Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice

Abstract

Alveolar cell apoptosis is involved in the pathogenesis of emphysema, a prevalent disease primarily caused by cigarette smoking. We report that ceramide, a second messenger lipid, is a crucial mediator of alveolar destruction in emphysema. Inhibition of enzymes controlling de novo ceramide synthesis prevented alveolar cell apoptosis, oxidative stress and emphysema caused by blockade of the vascular endothelial growth factor (VEGF) receptors in both rats and mice. Emphysema was reproduced with intratracheal instillation of ceramide in naive mice. Excessive ceramide triggers a feed-forward mechanism mediated by activation of secretory acid sphingomyelinase, as suggested by experiments with neutralizing ceramide antibody in mice and with acid sphingomyelinase-deficient fibroblasts. Concomitant augmentation of signaling initiated by a prosurvival metabolite, sphingosine-1-phosphate, prevented lung apoptosis, implying that a balance between ceramide and sphingosine-1-phosphate is required for maintenance of alveolar septal integrity. Finally, increased lung ceramides in individuals with smoking-induced emphysema suggests that ceramide upregulation may be a crucial pathogenic element and a promising target in this disease that currently lacks effective therapies.

Figure 1. Lung ceramide levels are increased by VEGFR blockade

(a) Mouse lung ceramide levels induced by VEGFR inhibitor (VEGFR-inh, SU5416) or vehicle (Ctl); n=4 mice/group; mean +SD. *p=0.01. Mouse lung ceramide expression (brown, arrows) at 3d after treatment (_a, alveoli, _b, bronchial epithelium, and _v large vessels). (b) Rat lung ceramide (n= 3–5 rats/group; mean +SD. *p<0.05). (c–e) Mouse lung enzymatic activities of ceramide synthase (c, n=4/group, +SD, *p=0.02); soluble (secretory) and lysosomal ASMase at 3d (d, n=4/group, +SE, p=0.04); and neutral sphingomyelinase (e, n=4/group; mean +SD). (f) Lung ceramides and ceramide metabolites at 7d after VEGFR-inh (n=4) or Ctl (n=3); mean +SE, *p<0.05.

Figure 1. Lung ceramide levels are increased by VEGFR blockade

(a) Mouse lung ceramide levels induced by VEGFR inhibitor (VEGFR-inh, SU5416) or vehicle (Ctl); n=4 mice/group; mean +SD. *p=0.01. Mouse lung ceramide expression (brown, arrows) at 3d after treatment (_a, alveoli, _b, bronchial epithelium, and _v large vessels). (b) Rat lung ceramide (n= 3–5 rats/group; mean +SD. *p<0.05). (c–e) Mouse lung enzymatic activities of ceramide synthase (c, n=4/group, +SD, *p=0.02); soluble (secretory) and lysosomal ASMase at 3d (d, n=4/group, +SE, p=0.04); and neutral sphingomyelinase (e, n=4/group; mean +SD). (f) Lung ceramides and ceramide metabolites at 7d after VEGFR-inh (n=4) or Ctl (n=3); mean +SE, *p<0.05.

Figure 1. Lung ceramide levels are increased by VEGFR blockade

(a) Mouse lung ceramide levels induced by VEGFR inhibitor (VEGFR-inh, SU5416) or vehicle (Ctl); n=4 mice/group; mean +SD. *p=0.01. Mouse lung ceramide expression (brown, arrows) at 3d after treatment (_a, alveoli, _b, bronchial epithelium, and _v large vessels). (b) Rat lung ceramide (n= 3–5 rats/group; mean +SD. *p<0.05). (c–e) Mouse lung enzymatic activities of ceramide synthase (c, n=4/group, +SD, *p=0.02); soluble (secretory) and lysosomal ASMase at 3d (d, n=4/group, +SE, p=0.04); and neutral sphingomyelinase (e, n=4/group; mean +SD). (f) Lung ceramides and ceramide metabolites at 7d after VEGFR-inh (n=4) or Ctl (n=3); mean +SE, *p<0.05.

