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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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CGSPGWVRC-GG-D(KLAKLAK)2 peptide causes lung cell apoptosis after 21 days of treatment. a, identification of apoptotic (detected by TUNEL; green) endothelial cells (detected by anti-CD34 antibody; red) and type II epithelial cells (detected by anti-SpC antibody; red) in the lungs of CGSPGWVRC-GG-D(KLAKLAK)2-, control peptide-, and vehicle-treated mice after 21 days of treatment. Shown are merged images, with co-localization of cell-specific markers and apoptosis: cytoplasmic marker alone (CD34 or SpC) (red cells) (yellow arrows), TUNEL-positive cells in CD34 or SpC-positive cells (yellow arrowheads), and TUNEL-positive cells without a cytoplasmic positive markers (large orange arrows). Cell nuclei were stained with DAPI (blue) (inset, top right). b, active caspase-3 expression in lung sections of mice 21 days after treatment with CGSPGWVRC-GG-D(KLAKLAK)2, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone. CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs show abundant active caspase-3-positive cells in the alveolar septa in contrast to control-treated lungs. Isotype control antibody on CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs was used as a negative control for the active caspase-3 staining. c, quantification of number of alveolar septal cells positive for active caspase-3. d, increased levels of active caspase-3 were detected in the lungs of CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated mice versus control-treated mice by Western blot analysis of lung tissue lysates with an active caspase-3-specific antibody. e, densitometric quantification of active caspase-3 expression levels obtained in Western blot analysis normalized to actin levels.
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fig5: CGSPGWVRC-GG-D(KLAKLAK)2 peptide causes lung cell apoptosis after 21 days of treatment. a, identification of apoptotic (detected by TUNEL; green) endothelial cells (detected by anti-CD34 antibody; red) and type II epithelial cells (detected by anti-SpC antibody; red) in the lungs of CGSPGWVRC-GG-D(KLAKLAK)2-, control peptide-, and vehicle-treated mice after 21 days of treatment. Shown are merged images, with co-localization of cell-specific markers and apoptosis: cytoplasmic marker alone (CD34 or SpC) (red cells) (yellow arrows), TUNEL-positive cells in CD34 or SpC-positive cells (yellow arrowheads), and TUNEL-positive cells without a cytoplasmic positive markers (large orange arrows). Cell nuclei were stained with DAPI (blue) (inset, top right). b, active caspase-3 expression in lung sections of mice 21 days after treatment with CGSPGWVRC-GG-D(KLAKLAK)2, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone. CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs show abundant active caspase-3-positive cells in the alveolar septa in contrast to control-treated lungs. Isotype control antibody on CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs was used as a negative control for the active caspase-3 staining. c, quantification of number of alveolar septal cells positive for active caspase-3. d, increased levels of active caspase-3 were detected in the lungs of CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated mice versus control-treated mice by Western blot analysis of lung tissue lysates with an active caspase-3-specific antibody. e, densitometric quantification of active caspase-3 expression levels obtained in Western blot analysis normalized to actin levels.

