Patent Application
20100050276
Apoptosis, or programmed cell death, is an integral component of tissue remodeling, such as tissue remodeling that occurs during development. Perturbations in apoptosis, such as excessive apoptosis or ill-timed apoptosis, have been implicated in a variety of diseases and conditions. Currently, the underlying causes and consequences of many diseases and conditions that are consequent to cell death, including programmed cell death, are unknown despite the availability of animal models for these diseases and conditions. Thus, there is a need to develop new, improved and effective models, in particular animal models for use in the study of apoptosis in disease pathology, and to identify therapeutic compounds that inhibit apoptosis to thereby prevent or treat diseases and conditions.
The present invention generally relates to transgenic non-human animals and methods of producing transgenic non-human animals; methods of screening for a compound that inhibits apoptosis; and methods of identifying a cell that is capable of differentiating into a target cell employing the transgenic, non-human animals.
In an embodiment, the invention is a transgenic non-human animal whose genome comprises a stable integration of at least one transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein operably-linked to at least one tetracycline-inducible promoter, wherein at least one cell of the transgenic non-human animal that expresses the transgene undergoes apoptosis, and wherein the non-human animal is not a rat.
In another embodiment, the invention is a transgenic non-human animal whose genome comprises a stable integration of at least one first transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein operably-linked to at least one tetracycline-inducible promoter and at least one second transgene that includes at least one second nucleic acid sequence encoding at least one member selected from the group consisting of a reverse tetracycline responsive transactivator protein and a tetracycline responsive transactivator protein.
In a further embodiment, the invention is a recombinant nucleic acid comprising a nucleotide sequence having at least about 75% identity to SEQ ID NO: 4 operably-linked to a tetracycline-inducible promoter, wherein the tetracycline-inducible promoter includes at least seven copies of a tetracycline operator nucleic acid sequence and a cytomegalovirus minimal promoter nucleic acid sequence.
In an additional embodiment, the invention includes a method for producing a transgenic non-human animal, comprising the step of crossing a first transgenic non-human animal whose genome comprises a stable integration of at least one first transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein operably-linked to a tetracycline-inducible promoter with a second transgenic non-human animal whose genome comprises a stable integration of at least one second transgene that includes at least one second nucleic acid sequence encoding at least one reverse tetracycline responsive transactivator protein.
In another embodiment, the invention is a method of screening for a compound that inhibits Fas-ligand mediated apoptosis, comprising the step of assessing Fas-ligand mediated apoptosis in a transgenic non-human animal whose genome comprises a stable integration of at least one first transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein operably-linked to at least one tetracycline-inducible promoter and at least one second transgene that includes at least one second nucleic acid sequence encoding at least one reverse tetracycline responsive transactivator protein, wherein the Fas-ligand mediated apoptosis is in response to administration of the compound in combination with at least one member selected from the group consisting of a tetracycline and a tetracycline analog to the transgenic non-human animal.
In a further embodiment, the invention is a method of identifying a cell that is capable of differentiating into a target cell, comprising the steps of inducing apoptosis of a population of target cells in a transgenic non-human animal whose genome comprises a stable integration of at least one first transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein operably-linked to at least one tetracycline-inducible promoter and at least one second transgene that includes a second nucleic acid sequence encoding at least one member selected from the group consisting of a reverse tetracycline responsive transactivator protein and a tetracycline responsive transactivator protein, and wherein the first transgene and the second transgene are co-expressed in the target cells; introducing at least one cell into the transgenic non-human animal, wherein the cell is selected form the group consisting of a stem cell, a progenitor cell and a bone marrow-derived cell; and detecting differentiation of the cell into a phenotype characteristic of the target cell.
The transgenic non-human animals of the invention can be employed to study the role of apoptosis in various diseases and to identify compounds that inhibit apoptosis. Advantages of the claimed invention include, for example, improved methods of screening for compounds having therapeutic utility in the treatment of apoptosis-mediated conditions.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The features and other details of the invention, either as steps of the invention or as combinations of parts of the invention, will now be more particularly described and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention.
