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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 phage binds specifically to lung endothelial cells. a, CGSPGWVRC phage binding to primary mouse lung microvascular endothelial cells cultured at either 33 or 37 °C detected by the BRASIL method. b, CGSPGWVRC phage binds specifically to lung endothelial cells but not to endothelial cells derived from control organs (brain, prostate, bone marrow, or kidney). c, CGSPGWVRC phage binding ex vivo to single-cell suspensions prepared from mouse lungs. CGSPGWVRC phage did not bind to single-cell suspensions prepared from the control organ (spleen). d, mutation of certain residues within CGSPGWVRC (to alanine) abolishes phage binding to lung endothelial cells. Bone marrow endothelial cells served as the negative control. Mutant residues are color-coded. e, CGSPGWVRC phage binding to lung endothelial cells is inhibited in a dose-dependent manner by the cognate synthetic peptide. An unrelated cyclic control peptide did not affect the phage binding. Shown are means ± S.E. from triplicate samples. Insertless control phage served as a negative control phage in a-d. Fd-tet phage values were set to 1 in a-c.
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fig1: CGSPGWVRC phage binds specifically to lung endothelial cells. a, CGSPGWVRC phage binding to primary mouse lung microvascular endothelial cells cultured at either 33 or 37 °C detected by the BRASIL method. b, CGSPGWVRC phage binds specifically to lung endothelial cells but not to endothelial cells derived from control organs (brain, prostate, bone marrow, or kidney). c, CGSPGWVRC phage binding ex vivo to single-cell suspensions prepared from mouse lungs. CGSPGWVRC phage did not bind to single-cell suspensions prepared from the control organ (spleen). d, mutation of certain residues within CGSPGWVRC (to alanine) abolishes phage binding to lung endothelial cells. Bone marrow endothelial cells served as the negative control. Mutant residues are color-coded. e, CGSPGWVRC phage binding to lung endothelial cells is inhibited in a dose-dependent manner by the cognate synthetic peptide. An unrelated cyclic control peptide did not affect the phage binding. Shown are means ± S.E. from triplicate samples. Insertless control phage served as a negative control phage in a-d. Fd-tet phage values were set to 1 in a-c.

Mentions: Validation of CGSPGWVRC Phage Binding to Lung Endothelial Cells—We performed a comprehensive characterization of the binding profile of the CGSPGWVRC phage relative to appropriate controls (Fig. 1). First we evaluated whether temperature (a surrogate for the level of the large T antigen in the lung endothelial cells) affected the binding of the dominant CGSPGWVRC phage clone to ImmortoMouse-derived lung endothelial cells. We performed phage-binding experiments with CGSPGWVRC phage or insertless control phage (fd-tet) on lung endothelial cells cultured either at 33 °C (permissive temperature) or 37 °C (Fig. 1a). We found no significant difference in CGSPGWVRC phage binding to lung endothelial cells cultured at either temperature (143 ± 13-fold binding at 33 °C versus 119 ± 2-fold binding at 37 °C relative to binding of negative control phage; Student's t test, p = 0.35). These data allowed us to use proliferating endothelial cells (cultured at 33 °C) for the subsequent experiments. Next, we evaluated the tissue of origin specificity of CGSPGWVRC phage binding to the lung endothelial cells. CGSPGWVRC phage or control phage were incubated with immortal endothelial cells derived from brain, prostate, bone marrow, kidney, or lung (27). We observed markedly increased binding of CGSPGWVRC phage to lung endothelial cells relative to control phage; moreover, binding of CGSPGWVRC phage to endothelial cells derived from brain, prostate, bone marrow, or kidney was detected only at background levels when compared with control phage (Fig. 1b). To rule out the possibility that the observed tissue-specific binding of CGSPGWVRC phage was related to adaptation to cell culture conditions, we also tested the CGSPGWVRC phage binding ex vivo to single-cell suspensions prepared from mouse lung or a negative control tissue (mouse spleen was used unless otherwise specified). Consistently, we observed strong binding (118 ± 12-fold) of CGSPGWVRC phage to lung single-cell suspension