Figure 1. Lung ceramide levels are increased by VEGFR blockade

(a) Mouse lung ceramide levels induced by VEGFR inhibitor (VEGFR-inh, SU5416) or vehicle (Ctl); n=4 mice/group; mean +SD. *p=0.01. Mouse lung ceramide expression (brown, arrows) at 3d after treatment (_a, alveoli, _b, bronchial epithelium, and _v large vessels). (b) Rat lung ceramide (n= 3–5 rats/group; mean +SD. *p<0.05). (c–e) Mouse lung enzymatic activities of ceramide synthase (c, n=4/group, +SD, *p=0.02); soluble (secretory) and lysosomal ASMase at 3d (d, n=4/group, +SE, p=0.04); and neutral sphingomyelinase (e, n=4/group; mean +SD). (f) Lung ceramides and ceramide metabolites at 7d after VEGFR-inh (n=4) or Ctl (n=3); mean +SE, *p<0.05.

Figure 1. Lung ceramide levels are increased by VEGFR blockade

(a) Mouse lung ceramide levels induced by VEGFR inhibitor (VEGFR-inh, SU5416) or vehicle (Ctl); n=4 mice/group; mean +SD. *p=0.01. Mouse lung ceramide expression (brown, arrows) at 3d after treatment (_a, alveoli, _b, bronchial epithelium, and _v large vessels). (b) Rat lung ceramide (n= 3–5 rats/group; mean +SD. *p<0.05). (c–e) Mouse lung enzymatic activities of ceramide synthase (c, n=4/group, +SD, *p=0.02); soluble (secretory) and lysosomal ASMase at 3d (d, n=4/group, +SE, p=0.04); and neutral sphingomyelinase (e, n=4/group; mean +SD). (f) Lung ceramides and ceramide metabolites at 7d after VEGFR-inh (n=4) or Ctl (n=3); mean +SE, *p<0.05.

Figure 1. Lung ceramide levels are increased by VEGFR blockade

(a) Mouse lung ceramide levels induced by VEGFR inhibitor (VEGFR-inh, SU5416) or vehicle (Ctl); n=4 mice/group; mean +SD. *p=0.01. Mouse lung ceramide expression (brown, arrows) at 3d after treatment (_a, alveoli, _b, bronchial epithelium, and _v large vessels). (b) Rat lung ceramide (n= 3–5 rats/group; mean +SD. *p<0.05). (c–e) Mouse lung enzymatic activities of ceramide synthase (c, n=4/group, +SD, *p=0.02); soluble (secretory) and lysosomal ASMase at 3d (d, n=4/group, +SE, p=0.04); and neutral sphingomyelinase (e, n=4/group; mean +SD). (f) Lung ceramides and ceramide metabolites at 7d after VEGFR-inh (n=4) or Ctl (n=3); mean +SE, *p<0.05.

Figure 2. Inhibition of de novo ceramide synthesis prevents VEGFR blockade-induced emphysema

Effect of ceramide synthase inhibition with FB1 on alveolar histology (a) and diameters (b) 21d after VEFGR-inh (arrow: alveolar destruction; bar 50 μm; mean + SD, *p <0.05 versus VEGFR-inh; **p <0.05 versus vehicle. Effect of ceramide synthase inhibition with FB1 on lung oxidative stress (c) detected by 8-hydroxyguanosine (arrows) and on apoptosis (d) detected by active caspase-3 (mean + SE; *p<0.05) at 7d following VEGFR-inh. (e) Effect of S1-P augmentation (S1-P analog FTY720) on mouse lung apoptosis 7d after VEGFR-inh. Mean + SE, *p=0.008 versus Ctl and *p=0.0002 versus VEGFR-inh + FTY720.

Figure 2. Inhibition of de novo ceramide synthesis prevents VEGFR blockade-induced emphysema

Effect of ceramide synthase inhibition with FB1 on alveolar histology (a) and diameters (b) 21d after VEFGR-inh (arrow: alveolar destruction; bar 50 μm; mean + SD, *p <0.05 versus VEGFR-inh; **p <0.05 versus vehicle. Effect of ceramide synthase inhibition with FB1 on lung oxidative stress (c) detected by 8-hydroxyguanosine (arrows) and on apoptosis (d) detected by active caspase-3 (mean + SE; *p<0.05) at 7d following VEGFR-inh. (e) Effect of S1-P augmentation (S1-P analog FTY720) on mouse lung apoptosis 7d after VEGFR-inh. Mean + SE, *p=0.008 versus Ctl and *p=0.0002 versus VEGFR-inh + FTY720.