In an embodiment, the invention is a transgenic non-human animal whose genome comprises a stable integration of at least one transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand (Fas-L) protein operably-linked to at least one tetracycline-inducible promoter, wherein at least one cell of the transgenic non-human animal that expresses the transgene undergoes apoptosis.
The transgenic non-human animal can be a non-human mammal, provided that the mammal is not a rat. Exemplary non-human mammals include mice, guinea pigs, hamsters, rabbits, goats, sheep, cattle, and pigs. Methods of generating transgenic animals are known to one of skill in the art (see, for example, Pinkert, C. A., Transgenic Animal Technology, Second Edition: A Laboratory Handbook (2002), Elsevier Science, USA).
In an embodiment, the non-human animal is a mouse.
In an embodiment, the cell of the transgenic non-human animal that expresses the transgene and undergoes apoptosis can be a somatic cell (e.g., an epithelial cell or non-epithelial cell, such as a muscle cell, a nerve cell or a connective tissue cell). In another embodiment, the cell of the transgenic non-human animal that expresses the transgene and undergoes apoptosis can be a germ cell.
In an embodiment, the transgenic non-human animal is fertile (e.g., capable of reproducing). In a further embodiment, the transgenic non-human animal survives (e.g., does not die) when apoptosis is induced.
The transgenic non-human animal has a genome that includes the stable integration of between about two to about thirty-five (e.g., about twenty) copies of the transgene. For example, the transgenic non-human animal can have a genome that includes the stable integration of about 5, about 10, about 15, about 20, about 25, about 30 or about 35 copies of the transgene.
In an embodiment, the transgenic non-human animal has between about a 10-fold to about a 200-fold (e.g., about a 30-fold) increase in Fas-ligand mRNA levels compared to a control cell. For example, the transgenic non-human animal can have about a 10-fold, about a 20-fold, about a 30-fold, about a 40-fold, about a 50-fold, about a 100-fold, about a 150-fold or about a 200-fold increase in Fas-ligand mRNA levels compared to a control cell.
“Control cell,” as used herein, refers to a cell that includes a reference level of Fas-ligand mRNA. For example, a control cell can be a cell (e.g., an isolated cell) from another animal, such as an animal whose genome does not include the transgene encoding a Fas-ligand protein (e.g., a transgenic animal having the genotype rtTA+/(tetOp)7-FasL−); an animal whose genome includes the transgene encoding a Fas-ligand protein, wherein the animal has not been exposed to tetracycline or a tetracycline analog; an animal whose genome includes the transgene encoding a Fas-ligand protein, but does not include a gene encoding a reverse tetracycline responsive transactivator protein or a tetracycline responsive transactivator protein (e.g., an animal having the genotype tTA−/rtTA−/(tetOp)7-FasL+). Alternatively, or additionally, the control cell can be a cell (e.g., an isolated cell) that does not express the transgene from the same non-human transgenic animal, or a cultured cell (e.g., a tissue culture cell).
Methods of determining Fas-ligand mRNA levels in a biological sample are well known in the art and include, for example, Northern blotting and real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994)).
The transgene can include a nucleic acid sequence encoding at least one mammalian (e.g., murine, rat, human) Fas-ligand protein. Exemplary Fas-ligand protein sequences encoding mammalian Fas-ligand proteins include the following:
FasL protein (mouse): GenPept Accession no. P41047
FasL protein (rat): GenPept Accession no. P36940
FasL protein (human): GenPept Accession no. P48023
Exemplary cDNA sequences encoding mammalian Fas-ligand proteins include the following:
FasL cDNA(mouse): GenBank Accession no. NM—010177
FasL cDNA(rat): GenBank Accession No. NM—012908
FasL cDNA (human): GenBank Accession No. X89102
In an embodiment, the transgene can include a nucleic acid sequence that has at least about 75.0% (e.g., about 76.0%, about 93.0%) identity to a murine Fas-ligand cDNA comprising SEQ ID NO:4. For example, the transgene can include a nucleic acid sequence encoding a Fas-ligand protein has at least about 75.0%, about 80.0%, about 85.0%, about 90.0%, about 95.0% or about 99.0% identity to a murine Fas-ligand cDNA comprising SEQ ID NO:4.
