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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 peptide mediates internalization of ligands into lung endothelial cells and cell apoptosis. a, CGSPGWVRC peptide mediates phage internalization into lung endothelial cells. CGSPGWVRC phage or control phage were incubated with lung endothelial cells at 37 °C to allow for phage internalization. Cells were stained with an anti-bacteriophage antibody after removal of the membrane-bound phage. The upper panels show staining of permeabilized cells revealing the internalized phage. The lower panels show staining of nonpermeabilized cells, demonstrating the successful removal of phage from the cell surface. Scale bar, 100 μm. b, lung endothelial cells were incubated with increasing concentrations (up to 100 μm) of proapoptotic peptide synthesized in conjunction with the targeting CGSPGWVRC peptide (CGSPGWVRC-GG-D(KLAKLAK)2) or negative control peptides (an equimolar mixture of CGSPGWVRC and D(KLAKLAK)2). Cell viability was determined by optical absorbance using a cell proliferation detection reagent. Shown are means ± S.E. from triplicate wells. c, lung endothelial cells were incubated with 100 μm proapoptotic peptide CGSPGWVRC-GG-D(KLAKLAK)2 or negative control peptides (equimolar mixture of CGSPGWVRC and D(KLAKLAK)2) for 2 h. Induction of cell apoptosis was detected by Annexin-V-fluorescein isothiocyanate (FITC) binding (top and middle panels) or TUNEL (bottom, yellow arrows).
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fig2: CGSPGWVRC peptide mediates internalization of ligands into lung endothelial cells and cell apoptosis. a, CGSPGWVRC peptide mediates phage internalization into lung endothelial cells. CGSPGWVRC phage or control phage were incubated with lung endothelial cells at 37 °C to allow for phage internalization. Cells were stained with an anti-bacteriophage antibody after removal of the membrane-bound phage. The upper panels show staining of permeabilized cells revealing the internalized phage. The lower panels show staining of nonpermeabilized cells, demonstrating the successful removal of phage from the cell surface. Scale bar, 100 μm. b, lung endothelial cells were incubated with increasing concentrations (up to 100 μm) of proapoptotic peptide synthesized in conjunction with the targeting CGSPGWVRC peptide (CGSPGWVRC-GG-D(KLAKLAK)2) or negative control peptides (an equimolar mixture of CGSPGWVRC and D(KLAKLAK)2). Cell viability was determined by optical absorbance using a cell proliferation detection reagent. Shown are means ± S.E. from triplicate wells. c, lung endothelial cells were incubated with 100 μm proapoptotic peptide CGSPGWVRC-GG-D(KLAKLAK)2 or negative control peptides (equimolar mixture of CGSPGWVRC and D(KLAKLAK)2) for 2 h. Induction of cell apoptosis was detected by Annexin-V-fluorescein isothiocyanate (FITC) binding (top and middle panels) or TUNEL (bottom, yellow arrows).

Mentions: Lung Endothelial Cell Binding Peptides Mediate Cell Internalization—We next set out to evaluate whether the ligand CGSPGWVRC peptide would mediate phage internalization into lung endothelial cells. CGSPGWVRC phage or control phage were incubated with either lung-derived or prostate-derived endothelial cells. Cells were then washed to remove noninternalized phage, permeabilized, and stained with an anti-bacteriophage antibody. A Cy3-conjugated secondary antibody was used to detect the presence and localization of phage particles. After 4 h at 37 °C, CGSPGWVRC phage particles were internalized into lung endothelial cells (Fig. 2a); only background staining was observed when control phage or nonpermeabilized cells were used as negative controls. In contrast, CGSPGWVRC phage was not internalized into prostate endothelial cells (data not shown). Internalization detection of CGSPGWVRC phage is time-dependent (weak at 30 min, moderate at 2-4 h, and strong at 8 h; data not shown). These data suggest that the CGSPGWVRC peptide can mediate the internalization of ligands such as phage into lung endothelial cells. In order to evaluate whether internalization of CGSPGWVRC-directed ligands can also occur outside of the phage context (i.e. the targeted delivery of pathobiologically relevant peptide), we generated and tested a synthetic chimeric peptide of the CGSPGWVRC ligand fused to the D(KLAKLAK)2 proapoptotic motif, an amphipathic α-helix-forming antimicrobial peptide that disrupts preferentially eukaryotic mitochondrial membranes upon ligand-directed internalization (25, 30-32). Increasing concentrations of the lung endothelial cell targeted CGSPGWVRC-GG-D(KLAKLAK)2 peptide or equimolar amounts of CGSPGWVRC plus D(KLAKLAK)2 were incubated with lung endothelial cells at 37 °C, and cell viability was assessed after 6 h. Treatment of lung endothelial cells resulted in a dose-dependent decrease in cell viability with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, whereas the equimolar combination of CGSPGWVRC plus D(KLAKLAK)2 peptides did not have any detectable effect on cell viability (Fig. 2b). The induction of endothelial cell apoptosis by CGSPGWVRC-GG-D(KLAKLAK)2 was further confirmed by the presence of two apoptotic markers (Annexin-V binding and TUNEL) (Fig. 2c). Taken together, these results indicate that the displayed CGSPGWVRC insert or the corresponding synthetic peptide can mediate ligand-directed internalization into lung endothelial cells both inside and outside of the phage particle context in a time- and dose-dependent manner and that internalization of the CGSPGWVRC-GG-D(KLAKLAK)2 proapoptotic peptide induces programmed cell death of the lung endothelial cells in vitro.