relative to control phage binding to the same lung single-cell suspension; in contrast, no binding or only minimal binding (1.7 ± 0.1-fold) of CGSPGWVRC phage to spleen single-cell suspension was observed when CGSPGWVRC phage binding was compared with the control phage binding to spleen-derived single-cell suspension (Fig. 1c). In order to characterize the role of individual amino acid residues in CGSPGWVRC peptide binding to lung endothelial cells, we used site-directed mutagenesis. We generated different alanine-scanning versions of the displayed peptide by cloning mutant phage inserts (CGSAGWVRC, CGSPGAVRC, CGSPGWVAC, and CGSPGAVAC) and comparing binding of each mutant insert to insertless control phage. Mutation of the tryptophan residue to alanine abolished phage binding to lung endothelial cells, and mutation of the arginine residue to alanine reduced the phage binding to lung endothelial cells by 92%. Mutation of both tryptophan and arginine residues to alanine abolished phage binding to lung endothelial cells. Although the proline to alanine mutation decreased phage binding to lung endothelial cells by 61%, binding of CGSAGWVRC peptide-displaying phage to lung endothelial cells was still significant relative to control phage binding to lung endothelial cells (p < 0.001). These results suggest critical roles for the tryptophan and arginine residues within the ligand insert CGSPGWVRC. None of the mutant phage clones showed binding to a control endothelial cell line derived from bone marrow (Fig. 1d). Finally, we found that CGSPGWVRC phage binding to lung endothelial cells is mediated specifically by the peptide, since phage binding was inhibited by the cognate synthetic CGSPGWVRC peptide; an unrelated negative control cyclic peptide at the equivalent molar concentrations had no inhibitory effect (Fig. 1e). Together, these results show that the peptide CGSPGWVRC is a potent and specific ligand to lung-derived endothelial 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 phage binds specifically to lung endothelial cells. a, CGSPGWVRC phage binding to primary mouse lung microvascular endothelial cells cultured at either 33 or 37 °C detected by the BRASIL method. b, CGSPGWVRC phage binds specifically to lung endothelial cells but not to endothelial cells derived from control organs (brain, prostate, bone marrow, or kidney). c, CGSPGWVRC phage binding ex vivo to single-cell suspensions prepared from mouse lungs. CGSPGWVRC phage did not bind to single-cell suspensions prepared from the control organ (spleen). d, mutation of certain residues within CGSPGWVRC (to alanine) abolishes phage binding to lung endothelial cells. Bone marrow endothelial cells served as the negative control. Mutant residues are color-coded. e, CGSPGWVRC phage binding to lung endothelial cells is inhibited in a dose-dependent manner by the cognate synthetic peptide. An unrelated cyclic control peptide did not affect the phage binding. Shown are means ± S.E. from triplicate samples. Insertless control phage served as a negative control phage in a-d. Fd-tet phage values were set to 1 in a-c.
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Related In: Results  -  Collection

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fig1: CGSPGWVRC phage binds specifically to lung endothelial cells. a, CGSPGWVRC phage binding to primary mouse lung microvascular endothelial cells cultured at either 33 or 37 °C detected by the BRASIL method. b, CGSPGWVRC phage binds specifically to lung endothelial cells but not to endothelial cells derived from control organs (brain, prostate, bone marrow, or kidney). c, CGSPGWVRC phage binding ex vivo to single-cell suspensions prepared from mouse lungs. CGSPGWVRC phage did not bind to single-cell suspensions prepared from the control organ (spleen). d, mutation of certain residues within CGSPGWVRC (to alanine) abolishes phage binding to lung endothelial cells. Bone marrow endothelial cells served as the negative control. Mutant residues are color-coded. e, CGSPGWVRC phage binding to lung endothelial cells is inhibited in a dose-dependent manner by the cognate synthetic peptide. An unrelated cyclic control peptide did not affect the phage binding. Shown are means ± S.E. from triplicate samples. Insertless control phage served as a negative control phage in a-d. Fd-tet phage values were set to 1 in a-c.