The percent identity of two nucleic acid sequences (or two amino acid sequences) can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The amino acid sequence or nucleic acid sequences at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions×100). The length of the protein or nucleic acid that can be aligned for comparison purposes is at least about 95% of the length of the reference sequence, for example, the murine Fas-ligand cDNA sequence (SEQ ID NO:4) or murine Fas-ligand protein (SEQ ID NO:1).
The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A preferred, non-limiting example of such a mathematical algorithm is described in Karlin et al. (Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993)). Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.2) as described in Schaffer et al. (Nucleic Acids Res., 29:2994-3005 (2001)). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN; available at the Internet site for the National Center for Biotechnology Information) can be used. In one embodiment, the database searched is a non-redundant (NR) database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and an Extension of 1.
Another mathematical algorithm employed for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG (Accelrys, San Diego, Calif.) sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 is used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti (Comput. Appl. Biosci., 10: 3-5 (1994)); and FASTA described in Pearson and Lipman (Proc. Natl. Acad Sci USA, 85: 2444-2448 (1988)).
The percent identity between two amino acid sequences can also be accomplished using the GAP program in the GCG software package (Accelrys, San Diego, Calif.) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yet another embodiment, the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, San Diego, Calif.), using a gap weight of 50 and a length weight of 3.
The transgene encoding at least one Fas-ligand protein can be operably-linked to at least one tetracycline-inducible promoter. In an embodiment, the tetracycline-inducible promoter can include at least about seven copies of a tet operator nucleic acid sequence. For example, the tetracycline-inducible promoter can include at least about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 tet operator nucleic acid sequences. In another embodiment, the tetracycline-inducible promoter can include seven copies of the tet operator nucleic acid sequence. An exemplary tet-responsive promoter nucleic acid sequence that includes seven tet operator nucleic acid sequences is SEQ ID NO:7, which is contained in the pTRE2 vector (BD Biosciences, Franklin Lakes, N.J.).
The tetracycline-inducible promoter can further include a cytomegalovirus minimal promoter nucleic acid sequence. An exemplary a cytomegalovirus minimal promoter nucleic acid sequence is SEQ ID NO:8.
The transgene encoding at least one Fas-ligand protein can further include a polyadenylation nucleic acid sequence, such as, for example, a beta-globin polyadenylation contained in the pTRE2 vector (BD Biosciences, Franklin Lakes, N.J.):
In another embodiment, the invention is a transgenic non-human animal whose genome comprises a stable integration of at least one first transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein operably-linked to at least one tetracycline-inducible promoter and at least one second transgene that includes at least one second nucleic acid sequence encoding at least one member selected from the group consisting of a reverse tetracycline responsive transactivator protein and a tetracycline responsive transactivator protein.
In an embodiment, the second transgene includes at least one second nucleic acid sequence encoding a reverse tetracycline responsive transactivator (rtTA) protein. In another embodiment, the second transgene includes at least one second nucleic acid sequence encoding a tetracycline responsive transactivator (tTA) protein.
In an embodiment, the second transgene includes at least one second nucleic acid sequence encoding a reverse tetracycline responsive transactivator protein, and apoptosis is induced in cells of the animal that express both the first and second transgene when tetracycline (tet) or a tetracycline analog is present. Tetracycline analogs are well known and include, for example, naturally occurring (e.g., chlortetracycline, oxytetracycline, demclocycline) and semi-synthetic (e.g., doxycyline (Dox), lymecycline, meclocycline, methacycline, minocycline, rolitetracycline) analogs.
The nucleic acid sequence encoding the reverse tetracycline responsive transactivator protein or the tetracycline responsive transactivator protein in the second transgene can be operably linked to a cell-specific and/or tissue-specific promoter. Exemplary promoters include, for example, a pancreatic β-cell promoter, an amyloid precursor protein gene promoter, a dystrophin gene promoter, a Clara cell secretory protein gene promoter (e.g., a Clara cell secretory protein-C promoter), a surfactant protein-B gene promoter, a surfactant protein-C gene promoter, an insulin gene promoter, an albumin gene promoter, an alpha Calcium/Calmodulin dependent Protein Kinase II (CamKII) gene promoter, a neuron-specific enolase gene promoter, a retinoblastoma gene promoter, a muscle creatine kinase gene promoter, an alpha myosin heavy chain gene promoter, a TEK tyrosine kinase gene promoter, a Tie receptor tyrosine kinase gene promoter, an immunoglobulin (Ig) heavy chain enhancer, a CD34 gene promoter, an SM22alpha gene promoter, and a glial fibrillary acidic gene promoter.