Mentions: Targeted CGSPGWVRC-GG-D(KLAKLAK)2 Induces both Lung Cell Apoptosis and Decreased Lung Cell Proliferation—As shown above, CGSPGWVRC-GG-D(KLAKLAK)2 peptide treatment led to lung endothelial cell apoptosis in vitro. Given that peptide treatment with CGSPGWVRC-GG-D(KLAKLAK)2 caused alveolar destruction in mice, we determined whether this destruction was associated with directed apoptosis of alveolar endothelial cells. After 4 days of treatment with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, staining of lung sections with DAPI and TUNEL showed abundant TUNEL-positive cells compared with staining of lung sections from mice receiving control peptides (supplemental Fig. S2a). TUNEL-positive cells in lung sections were abundant after 21 days of CGSPGWVRC-GG-D(KLAKLAK)2 peptide treatment (Fig. 5a) (CGSPGWVRC-GG-D(KLAKLAK)2 = 0.0288 TUNEL+/DAPI+ versus peptide = 0.0146 TUNEL+/DAPI+, p < 0.03; Mann-Whitney rank sum test). Alveolar endothelial, type II, and myofibroblastic cells were equally affected at day 4 (Fig. S2a). A similar proportion of endothelial and type II cell death was also identified on day 21 after treatment with CGSPGWVRC-GG-D(KLAKLAK)2 peptide (Fig. 5a), as detected by staining of the TUNEL-labeled lung sections with either a CD34-specific antibody (an endothelial cell marker) or an SpC-specific antibody (a type II alveolar epithelial cell marker), or smooth muscle cell α-actin-antibody (for myofibroblasts). The TUNEL findings were also confirmed by immunohistochemical staining for active caspase-3, which revealed an increased number of apoptotic cells in the alveolar septa of the CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated mice; in contrast, lungs from mice treated with control peptide or vehicle showed much less active caspase-3-positive cells (Fig. 5b). Increased active caspase-3-positive cells in the alveolar septa of the CGSPGWVRC-GG-D(KLAKLAK)2 peptidetreated mice were evident after only 4 days of treatment (Fig. S2, b and c). Enhanced activation of caspase-3 in lungs of mice after 4 days (Fig. S2, b-e) and after 21 days (Fig. 5, c and d) of treatment with CGSPGWVRC-GG-D(KLAKLAK)2 peptide was further confirmed by the detection of increased levels of the 19-kDa active caspase-3 peptide in whole-lung lysates by Western blot analysis. Densitometric assessment of the Western blot reaction at day 21 (Figs. 5, d and e, and S3) showed 3.36 ± 0.89 densitometric units of active caspase-3 in lungs of mice receiving CGSPGWVRC-GG-D(KLAKLAK)2 peptide (n = 5) compared with 0.66 ± 0.19 densitometric units in the lungs of mice receiving control peptide (n = 6). In addition to increased apoptosis of alveolar cells, we also detected decreased cell proliferation in the lungs of mice treated with CGSPGWVRC-GG-D(KLAKLAK)2 peptide for 21 days when compared with the lungs of mice treated with vehicle or control peptide (Fig. 6, a and b). In sum, these data indicate that systemic administration of CGSPGWVRC-GG-D(KLAKLAK)2 peptide caused apoptosis of alveolar cells.


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)

CGSPGWVRC-GG-D(KLAKLAK)2 peptide causes lung cell apoptosis after 21 days of treatment. a, identification of apoptotic (detected by TUNEL; green) endothelial cells (detected by anti-CD34 antibody; red) and type II epithelial cells (detected by anti-SpC antibody; red) in the lungs of CGSPGWVRC-GG-D(KLAKLAK)2-, control peptide-, and vehicle-treated mice after 21 days of treatment. Shown are merged images, with co-localization of cell-specific markers and apoptosis: cytoplasmic marker alone (CD34 or SpC) (red cells) (yellow arrows), TUNEL-positive cells in CD34 or SpC-positive cells (yellow arrowheads), and TUNEL-positive cells without a cytoplasmic positive markers (large orange arrows). Cell nuclei were stained with DAPI (blue) (inset, top right). b, active caspase-3 expression in lung sections of mice 21 days after treatment with CGSPGWVRC-GG-D(KLAKLAK)2, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone. CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs show abundant active caspase-3-positive cells in the alveolar septa in contrast to control-treated lungs. Isotype control antibody on CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs was used as a negative control for the active caspase-3 staining. c, quantification of number of alveolar septal cells positive for active caspase-3. d, increased levels of active caspase-3 were detected in the lungs of CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated mice versus control-treated mice by Western blot analysis of lung tissue lysates with an active caspase-3-specific antibody. e, densitometric quantification of active caspase-3 expression levels obtained in Western blot analysis normalized to actin levels.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2570855&req=5