Figure 2. Inhibition of de novo ceramide synthesis prevents VEGFR blockade-induced emphysema

Effect of ceramide synthase inhibition with FB1 on alveolar histology (a) and diameters (b) 21d after VEFGR-inh (arrow: alveolar destruction; bar 50 μm; mean + SD, *p <0.05 versus VEGFR-inh; **p <0.05 versus vehicle. Effect of ceramide synthase inhibition with FB1 on lung oxidative stress (c) detected by 8-hydroxyguanosine (arrows) and on apoptosis (d) detected by active caspase-3 (mean + SE; *p<0.05) at 7d following VEGFR-inh. (e) Effect of S1-P augmentation (S1-P analog FTY720) on mouse lung apoptosis 7d after VEGFR-inh. Mean + SE, *p=0.008 versus Ctl and *p=0.0002 versus VEGFR-inh + FTY720.

Figure 2. Inhibition of de novo ceramide synthesis prevents VEGFR blockade-induced emphysema

Effect of ceramide synthase inhibition with FB1 on alveolar histology (a) and diameters (b) 21d after VEFGR-inh (arrow: alveolar destruction; bar 50 μm; mean + SD, *p <0.05 versus VEGFR-inh; **p <0.05 versus vehicle. Effect of ceramide synthase inhibition with FB1 on lung oxidative stress (c) detected by 8-hydroxyguanosine (arrows) and on apoptosis (d) detected by active caspase-3 (mean + SE; *p<0.05) at 7d following VEGFR-inh. (e) Effect of S1-P augmentation (S1-P analog FTY720) on mouse lung apoptosis 7d after VEGFR-inh. Mean + SE, *p=0.008 versus Ctl and *p=0.0002 versus VEGFR-inh + FTY720.

Figure 2. Inhibition of de novo ceramide synthesis prevents VEGFR blockade-induced emphysema

Effect of ceramide synthase inhibition with FB1 on alveolar histology (a) and diameters (b) 21d after VEFGR-inh (arrow: alveolar destruction; bar 50 μm; mean + SD, *p <0.05 versus VEGFR-inh; **p <0.05 versus vehicle. Effect of ceramide synthase inhibition with FB1 on lung oxidative stress (c) detected by 8-hydroxyguanosine (arrows) and on apoptosis (d) detected by active caspase-3 (mean + SE; *p<0.05) at 7d following VEGFR-inh. (e) Effect of S1-P augmentation (S1-P analog FTY720) on mouse lung apoptosis 7d after VEGFR-inh. Mean + SE, *p=0.008 versus Ctl and *p=0.0002 versus VEGFR-inh + FTY720.

Figure 3. Ceramide augmentation triggers lung apoptosis and emphysema

Effect of i-t C₁₂ ceramide (C₁₂Cer) or inactive dihydroceramide (DH-Cer) instillation at 24h on (a) total lung ceramide (mean +SD, *p =0.002); (b) alveolar diameters (mean +SD, *p <0.05); and (c) apoptosis (arrows: active caspase-3; bar 50 μm) quantified as cells per field (mean +SD, ^#p =0.001; *p <0.05). (d) Mouse lung co-stained for nuclei (DAPI, blue), apoptotic nuclei (TUNEL, green, arrows, arrowheads) and alveolar epithelial II (SPC) or endothelial cells (CD₃₄) in red. C₁₂Cer triggered mostly endothelial, but also epithelial cell apoptosis (arrows). (e) Effect of caspase inhibition (z-Asp-CH₍₂₎-DCB) on C₁₂Cer-triggered lung caspase-3 activation (arrows; bar 25 μm). Effect of C₁₂Cer on lung (f) MMP-12 activation (densitometric units normalized by actin + SD; *p<0.05) detected by immunoblot (inset; HT1080 cells positive control), (g) matrix proteolysis detected by gelatin zymography (lanes 2–4: 24h; 5–7: 72h), and (h) macrophage accumulation (arrows).