In an embodiment, the second transgene includes the Clara cell secretory protein gene (e.g., Clara cell secretory protein C gene) promoter and the transgenic non-human animal has a phenotype that includes, for example, an alveolar type II cell apoptosis, a nonciliated bronchial epithelial cell apoptosis, a disrupted alveolar development, a decreased vascular density and/or a postnatal lethality (e.g., the animal dies during the postnatal period) consequent to apoptosis.
Standard techniques for assessing apoptosis are well known and include, for example, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end (TUNEL) labeling, ultrastructural analysis (e.g., electron microscopy) and detection of caspase (e.g., caspase-3) cleavage (e.g., by Western blot analysis), as described herein.
“Disrupted alveolar development,” as used herein, refers to alveolar simplification that resembles the pulmonary pathology of human bronchopulmonary dysplasia. Exemplary features of disrupted alveolar development include large simplified airspaces, a paucity of alveolar septation, and secondary crest formation. Disrupted alveolar development can be detected using standard techniques, such as, for example, microscopy, stereological volumetric techniques and morphometric analysis (e.g., computer-assisted morphometric analysis) of mean cord length (MCL) and radial alveolar count (RAC) in lung samples.
“Decreased vascular density,” as used herein, refers to a vessel density that is at least about 25% reduced in the lungs of the double transgenic non-human animal (i.e., the non-human transgenic animal whose genome comprises the first and second transgenes (e.g., an animal having the genotype CCSP-rtTA+/(tetOp)7-FasL+)) compared to a control animal (e.g., a single-transgenic littermate, such as an animal having the genotype CCSP-rtTA+/(tetOp)7-FasL−). Standard methods of measuring vascular density have been described (Balasubramaniam V, Mervis CF, Maxey A M, Markham N E, Abman SH: Hyperoxia reduces bone marrow, circulating, and lung endothelial progenitor cells in the developing lung: implications for the patho-genesis of bronchopulmonary dysplasia. Am J Physiol 2007, 292:L1073-L1084).
In another embodiment, the second transgene includes a pancreatic β-cell promoter (e.g., a mouse insulin gene promoter, a rat insulin gene promoter); and the transgenic non-human animal has a phenotype that resembles a diabetic condition consequent to apoptosis.
In a further embodiment, the second transgene includes an amyloid precursor protein gene promoter; and the transgenic non-human animal has a phenotype that resembles an Alzheimer's condition consequent to apoptosis.
In an additional embodiment, the second transgene includes a dystrophin gene promoter; and the transgenic non-human animal has a phenotype that resembles a muscular dystrophy condition consequent to apoptosis.
In another embodiment, the second transgene includes an alpha myosin heavy chain gene promoter; and the transgenic non-human animal has a phenotype that resembles a myocardial infarction consequent to apoptosis.
In another embodiment, the second transgene includes a surfactant protein B gene promoter or a surfactant protein C gene promoter; and the transgenic non-human animal has a phenotype that resembles lung injury consequent to apoptosis.
In an additional embodiment, the second transgene includes an albumin gene promoter; and the transgenic non-human animal has a phenotype that resembles liver failure consequent to apoptosis.
The second transgene in the transgenic non-human animals described herein can have at least one member selected from the group consisting of an alphaCaMKII gene promoter, a neuron-specific enolase gene promoter, a glial fibrillary acidic protein gene promoter and a retinoblastoma gene promoter; and the transgenic non-human animal can have a phenotype that resembles a neurodegenerative brain disorder consequent to apoptosis.
In another embodiment, the second transgene includes a muscle creatine kinase gene promoter; and the transgenic non-human animal has a phenotype that resembles a degenerative muscle disorder consequent to apoptosis.
In still another embodiment, the second transgene includes a TEK gene promoter or Tie gene promoter; and the transgenic non-human animal has a phenotype that resembles endothelial injury consequent to apoptosis.