Mentions: Validation of CGSPGWVRC Phage Binding to Lung Endothelial Cells—We performed a comprehensive characterization of the binding profile of the CGSPGWVRC phage relative to appropriate controls (Fig. 1). First we evaluated whether temperature (a surrogate for the level of the large T antigen in the lung endothelial cells) affected the binding of the dominant CGSPGWVRC phage clone to ImmortoMouse-derived lung endothelial cells. We performed phage-binding experiments with CGSPGWVRC phage or insertless control phage (fd-tet) on lung endothelial cells cultured either at 33 °C (permissive temperature) or 37 °C (Fig. 1a). We found no significant difference in CGSPGWVRC phage binding to lung endothelial cells cultured at either temperature (143 ± 13-fold binding at 33 °C versus 119 ± 2-fold binding at 37 °C relative to binding of negative control phage; Student's t test, p = 0.35). These data allowed us to use proliferating endothelial cells (cultured at 33 °C) for the subsequent experiments. Next, we evaluated the tissue of origin specificity of CGSPGWVRC phage binding to the lung endothelial cells. CGSPGWVRC phage or control phage were incubated with immortal endothelial cells derived from brain, prostate, bone marrow, kidney, or lung (27). We observed markedly increased binding of CGSPGWVRC phage to lung endothelial cells relative to control phage; moreover, binding of CGSPGWVRC phage to endothelial cells derived from brain, prostate, bone marrow, or kidney was detected only at background levels when compared with control phage (Fig. 1b). To rule out the possibility that the observed tissue-specific binding of CGSPGWVRC phage was related to adaptation to cell culture conditions, we also tested the CGSPGWVRC phage binding ex vivo to single-cell suspensions prepared from mouse lung or a negative control tissue (mouse spleen was used unless otherwise specified). Consistently, we observed strong binding (118 ± 12-fold) of CGSPGWVRC phage to lung single-cell suspension relative to control phage binding to the same lung single-cell suspension; in contrast, no binding or only minimal binding (1.7 ± 0.1-fold) of CGSPGWVRC phage to spleen single-cell suspension was observed when CGSPGWVRC phage binding was compared with the control phage binding to spleen-derived single-cell suspension (Fig. 1c). In order to characterize the role of individual amino acid residues in CGSPGWVRC peptide binding to lung endothelial cells, we used site-directed mutagenesis. We generated different alanine-scanning versions of the displayed peptide by cloning mutant phage inserts (CGSAGWVRC, CGSPGAVRC, CGSPGWVAC, and CGSPGAVAC) and comparing binding of each mutant insert to insertless control phage. Mutation of the tryptophan residue to alanine abolished phage binding to lung endothelial cells, and mutation of the arginine residue to alanine reduced the phage binding to lung endothelial cells by 92%. Mutation of both tryptophan and arginine residues to alanine abolished phage binding to lung endothelial cells. Although the proline to alanine mutation decreased phage binding to lung endothelial cells by 61%, binding of CGSAGWVRC peptide-displaying phage to lung endothelial cells was still significant relative to control phage binding to lung endothelial cells (p < 0.001). These results suggest critical roles for the tryptophan and arginine residues within the ligand insert CGSPGWVRC. None of the mutant phage clones showed binding to a control endothelial cell line derived from bone marrow (Fig. 1d). Finally, we found that CGSPGWVRC phage binding to lung endothelial cells is mediated specifically by the peptide, since phage binding was inhibited by the cognate synthetic CGSPGWVRC peptide; an unrelated negative control cyclic peptide at the equivalent molar concentrations had no inhibitory effect (Fig. 1e). Together, these results show that the peptide CGSPGWVRC is a potent and specific ligand to lung-derived endothelial 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