Figure 3. Ceramide augmentation triggers lung apoptosis and emphysema

Effect of i-t C₁₂ ceramide (C₁₂Cer) or inactive dihydroceramide (DH-Cer) instillation at 24h on (a) total lung ceramide (mean +SD, *p =0.002); (b) alveolar diameters (mean +SD, *p <0.05); and (c) apoptosis (arrows: active caspase-3; bar 50 μm) quantified as cells per field (mean +SD, ^#p =0.001; *p <0.05). (d) Mouse lung co-stained for nuclei (DAPI, blue), apoptotic nuclei (TUNEL, green, arrows, arrowheads) and alveolar epithelial II (SPC) or endothelial cells (CD₃₄) in red. C₁₂Cer triggered mostly endothelial, but also epithelial cell apoptosis (arrows). (e) Effect of caspase inhibition (z-Asp-CH₍₂₎-DCB) on C₁₂Cer-triggered lung caspase-3 activation (arrows; bar 25 μm). Effect of C₁₂Cer on lung (f) MMP-12 activation (densitometric units normalized by actin + SD; *p<0.05) detected by immunoblot (inset; HT1080 cells positive control), (g) matrix proteolysis detected by gelatin zymography (lanes 2–4: 24h; 5–7: 72h), and (h) macrophage accumulation (arrows).

Figure 3. Ceramide augmentation triggers lung apoptosis and emphysema

Effect of i-t C₁₂ ceramide (C₁₂Cer) or inactive dihydroceramide (DH-Cer) instillation at 24h on (a) total lung ceramide (mean +SD, *p =0.002); (b) alveolar diameters (mean +SD, *p <0.05); and (c) apoptosis (arrows: active caspase-3; bar 50 μm) quantified as cells per field (mean +SD, ^#p =0.001; *p <0.05). (d) Mouse lung co-stained for nuclei (DAPI, blue), apoptotic nuclei (TUNEL, green, arrows, arrowheads) and alveolar epithelial II (SPC) or endothelial cells (CD₃₄) in red. C₁₂Cer triggered mostly endothelial, but also epithelial cell apoptosis (arrows). (e) Effect of caspase inhibition (z-Asp-CH₍₂₎-DCB) on C₁₂Cer-triggered lung caspase-3 activation (arrows; bar 25 μm). Effect of C₁₂Cer on lung (f) MMP-12 activation (densitometric units normalized by actin + SD; *p<0.05) detected by immunoblot (inset; HT1080 cells positive control), (g) matrix proteolysis detected by gelatin zymography (lanes 2–4: 24h; 5–7: 72h), and (h) macrophage accumulation (arrows).

Figure 3. Ceramide augmentation triggers lung apoptosis and emphysema

Effect of i-t C₁₂ ceramide (C₁₂Cer) or inactive dihydroceramide (DH-Cer) instillation at 24h on (a) total lung ceramide (mean +SD, *p =0.002); (b) alveolar diameters (mean +SD, *p <0.05); and (c) apoptosis (arrows: active caspase-3; bar 50 μm) quantified as cells per field (mean +SD, ^#p =0.001; *p <0.05). (d) Mouse lung co-stained for nuclei (DAPI, blue), apoptotic nuclei (TUNEL, green, arrows, arrowheads) and alveolar epithelial II (SPC) or endothelial cells (CD₃₄) in red. C₁₂Cer triggered mostly endothelial, but also epithelial cell apoptosis (arrows). (e) Effect of caspase inhibition (z-Asp-CH₍₂₎-DCB) on C₁₂Cer-triggered lung caspase-3 activation (arrows; bar 25 μm). Effect of C₁₂Cer on lung (f) MMP-12 activation (densitometric units normalized by actin + SD; *p<0.05) detected by immunoblot (inset; HT1080 cells positive control), (g) matrix proteolysis detected by gelatin zymography (lanes 2–4: 24h; 5–7: 72h), and (h) macrophage accumulation (arrows).