In a further embodiment, the second transgene includes an immunoglobulin heavy chain enhancer; and the transgenic non-human animal has a phenotype that resembles aplastic anemia and/or bone marrow ablation consequent to apoptosis.
In an additional embodiment, the second transgene includes a CD34 gene promoter; and the transgenic non-human animal has a phenotype that resembles bone marrow ablation, aplastic anemia and/or a hematologic condition consequent to apoptosis.
In another embodiment, the second transgene includes an SM22 alpha gene promoter; and the transgenic non-human animal has a phenotype that resembles a condition associated with dissolution of vascular smooth muscle cells consequent to apoptosis.
The transgenic non-human animal can include at least one cell that co-expresses both the first transgene and the second transgene and subsequently undergoes apoptosis.
In an embodiment, the cell of the transgenic non-human animal that expresses a transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein and undergoes apoptosis is an epithelial tissue cell. For example, the epithelial tissue cell can be a lung epithelial cell, such as at least one member selected from the group consisting of a ciliated lung epithelial cell and a non-ciliated lung epithelial cell (e.g., a nonciliated bronchial epithelial cell). In another embodiment, the epithelial tissue cell can be an alveolar lung epithelial cell (e.g., a type II alveolar lung epithelial cell).
In an embodiment, the cell of the transgenic non-human animal that expresses a transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein and undergoes apoptosis is a cell from a tissue other than epithelium (also referred to as a “non-epithelial cell”). For example, in an embodiment, the cell is a connective tissue cell. Exemplary connective tissue cells include, for example, a chondrocyte.
In an additional embodiment, the cell of the transgenic non-human animal that expresses a transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein and undergoes apoptosis is a nervous tissue cell. Exemplary nervous tissue cells include, for example, a central nervous tissue cell (a brain cell or a spinal cord cell) and a peripheral nervous tissue cell.
In another embodiment, the cell of the transgenic non-human animal that expresses a transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein and undergoes apoptosis is a muscle tissue cell (e.g., a smooth muscle cell, a skeletal muscle cell, a cardiac muscle cell).
In yet another embodiment, the cell of the transgenic non-human animal that expresses a transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein and undergoes apoptosis is a retinal cell (e.g., a retinal epithelial cell, a retinal ganglion cell).
In another embodiment, the invention is a recombinant nucleic acid comprising a nucleotide sequence having at least about 75.0% (e.g., about 76.0%, about 93.0%) identity to a murine Fas-ligand cDNA comprising SEQ ID NO:4 operably-linked to a tetracycline-inducible promoter, wherein the tetracycline-inducible promoter includes at least seven copies of a tetracycline operator nucleic acid sequence and a cytomegalovirus minimal promoter nucleic acid sequence. For example, the recombinant nucleic acid can include a nucleic acid sequence encoding a Fas-ligand protein having at least about 75.0%, about 80.0%, about 85.0%, about 90.0%, about 95.0% or about 99.0% identity to SEQ ID NO:4.
The recombinant nucleic acid also can include at least about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 tet operator nucleic acid sequences. In an embodiment, the tetracycline-inducible promoter can include seven copies of the tet operator nucleic acid sequence (e.g., SEQ ID NO:7).
The recombinant nucleic acid can further include a cytomegalovirus minimal promoter nucleic acid sequence. An exemplary cytomegalovirus minimal promoter nucleic acid sequence is SEQ ID NO:8.
In addition, the recombinant nucleic acid can include a polyadenylation nucleic acid sequence (e.g., SEQ ID NO:9).
In an embodiment, the recombinant nucleic acid can include SEQ ID NO: 15, which includes the full-length mouse FasL cDNA and restriction sites used to generate the FasL transgene construct described herein:
In an additional embodiment, the recombinant nucleic acid can include a vector nucleic acid sequence (e.g., a pTRE2 vector (BD Biosciences, Franklin Lakes, N.J.) nucleic acid sequence).
In a further embodiment, the invention is a method for producing a transgenic non-human animal, comprising the step of crossing a first transgenic non-human animal whose genome comprises a stable integration of at least one first transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein operably-linked to a tetracycline-inducible promoter with a second transgenic non-human animal whose genome comprises a stable integration of at least one second transgene that includes at least one second nucleic acid sequence encoding at least one reverse tetracycline responsive transactivator protein.
