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Targeted induction of lung endothelial cell apoptosis causes emphysema-like changes in the mouse.

Giordano RJ, Lahdenranta J, Zhen L, Chukwueke U, Petrache I, Langley RR, Fidler IJ, Pasqualini R, Tuder RM, Arap W - J. Biol. Chem. (2008)

Bottom Line: As early as 4 days following peptide administration, mice developed air space enlargement associated with enhanced oxidative stress, influx of macrophages, and up-regulation of ceramide.Thus, our data enable the generation of a convenient mouse model of human emphysema.Finally, combinatorial screenings on immortalized cells followed by in vivo targeting establishes an experimental framework for discovery and validation of additional ligand-directed pharmacodelivery systems.

View Article: PubMed Central - PubMed

Affiliation: University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
Pulmonary gas exchange relies on a rich capillary network, which, together with alveolar epithelial type I and II cells, form alveolar septa, the functional units in the lung. Alveolar capillary endothelial cells are critical in maintaining alveolar structure, because disruption of endothelial cell integrity underlies several lung diseases. Here we show that targeted ablation of lung capillary endothelial cells recapitulates the cellular events involved in cigarette smoke-induced emphysema, one of the most prevalent nonneoplastic lung diseases. Based on phage library screening on an immortalized lung endothelial cell line, we identified a lung endothelial cell-binding peptide, which preferentially homes to lung blood vessels. This peptide fused to a proapoptotic motif specifically induced programmed cell death of lung endothelial cells in vitro as well as targeted apoptosis of the lung microcirculation in vivo. As early as 4 days following peptide administration, mice developed air space enlargement associated with enhanced oxidative stress, influx of macrophages, and up-regulation of ceramide. Given that these are all critical elements of the corresponding human emphysema caused by cigarette smoke, these data provide evidence for a central role for the alveolar endothelial cells in the maintenance of lung structure and of endothelial cell apoptosis in the pathogenesis of emphysema-like changes. Thus, our data enable the generation of a convenient mouse model of human emphysema. Finally, combinatorial screenings on immortalized cells followed by in vivo targeting establishes an experimental framework for discovery and validation of additional ligand-directed pharmacodelivery systems.

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Levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine and ceramide are increased in CGSPGWVRC-GG-D(KLAKLAK)2-treated lungs (21 days of treatment). a, CGSPGWVRC-GG-D(KLAKLAK)2 peptide induces oxidative damage to mouse lungs, as indicated by increasing 8-oxo-dG expression. Immunohistochemical staining of 8-oxo-dG in lung sections from mice treated for 21 days with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone. Isotype control antibody on CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs served as a negative control for anti-8-oxo-dG staining. Lung sections from the CGSPGWVRC-GG-D(KLAKLAK)2-treated mice show elevated 8-oxo-dG expression compared with control-treated mice. Scale bar (a and c), 25 μm. b, quantification of the 8-oxo-dG intensity in the lung tissues from CGSPGWVRC-GG-D(KLAKLAK)2-treated mice. c and d, analysis of ceramide species by mass spectrometry shows that lungs of mice treated with the CGSPGWVRC-GG-D(KLAKLAK)2 peptide for 7 days have increased levels of ceramides (c) as well as dihydroceramides, ceramide precursors in the de novo pathway of ceramide synthesis (d), compared with the lungs from control peptide (CGSPGWVRC or D(KLAKLAK)2)-treated mice. e, CGSPGWVRC-GG-D(KLAKLAK)2 peptide induces elevation of ceramide levels in mouse lungs. Lung sections from mice after 21 days of treatment with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone were subjected to immunohistochemical staining for ceramide. Ceramide staining in CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs show numerous ceramide-positive alveolar cells, whereas lungs treated with control peptides or vehicle show only sporadic ceramide-positive cells. f, quantification of ceramide expression detected by immunohistochemistry is described under “Materials and Methods.”
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fig7: Levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine and ceramide are increased in CGSPGWVRC-GG-D(KLAKLAK)2-treated lungs (21 days of treatment). a, CGSPGWVRC-GG-D(KLAKLAK)2 peptide induces oxidative damage to mouse lungs, as indicated by increasing 8-oxo-dG expression. Immunohistochemical staining of 8-oxo-dG in lung sections from mice treated for 21 days with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone. Isotype control antibody on CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs served as a negative control for anti-8-oxo-dG staining. Lung sections from the CGSPGWVRC-GG-D(KLAKLAK)2-treated mice show elevated 8-oxo-dG expression compared with control-treated mice. Scale bar (a and c), 25 μm. b, quantification of the 8-oxo-dG intensity in the lung tissues from CGSPGWVRC-GG-D(KLAKLAK)2-treated mice. c and d, analysis of ceramide species by mass spectrometry shows that lungs of mice treated with the CGSPGWVRC-GG-D(KLAKLAK)2 peptide for 7 days have increased levels of ceramides (c) as well as dihydroceramides, ceramide precursors in the de novo pathway of ceramide synthesis (d), compared with the lungs from control peptide (CGSPGWVRC or D(KLAKLAK)2)-treated mice. e, CGSPGWVRC-GG-D(KLAKLAK)2 peptide induces elevation of ceramide levels in mouse lungs. Lung sections from mice after 21 days of treatment with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone were subjected to immunohistochemical staining for ceramide. Ceramide staining in CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs show numerous ceramide-positive alveolar cells, whereas lungs treated with control peptides or vehicle show only sporadic ceramide-positive cells. f, quantification of ceramide expression detected by immunohistochemistry is described under “Materials and Methods.”