Figure 3. Ceramide augmentation triggers lung apoptosis and emphysema

Effect of i-t C₁₂ ceramide (C₁₂Cer) or inactive dihydroceramide (DH-Cer) instillation at 24h on (a) total lung ceramide (mean +SD, *p =0.002); (b) alveolar diameters (mean +SD, *p <0.05); and (c) apoptosis (arrows: active caspase-3; bar 50 μm) quantified as cells per field (mean +SD, ^#p =0.001; *p <0.05). (d) Mouse lung co-stained for nuclei (DAPI, blue), apoptotic nuclei (TUNEL, green, arrows, arrowheads) and alveolar epithelial II (SPC) or endothelial cells (CD₃₄) in red. C₁₂Cer triggered mostly endothelial, but also epithelial cell apoptosis (arrows). (e) Effect of caspase inhibition (z-Asp-CH₍₂₎-DCB) on C₁₂Cer-triggered lung caspase-3 activation (arrows; bar 25 μm). Effect of C₁₂Cer on lung (f) MMP-12 activation (densitometric units normalized by actin + SD; *p<0.05) detected by immunoblot (inset; HT1080 cells positive control), (g) matrix proteolysis detected by gelatin zymography (lanes 2–4: 24h; 5–7: 72h), and (h) macrophage accumulation (arrows).

Figure 3. Ceramide augmentation triggers lung apoptosis and emphysema

Effect of i-t C₁₂ ceramide (C₁₂Cer) or inactive dihydroceramide (DH-Cer) instillation at 24h on (a) total lung ceramide (mean +SD, *p =0.002); (b) alveolar diameters (mean +SD, *p <0.05); and (c) apoptosis (arrows: active caspase-3; bar 50 μm) quantified as cells per field (mean +SD, ^#p =0.001; *p <0.05). (d) Mouse lung co-stained for nuclei (DAPI, blue), apoptotic nuclei (TUNEL, green, arrows, arrowheads) and alveolar epithelial II (SPC) or endothelial cells (CD₃₄) in red. C₁₂Cer triggered mostly endothelial, but also epithelial cell apoptosis (arrows). (e) Effect of caspase inhibition (z-Asp-CH₍₂₎-DCB) on C₁₂Cer-triggered lung caspase-3 activation (arrows; bar 25 μm). Effect of C₁₂Cer on lung (f) MMP-12 activation (densitometric units normalized by actin + SD; *p<0.05) detected by immunoblot (inset; HT1080 cells positive control), (g) matrix proteolysis detected by gelatin zymography (lanes 2–4: 24h; 5–7: 72h), and (h) macrophage accumulation (arrows).

Figure 3. Ceramide augmentation triggers lung apoptosis and emphysema

Effect of i-t C₁₂ ceramide (C₁₂Cer) or inactive dihydroceramide (DH-Cer) instillation at 24h on (a) total lung ceramide (mean +SD, *p =0.002); (b) alveolar diameters (mean +SD, *p <0.05); and (c) apoptosis (arrows: active caspase-3; bar 50 μm) quantified as cells per field (mean +SD, ^#p =0.001; *p <0.05). (d) Mouse lung co-stained for nuclei (DAPI, blue), apoptotic nuclei (TUNEL, green, arrows, arrowheads) and alveolar epithelial II (SPC) or endothelial cells (CD₃₄) in red. C₁₂Cer triggered mostly endothelial, but also epithelial cell apoptosis (arrows). (e) Effect of caspase inhibition (z-Asp-CH₍₂₎-DCB) on C₁₂Cer-triggered lung caspase-3 activation (arrows; bar 25 μm). Effect of C₁₂Cer on lung (f) MMP-12 activation (densitometric units normalized by actin + SD; *p<0.05) detected by immunoblot (inset; HT1080 cells positive control), (g) matrix proteolysis detected by gelatin zymography (lanes 2–4: 24h; 5–7: 72h), and (h) macrophage accumulation (arrows).