In an embodiment, at least one cell of the transgenic non-human animal that co-expresses both the first transgene and the second transgene undergoes apoptosis.
The first and second transgenic non-human animals employed in the methods described herein can have a genotype that is hemizygous or homozygous for their respective transgenes.
In an embodiment, both the first transgenic non-human animal and second transgenic non-human animal are mice, such as mice with an FVB/N genetic background.
When the first transgenic non-human animal and second transgenic non-human animal are mice, the second transgenic mouse can be a tet-activator mouse, including mouse lines listed in Table 1.
β-cells of
In an additional embodiment, the invention is a method of screening for a compound that inhibits Fas-ligand mediated apoptosis, comprising the step of assessing Fas-ligand mediated apoptosis in a transgenic non-human animal whose genome comprises a stable integration of at least one first transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein operably-linked to at least one tetracycline-inducible promoter and at least one second transgene that includes at least one second nucleic acid sequence encoding at least one reverse tetracycline responsive transactivator protein, wherein the Fas-ligand mediated apoptosis is in response to administration of the compound in combination with at least one member selected from the group consisting of a tetracycline and a tetracycline analog to the transgenic non-human animal.
Exemplary compounds for use in the method include, for example, small molecules (e.g., small organic molecules, small inorganic molecules), peptides, peptidomimetics, polypeptides (e.g., fusion proteins, antibodies, antibody fragments), nucleic acids (e.g., siRNA, aptamers). In different embodiments, the compound can be FasL siRNA, caspase (e.g., caspase-3, caspase-6) siRNA, caspase small molecule inhibitors, or a FasL fusion protein.
In another embodiment, the invention is a method of screening for a compound that promotes Fas-ligand mediated apoptosis, comprising the step of assessing Fas-ligand mediated apoptosis in a transgenic non-human animal whose genome comprises a stable integration of at least one first transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein operably-linked to at least one tetracycline-inducible promoter and at least one second transgene that includes at least one second nucleic acid sequence encoding at least one reverse tetracycline responsive transactivator protein, wherein the Fas-ligand mediated apoptosis is in response to administration of the compound in combination with at least one member selected from the group consisting of a tetracycline and a tetracycline analog to the transgenic non-human animal.
In a further embodiment, the invention is a method of identifying a cell that is capable of differentiating into a target cell, comprising the steps of inducing apoptosis of a population of target cells in a transgenic non-human animal whose genome comprises a stable integration of at least one first transgene that includes at least one nucleic acid sequence encoding at least one Fas-ligand protein operably-linked to at least one tetracycline-inducible promoter and at least one second transgene that includes a second nucleic acid sequence encoding at least one member selected from the group consisting of a reverse tetracycline responsive transactivator protein and a tetracycline responsive transactivator protein, and wherein the first transgene and the second transgene are co-expressed in the target cells; introducing at least one cell into the transgenic non-human animal, wherein the cell is selected form the group consisting of a stem cell, a progenitor cell and a bone marrow-derived cell; and detecting differentiation of the cell into a phenotype characteristic of the target cell.
Exemplary target cells include pancreatic β-cells, cardiac muscle cells, skeletal muscle cells, neurons, glial cells and lung epithelial cells.
In an embodiment, the cell introduced into the transgenic non-human animal can be a stem cell. The stem cell can be a bone marrow-derived stem cell, such as a hematopoietic stem cell (e.g., a hemocytoblast) or a nonhematopoietic stem cell (e.g., a bone marrow-derived mesenchymal cell, a bone marrow-derived stromal cell, a bone marrow-derived macrophage cell, a bone marrow-derived dendritic cell).
In another embodiment, the cell introduced into the transgenic non-human animal can be a progenitor cell. Exemplary progenitor cells include a satellite cell, an intermediate progenitor cell, a neural progenitor cell, a periosteal cell, a pancreatic progenitor cell, and endothelial progenitor cell.
Differentiation of the cell into a phenotype characteristic of the target cell can be determined by standard techniques (e.g., microscopy, histological evaluation, immunocytochemical analysis).