Mentions: Targeted CGSPGWVRC-GG-D(KLAKLAK)2 Increases Pulmonary Oxidative Stress—We have shown that oxidative stress is critically involved in the pathogenesis of experimental cigarette smoke-induced emphysema (17). Furthermore, we demonstrated that emphysema due to VEGF receptor blockade depends on the mutual interaction between apoptosis and oxidative stress, since either apoptosis blockade or inhibition of superoxide anion prevents emphysema development (6). Immunohistochemical detection of 8-oxo-dG was used to evaluate oxidative stress in the lungs of mice receiving CGSPGWVRC-GG-D(KLAKLAK)2 for 4 and for 21 days. After 4 days of treatment, lung sections from CGSPGWVRC-GG-D(KLAKLAK)2-treated mice showed increased 8-oxo-dG expression when compared with the lung sections from the control peptide-treated mice (Fig. S4). After 21 days of treatment with vehicle or control peptides, a small number of alveolar septal cells showed staining for 8-oxo-dG (355 ± 20 and 346 ± 17 expression intensity units, respectively), whereas significantly more cells (617 ± 28 expression intensity units; p < 0.05) were stained in CGSPGWVRC-GG-D(KLAKLAK)2-treated lungs (Fig. 7, a and b). These results show that emphysema and alveolar cell apoptosis due to systemic administration of CGSPGWVRC-GG-D(KLAKLAK)2 are associated with enhanced oxidative damage to the lungs.


Targeted induction of lung endothelial cell apoptosis causes emphysema-like changes in the mouse.

Giordano RJ, Lahdenranta J, Zhen L, Chukwueke U, Petrache I, Langley RR, Fidler IJ, Pasqualini R, Tuder RM, Arap W - J. Biol. Chem. (2008)

Levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine and ceramide are increased in CGSPGWVRC-GG-D(KLAKLAK)2-treated lungs (21 days of treatment). a, CGSPGWVRC-GG-D(KLAKLAK)2 peptide induces oxidative damage to mouse lungs, as indicated by increasing 8-oxo-dG expression. Immunohistochemical staining of 8-oxo-dG in lung sections from mice treated for 21 days with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone. Isotype control antibody on CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs served as a negative control for anti-8-oxo-dG staining. Lung sections from the CGSPGWVRC-GG-D(KLAKLAK)2-treated mice show elevated 8-oxo-dG expression compared with control-treated mice. Scale bar (a and c), 25 μm. b, quantification of the 8-oxo-dG intensity in the lung tissues from CGSPGWVRC-GG-D(KLAKLAK)2-treated mice. c and d, analysis of ceramide species by mass spectrometry shows that lungs of mice treated with the CGSPGWVRC-GG-D(KLAKLAK)2 peptide for 7 days have increased levels of ceramides (c) as well as dihydroceramides, ceramide precursors in the de novo pathway of ceramide synthesis (d), compared with the lungs from control peptide (CGSPGWVRC or D(KLAKLAK)2)-treated mice. e, CGSPGWVRC-GG-D(KLAKLAK)2 peptide induces elevation of ceramide levels in mouse lungs. Lung sections from mice after 21 days of treatment with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone were subjected to immunohistochemical staining for ceramide. Ceramide staining in CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs show numerous ceramide-positive alveolar cells, whereas lungs treated with control peptides or vehicle show only sporadic ceramide-positive cells. f, quantification of ceramide expression detected by immunohistochemistry is described under “Materials and Methods.”
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Related In: Results  -  Collection

