in cells treated with Cer16 (10 μM; 2 hours in [a] and indicated time in [b]) or Veh, and effect of autophagy inhibitor, 3-MA (5 mM, 1 h pretreatment). Mean + SEM (n = 4, *P < 0.05). (c). Levels of high-mobility group box 1 (HMGB1) or LC3-I and LC3-II measured by Western blot in cells treated with Cer16 (10 μM, for the indicated time) or Veh and either protein synthesis inhibitor, cycloheximide (CHX; 1 μg/ml, 1 h pretreatment), ERK1/2 inhibitor, PD98059 (PD; 50 μM, 1 h pretreatment), or Akt inhibitor LY294002 (LY; 30 μM, 1 h pretreatment).

Figure 6.

Modulation of intracellular sphingolipids by Cer16 in human lung microvascular endothelial cells. (a–d) Abundance of indicated sphingolipid species measured by combined liquid chromatography–tandem mass spectrometry in cells treated with Cer16 (10 μM, 2 h in [a and b]; 6 h in [c and d]) or Veh, and effect of Akt inhibitor LY294002 (30 μM, 1 h pretreatment) or autophagosome formation inhibitor, 3-MA (5 mM, 1 h pretreatment). Mean + SEM (n = 2; *P < 0.05, #P < 0.005). (e) Cell metabolic activity assessed by MTT assay in cells treated with Cer16 (10 μM, 24 h) or Veh, and effect of ceramidase inhibitor, (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (MAPP; 1 μM, 2 h pretreatment). Mean + SEM (n = 3; *P < 0.001, #P < 0.05). (f) HMGB1, LC3-I, and LC3-II expression detected in protein lysate from cells treated with Cer16 (10 μM, 6 h) or Veh, and effect of MAPP. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Densitometry of HMGB1 expression normalized by loading control is indicated numerically in between blots. S1P, sphingosine 1-phosphate; SPH, sphingosine.

Figure 7.

Schematic summarizing findings of Cer16 effect on human LMVECs isolated from nonsmokers compared with those isolated from smokers. Human naive LMVECs respond to ceramide by increasing ER stress and mitochondrial depolarization, which is associated with AIF-mediated, caspase-independent (Akt-inhibited caspases) apoptosis and autophagy (top). Chronic exposure to cigarette smoke (CS) is associated with Akt hyperphosphorylation and increased levels of SPH, which contributes to robust induction of autophagy associated with high HMGB1 levels, and which, together, contribute to develop an apoptotic-resistant phenotype (bottom).