fig5: CGSPGWVRC-GG-D(KLAKLAK)2 peptide causes lung cell apoptosis after 21 days of treatment. a, identification of apoptotic (detected by TUNEL; green) endothelial cells (detected by anti-CD34 antibody; red) and type II epithelial cells (detected by anti-SpC antibody; red) in the lungs of CGSPGWVRC-GG-D(KLAKLAK)2-, control peptide-, and vehicle-treated mice after 21 days of treatment. Shown are merged images, with co-localization of cell-specific markers and apoptosis: cytoplasmic marker alone (CD34 or SpC) (red cells) (yellow arrows), TUNEL-positive cells in CD34 or SpC-positive cells (yellow arrowheads), and TUNEL-positive cells without a cytoplasmic positive markers (large orange arrows). Cell nuclei were stained with DAPI (blue) (inset, top right). b, active caspase-3 expression in lung sections of mice 21 days after treatment with CGSPGWVRC-GG-D(KLAKLAK)2, control peptides (CGSPGWVRC and D(KLAKLAK)2), or vehicle alone. CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs show abundant active caspase-3-positive cells in the alveolar septa in contrast to control-treated lungs. Isotype control antibody on CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated lungs was used as a negative control for the active caspase-3 staining. c, quantification of number of alveolar septal cells positive for active caspase-3. d, increased levels of active caspase-3 were detected in the lungs of CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated mice versus control-treated mice by Western blot analysis of lung tissue lysates with an active caspase-3-specific antibody. e, densitometric quantification of active caspase-3 expression levels obtained in Western blot analysis normalized to actin levels.
Mentions: Targeted CGSPGWVRC-GG-D(KLAKLAK)2 Induces both Lung Cell Apoptosis and Decreased Lung Cell Proliferation—As shown above, CGSPGWVRC-GG-D(KLAKLAK)2 peptide treatment led to lung endothelial cell apoptosis in vitro. Given that peptide treatment with CGSPGWVRC-GG-D(KLAKLAK)2 caused alveolar destruction in mice, we determined whether this destruction was associated with directed apoptosis of alveolar endothelial cells. After 4 days of treatment with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, staining of lung sections with DAPI and TUNEL showed abundant TUNEL-positive cells compared with staining of lung sections from mice receiving control peptides (supplemental Fig. S2a). TUNEL-positive cells in lung sections were abundant after 21 days of CGSPGWVRC-GG-D(KLAKLAK)2 peptide treatment (Fig. 5a) (CGSPGWVRC-GG-D(KLAKLAK)2 = 0.0288 TUNEL+/DAPI+ versus peptide = 0.0146 TUNEL+/DAPI+, p < 0.03; Mann-Whitney rank sum test). Alveolar endothelial, type II, and myofibroblastic cells were equally affected at day 4 (Fig. S2a). A similar proportion of endothelial and type II cell death was also identified on day 21 after treatment with CGSPGWVRC-GG-D(KLAKLAK)2 peptide (Fig. 5a), as detected by staining of the TUNEL-labeled lung sections with either a CD34-specific antibody (an endothelial cell marker) or an SpC-specific antibody (a type II alveolar epithelial cell marker), or smooth muscle cell α-actin-antibody (for myofibroblasts). The TUNEL findings were also confirmed by immunohistochemical staining for active caspase-3, which revealed an increased number of apoptotic cells in the alveolar septa of the CGSPGWVRC-GG-D(KLAKLAK)2 peptide-treated mice; in contrast, lungs from mice treated with control peptide or vehicle showed much less active caspase-3-positive cells (Fig. 5b). Increased active caspase-3-positive cells in the alveolar septa of the CGSPGWVRC-GG-D(KLAKLAK)2 peptidetreated mice were evident after only 4 days of treatment (Fig. S2, b and c). Enhanced activation of caspase-3 in lungs of mice after 4 days (Fig. S2, b-e) and after 21 days (Fig. 5, c and d) of treatment with CGSPGWVRC-GG-D(KLAKLAK)2 peptide was further confirmed by the detection of increased levels of the 19-kDa active caspase-3 peptide in whole-lung lysates by Western blot analysis. Densitometric assessment of the Western blot reaction at day 21 (Figs. 5, d and e, and S3) showed 3.36 ± 0.89 densitometric units of active caspase-3 in lungs of mice receiving CGSPGWVRC-GG-D(KLAKLAK)2 peptide (n = 5) compared with 0.66 ± 0.19 densitometric units in the lungs of mice receiving control peptide (n = 6). In addition to increased apoptosis of alveolar cells, we also detected decreased cell proliferation in the lungs of mice treated with CGSPGWVRC-GG-D(KLAKLAK)2 peptide for 21 days when compared with the lungs of mice treated with vehicle or control peptide (Fig. 6, a and b). In sum, these data indicate that systemic administration of CGSPGWVRC-GG-D(KLAKLAK)2 peptide caused apoptosis of alveolar cells.

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