Figure 3. Ceramide augmentation triggers lung apoptosis and emphysema

Effect of i-t C₁₂ ceramide (C₁₂Cer) or inactive dihydroceramide (DH-Cer) instillation at 24h on (a) total lung ceramide (mean +SD, *p =0.002); (b) alveolar diameters (mean +SD, *p <0.05); and (c) apoptosis (arrows: active caspase-3; bar 50 μm) quantified as cells per field (mean +SD, ^#p =0.001; *p <0.05). (d) Mouse lung co-stained for nuclei (DAPI, blue), apoptotic nuclei (TUNEL, green, arrows, arrowheads) and alveolar epithelial II (SPC) or endothelial cells (CD₃₄) in red. C₁₂Cer triggered mostly endothelial, but also epithelial cell apoptosis (arrows). (e) Effect of caspase inhibition (z-Asp-CH₍₂₎-DCB) on C₁₂Cer-triggered lung caspase-3 activation (arrows; bar 25 μm). Effect of C₁₂Cer on lung (f) MMP-12 activation (densitometric units normalized by actin + SD; *p<0.05) detected by immunoblot (inset; HT1080 cells positive control), (g) matrix proteolysis detected by gelatin zymography (lanes 2–4: 24h; 5–7: 72h), and (h) macrophage accumulation (arrows).

Figure 4. Self-amplification of lung ceramide synthesis

Effect of short-chain exogenous ceramide on endogenous ceramide species profile in the (a) lung after C₁₂Cer (1mg/kg, 24h, black) or vehicle (gray) and in (b) bovine pulmonary artery endothelial cells (thin layer chromatography of intra-cellular ceramides detected by DAG kinase assay) after C₈Cer (10 μM; n=2). (c) Effect of C₁₂Cer on the ASMase activity in the mouse lung (24h, mean +SD, *p =0.02). Effect of neutralizing ceramide-specific antibody (4mg/kg/d) on (d) lung ceramide species (mean +SE; *p =0.002) and on (e) lung apoptosis detected by active caspase-3 (arrows, bar 50 μm) induced by VEGFR-inh (3d).

Figure 4. Self-amplification of lung ceramide synthesis

Effect of short-chain exogenous ceramide on endogenous ceramide species profile in the (a) lung after C₁₂Cer (1mg/kg, 24h, black) or vehicle (gray) and in (b) bovine pulmonary artery endothelial cells (thin layer chromatography of intra-cellular ceramides detected by DAG kinase assay) after C₈Cer (10 μM; n=2). (c) Effect of C₁₂Cer on the ASMase activity in the mouse lung (24h, mean +SD, *p =0.02). Effect of neutralizing ceramide-specific antibody (4mg/kg/d) on (d) lung ceramide species (mean +SE; *p =0.002) and on (e) lung apoptosis detected by active caspase-3 (arrows, bar 50 μm) induced by VEGFR-inh (3d).

Figure 4. Self-amplification of lung ceramide synthesis

Effect of short-chain exogenous ceramide on endogenous ceramide species profile in the (a) lung after C₁₂Cer (1mg/kg, 24h, black) or vehicle (gray) and in (b) bovine pulmonary artery endothelial cells (thin layer chromatography of intra-cellular ceramides detected by DAG kinase assay) after C₈Cer (10 μM; n=2). (c) Effect of C₁₂Cer on the ASMase activity in the mouse lung (24h, mean +SD, *p =0.02). Effect of neutralizing ceramide-specific antibody (4mg/kg/d) on (d) lung ceramide species (mean +SE; *p =0.002) and on (e) lung apoptosis detected by active caspase-3 (arrows, bar 50 μm) induced by VEGFR-inh (3d).

Figure 4. Self-amplification of lung ceramide synthesis

Effect of short-chain exogenous ceramide on endogenous ceramide species profile in the (a) lung after C₁₂Cer (1mg/kg, 24h, black) or vehicle (gray) and in (b) bovine pulmonary artery endothelial cells (thin layer chromatography of intra-cellular ceramides detected by DAG kinase assay) after C₈Cer (10 μM; n=2). (c) Effect of C₁₂Cer on the ASMase activity in the mouse lung (24h, mean +SD, *p =0.02). Effect of neutralizing ceramide-specific antibody (4mg/kg/d) on (d) lung ceramide species (mean +SE; *p =0.002) and on (e) lung apoptosis detected by active caspase-3 (arrows, bar 50 μm) induced by VEGFR-inh (3d).