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fig7: Levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine and ceramide are increased in CGSPGWVRC-GG-D(KLAKLAK)2-treated lungs (21 days of treatment). a, CGSPGWVRC-GG-D(KLAKLAK)2 peptide induces oxidative damage to mouse lungs, as indicated by increasing 8-oxo-dG expression. Immunohistochemical staining of 8-oxo-dG in lung sections from mice treated for 21 days with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone. Isotype control antibody on CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs served as a negative control for anti-8-oxo-dG staining. Lung sections from the CGSPGWVRC-GG-D(KLAKLAK)2-treated mice show elevated 8-oxo-dG expression compared with control-treated mice. Scale bar (a and c), 25 μm. b, quantification of the 8-oxo-dG intensity in the lung tissues from CGSPGWVRC-GG-D(KLAKLAK)2-treated mice. c and d, analysis of ceramide species by mass spectrometry shows that lungs of mice treated with the CGSPGWVRC-GG-D(KLAKLAK)2 peptide for 7 days have increased levels of ceramides (c) as well as dihydroceramides, ceramide precursors in the de novo pathway of ceramide synthesis (d), compared with the lungs from control peptide (CGSPGWVRC or D(KLAKLAK)2)-treated mice. e, CGSPGWVRC-GG-D(KLAKLAK)2 peptide induces elevation of ceramide levels in mouse lungs. Lung sections from mice after 21 days of treatment with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone were subjected to immunohistochemical staining for ceramide. Ceramide staining in CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs show numerous ceramide-positive alveolar cells, whereas lungs treated with control peptides or vehicle show only sporadic ceramide-positive cells. f, quantification of ceramide expression detected by immunohistochemistry is described under “Materials and Methods.”
Mentions: Targeted CGSPGWVRC-GG-D(KLAKLAK)2 Increases Pulmonary Oxidative Stress—We have shown that oxidative stress is critically involved in the pathogenesis of experimental cigarette smoke-induced emphysema (17). Furthermore, we demonstrated that emphysema due to VEGF receptor blockade depends on the mutual interaction between apoptosis and oxidative stress, since either apoptosis blockade or inhibition of superoxide anion prevents emphysema development (6). Immunohistochemical detection of 8-oxo-dG was used to evaluate oxidative stress in the lungs of mice receiving CGSPGWVRC-GG-D(KLAKLAK)2 for 4 and for 21 days. After 4 days of treatment, lung sections from CGSPGWVRC-GG-D(KLAKLAK)2-treated mice showed increased 8-oxo-dG expression when compared with the lung sections from the control peptide-treated mice (Fig. S4). After 21 days of treatment with vehicle or control peptides, a small number of alveolar septal cells showed staining for 8-oxo-dG (355 ± 20 and 346 ± 17 expression intensity units, respectively), whereas significantly more cells (617 ± 28 expression intensity units; p < 0.05) were stained in CGSPGWVRC-GG-D(KLAKLAK)2-treated lungs (Fig. 7, a and b). These results show that emphysema and alveolar cell apoptosis due to systemic administration of CGSPGWVRC-GG-D(KLAKLAK)2 are associated with enhanced oxidative damage to the lungs.

Bottom Line: As early as 4 days following peptide administration, mice developed air space enlargement associated with enhanced oxidative stress, influx of macrophages, and up-regulation of ceramide.Thus, our data enable the generation of a convenient mouse model of human emphysema.Finally, combinatorial screenings on immortalized cells followed by in vivo targeting establishes an experimental framework for discovery and validation of additional ligand-directed pharmacodelivery systems.

View Article: PubMed Central - PubMed

Affiliation: University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
Pulmonary gas exchange relies on a rich capillary network, which, together with alveolar epithelial type I and II cells, form alveolar septa, the functional units in the lung. Alveolar capillary endothelial cells are critical in maintaining alveolar structure, because disruption of endothelial cell integrity underlies several lung diseases. Here we show that targeted ablation of lung capillary endothelial cells recapitulates the cellular events involved in cigarette smoke-induced emphysema, one of the most prevalent nonneoplastic lung diseases. Based on phage library screening on an immortalized lung endothelial cell line, we identified a lung endothelial cell-binding peptide, which preferentially homes to lung blood vessels. This peptide fused to a proapoptotic motif specifically induced programmed cell death of lung endothelial cells in vitro as well as targeted apoptosis of the lung microcirculation in vivo. As early as 4 days following peptide administration, mice developed air space enlargement associated with enhanced oxidative stress, influx of macrophages, and up-regulation of ceramide. Given that these are all critical elements of the corresponding human emphysema caused by cigarette smoke, these data provide evidence for a central role for the alveolar endothelial cells in the maintenance of lung structure and of endothelial cell apoptosis in the pathogenesis of emphysema-like changes. Thus, our data enable the generation of a convenient mouse model of human emphysema. Finally, combinatorial screenings on immortalized cells followed by in vivo targeting establishes an experimental framework for discovery and validation of additional ligand-directed pharmacodelivery systems.

Show MeSH
Related in: MedlinePlus