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 peptide mediates internalization of ligands into lung endothelial cells and cell apoptosis. a, CGSPGWVRC peptide mediates phage internalization into lung endothelial cells. CGSPGWVRC phage or control phage were incubated with lung endothelial cells at 37 °C to allow for phage internalization. Cells were stained with an anti-bacteriophage antibody after removal of the membrane-bound phage. The upper panels show staining of permeabilized cells revealing the internalized phage. The lower panels show staining of nonpermeabilized cells, demonstrating the successful removal of phage from the cell surface. Scale bar, 100 μm. b, lung endothelial cells were incubated with increasing concentrations (up to 100 μm) of proapoptotic peptide synthesized in conjunction with the targeting CGSPGWVRC peptide (CGSPGWVRC-GG-D(KLAKLAK)2) or negative control peptides (an equimolar mixture of CGSPGWVRC and D(KLAKLAK)2). Cell viability was determined by optical absorbance using a cell proliferation detection reagent. Shown are means ± S.E. from triplicate wells. c, lung endothelial cells were incubated with 100 μm proapoptotic peptide CGSPGWVRC-GG-D(KLAKLAK)2 or negative control peptides (equimolar mixture of CGSPGWVRC and D(KLAKLAK)2) for 2 h. Induction of cell apoptosis was detected by Annexin-V-fluorescein isothiocyanate (FITC) binding (top and middle panels) or TUNEL (bottom, yellow arrows).
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fig2: CGSPGWVRC peptide mediates internalization of ligands into lung endothelial cells and cell apoptosis. a, CGSPGWVRC peptide mediates phage internalization into lung endothelial cells. CGSPGWVRC phage or control phage were incubated with lung endothelial cells at 37 °C to allow for phage internalization. Cells were stained with an anti-bacteriophage antibody after removal of the membrane-bound phage. The upper panels show staining of permeabilized cells revealing the internalized phage. The lower panels show staining of nonpermeabilized cells, demonstrating the successful removal of phage from the cell surface. Scale bar, 100 μm. b, lung endothelial cells were incubated with increasing concentrations (up to 100 μm) of proapoptotic peptide synthesized in conjunction with the targeting CGSPGWVRC peptide (CGSPGWVRC-GG-D(KLAKLAK)2) or negative control peptides (an equimolar mixture of CGSPGWVRC and D(KLAKLAK)2). Cell viability was determined by optical absorbance using a cell proliferation detection reagent. Shown are means ± S.E. from triplicate wells. c, lung endothelial cells were incubated with 100 μm proapoptotic peptide CGSPGWVRC-GG-D(KLAKLAK)2 or negative control peptides (equimolar mixture of CGSPGWVRC and D(KLAKLAK)2) for 2 h. Induction of cell apoptosis was detected by Annexin-V-fluorescein isothiocyanate (FITC) binding (top and middle panels) or TUNEL (bottom, yellow arrows).
Mentions: Lung Endothelial Cell Binding Peptides Mediate Cell Internalization—We next set out to evaluate whether the ligand CGSPGWVRC peptide would mediate phage internalization into lung endothelial cells. CGSPGWVRC phage or control phage were incubated with either lung-derived or prostate-derived endothelial cells. Cells were then washed to remove noninternalized phage, permeabilized, and stained with an anti-bacteriophage antibody. A Cy3-conjugated secondary antibody was used to detect the presence and localization of phage particles. After 4 h at 37 °C, CGSPGWVRC phage particles were internalized into lung endothelial cells (Fig. 2a); only background staining was observed when control phage or nonpermeabilized cells were used as negative controls. In contrast, CGSPGWVRC phage was not internalized into prostate endothelial cells (data not shown). Internalization detection of CGSPGWVRC phage is time-dependent (weak at 30 min, moderate at 2-4 h, and strong at 8 h; data not shown). These data suggest that the CGSPGWVRC peptide can mediate the internalization of ligands such as phage into lung endothelial cells. In order to evaluate whether internalization of CGSPGWVRC-directed ligands can also occur outside of the phage context (i.e. the targeted delivery of pathobiologically relevant peptide), we generated and tested a synthetic chimeric peptide of the CGSPGWVRC ligand fused to the D(KLAKLAK)2 proapoptotic motif, an amphipathic α-helix-forming antimicrobial peptide that disrupts preferentially eukaryotic mitochondrial membranes upon ligand-directed internalization (25, 30-32). Increasing concentrations of the lung endothelial cell targeted CGSPGWVRC-GG-D(KLAKLAK)2 peptide or equimolar amounts of CGSPGWVRC plus D(KLAKLAK)2 were incubated with lung endothelial cells at 37 °C, and cell viability was assessed after 6 h. Treatment of lung endothelial cells resulted in a dose-dependent decrease in cell viability with CGSPGWVRC-GG-D(KLAKLAK)2 peptide, whereas the equimolar combination of CGSPGWVRC plus D(KLAKLAK)2 peptides did not have any detectable effect on cell viability (Fig. 2b). The induction of endothelial cell apoptosis by CGSPGWVRC-GG-D(KLAKLAK)2 was further confirmed by the presence of two apoptotic markers (Annexin-V binding and TUNEL) (Fig. 2c). Taken together, these results indicate that the displayed CGSPGWVRC insert or the corresponding synthetic peptide can mediate ligand-directed internalization into lung endothelial cells both inside and outside of the phage particle context in a time- and dose-dependent manner and that internalization of the CGSPGWVRC-GG-D(KLAKLAK)2 proapoptotic peptide induces programmed cell death of the lung endothelial cells in vitro.

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