Figure 4. Self-amplification of lung ceramide synthesis

Effect of short-chain exogenous ceramide on endogenous ceramide species profile in the (a) lung after C₁₂Cer (1mg/kg, 24h, black) or vehicle (gray) and in (b) bovine pulmonary artery endothelial cells (thin layer chromatography of intra-cellular ceramides detected by DAG kinase assay) after C₈Cer (10 μM; n=2). (c) Effect of C₁₂Cer on the ASMase activity in the mouse lung (24h, mean +SD, *p =0.02). Effect of neutralizing ceramide-specific antibody (4mg/kg/d) on (d) lung ceramide species (mean +SE; *p =0.002) and on (e) lung apoptosis detected by active caspase-3 (arrows, bar 50 μm) induced by VEGFR-inh (3d).

Figure 5. Increased lung ceramide in patients with emphysema

(a) Effect of cigarette smoke (1%) or TNF-α (10 ng/ml; 40 min) ceramide in lung endothelial cells. Ceramide analysis in human emphysema (patient data in Supplementary table 1) by (b) immunostaining (in patients without emphysema (Ctl) ceramide was expressed mostly by alveolar macrophages (arrow), while in emphysema, ceramide was increased in alveolar septal endothelial and epithelial II and I (arrowhead) cells and macrophages; *p =0.02); by (c) DAG kinase assay (means ±SD; *p =0.01); and by (d, e) LC-MS/MS (emphysema, black; non-emphysema non-smokers, light gray; non-emphysema smoker (Smk, gray); *p <0.05.

Figure 5. Increased lung ceramide in patients with emphysema

(a) Effect of cigarette smoke (1%) or TNF-α (10 ng/ml; 40 min) ceramide in lung endothelial cells. Ceramide analysis in human emphysema (patient data in Supplementary table 1) by (b) immunostaining (in patients without emphysema (Ctl) ceramide was expressed mostly by alveolar macrophages (arrow), while in emphysema, ceramide was increased in alveolar septal endothelial and epithelial II and I (arrowhead) cells and macrophages; *p =0.02); by (c) DAG kinase assay (means ±SD; *p =0.01); and by (d, e) LC-MS/MS (emphysema, black; non-emphysema non-smokers, light gray; non-emphysema smoker (Smk, gray); *p <0.05.

Figure 5. Increased lung ceramide in patients with emphysema

(a) Effect of cigarette smoke (1%) or TNF-α (10 ng/ml; 40 min) ceramide in lung endothelial cells. Ceramide analysis in human emphysema (patient data in Supplementary table 1) by (b) immunostaining (in patients without emphysema (Ctl) ceramide was expressed mostly by alveolar macrophages (arrow), while in emphysema, ceramide was increased in alveolar septal endothelial and epithelial II and I (arrowhead) cells and macrophages; *p =0.02); by (c) DAG kinase assay (means ±SD; *p =0.01); and by (d, e) LC-MS/MS (emphysema, black; non-emphysema non-smokers, light gray; non-emphysema smoker (Smk, gray); *p <0.05.

Figure 5. Increased lung ceramide in patients with emphysema

(a) Effect of cigarette smoke (1%) or TNF-α (10 ng/ml; 40 min) ceramide in lung endothelial cells. Ceramide analysis in human emphysema (patient data in Supplementary table 1) by (b) immunostaining (in patients without emphysema (Ctl) ceramide was expressed mostly by alveolar macrophages (arrow), while in emphysema, ceramide was increased in alveolar septal endothelial and epithelial II and I (arrowhead) cells and macrophages; *p =0.02); by (c) DAG kinase assay (means ±SD; *p =0.01); and by (d, e) LC-MS/MS (emphysema, black; non-emphysema non-smokers, light gray; non-emphysema smoker (Smk, gray); *p <0.05.

Figure 5. Increased lung ceramide in patients with emphysema

(a) Effect of cigarette smoke (1%) or TNF-α (10 ng/ml; 40 min) ceramide in lung endothelial cells. Ceramide analysis in human emphysema (patient data in Supplementary table 1) by (b) immunostaining (in patients without emphysema (Ctl) ceramide was expressed mostly by alveolar macrophages (arrow), while in emphysema, ceramide was increased in alveolar septal endothelial and epithelial II and I (arrowhead) cells and macrophages; *p =0.02); by (c) DAG kinase assay (means ±SD; *p =0.01); and by (d, e) LC-MS/MS (emphysema, black; non-emphysema non-smokers, light gray; non-emphysema smoker (Smk, gray); *p <0.05.