In an embodiment, differentiation of the cell into a phenotype characteristic of the target cell can be determined, for example, by detecting repopulation of the target cells in the animal.
Premature infants are at risk for bronchopulmonary dysplasia, a complex condition characterized by impaired alveolar development and increased alveolar epithelial apoptosis. The functional involvement of pulmonary apoptosis in bronchopulmonary dysplasia-associated alveolar disruption remains undetermined. The aims of this study were to generate conditional lung-specific Fas-ligand (FasL) transgenic mice and to determine the effects of FasL-induced respiratory epithelial apoptosis on alveolar remodeling in postcanalicular lungs. Transgenic (TetOp)7-FasL responder mice, generated by pronuclear microinjection, were bred with Clara cell secretory protein (CCSP)-rtTA activator mice. Doxycycline (Dox) was administered from embryonal day 14 to postnatal day 7, and lungs were studied between embryonal day 19 and postnatal day 21. Dox administration induced marked respiratory epithelium-specific FasL mRNA and protein up-regulation in double-transgenic CCSP-rtTA+/(TetOp)7-FasL+ mice compared with single-transgenic CCSP-rtTA+ littermates. The Dox-induced FasL up-regulation was associated with dramatically increased apoptosis of alveolar type II cells and Clara cells, disrupted alveolar development, decreased vascular density, and increased postnatal lethality. The data described herein demonstrate that FasL-induced alveolar epithelial apoptosis during postcanalicular lung remodeling is sufficient to disrupt alveolar development after birth. The availability of inducible lung-specific FasL transgenic mice will facilitate studies of the role of apoptosis in normal and disrupted alveologenesis and may lead to novel therapeutic approaches for perinatal and adult pulmonary diseases characterized by dysregulated apoptosis.
Preterm infants who require assisted ventilation and supplemental oxygen are at risk for bronchopulmonary dysplasia (BPD), a chronic lung disease of newborn infants associated with significant mortality and long-term morbidity.1,2 The pathological hallmark of BPD in the postsurfactant era is an impairment of alveolar development, resulting in large and simplified airspaces that show little evidence of vascularized ridges (secondary crests) or alveolar septa.3,4 In addition, the lungs of infants with BPD show structurally abnormal microvasculature and variable degrees of interstitial fibrosis.5,6 The BPD currently observed in extremely premature infants has been termed “new” BPD to differentiate this condition from the pathologically and epidemiologically distinct historical BPD, originally described in the late 1960s by Northway and colleagues.7 The latter occurred in less premature infants and was characterized by more severe patterns of acute lung and airway injury.
Many risk factors have been implicated in the pathogenesis of BPD. Among these, prematurity, oxygen toxicity, and barotrauma are considered central to a final common outcome.1,8 There are variable contributions of infection/inflammation, glucocorticoid exposure, chorioamnionitis, and genetic polymorphisms.1,9 The precise mechanisms whereby these predisposing conditions result in disrupted alveolar development remain primarily unknown.
Our research efforts in recent years have focused on the role of alveolar epithelial apoptosis in postcanalicular alveolar development. It has been demonstrated that moderate and precisely timed alveolar epithelial type II cell apoptosis is an integral component of physiological postcanalicular lung remodeling in mice, rats, and rabbits. Although the exact biological role of apoptosis in alveologenesis remains uncertain, its choreographed occurrence across mammalian species strongly suggests apoptotic elimination of surplus type II cells during perinatal alveolar remodeling is a naturally occurring and developmentally relevant event.
Although moderate and well timed apoptosis appears to represent a physiological phenomenon during postcanalicular lung development, exaggerated and/or premature alveolar epithelial apoptosis may play a critical role in the pathogenesis of BPD-associated alveolar disruption. Several recent reports described increased levels of alveolar epithelial apoptosis in the lungs of ventilated preterm infants with respiratory distress syndrome or early BPD.15-17 The temporal patterns of apoptosis and alveolar disruption in ventilated preterm lungs are suggestive of a causative relationship; however, functional involvement of alveolar epithelial apoptosis in disrupted alveologenesis has not been demonstrated thus far.
As described herein, a gain-of-function approach was used to determine the functional role of alveolar epithelial apoptosis in alveolar remodeling. Enhanced respiratory epithelial apoptosis was achieved by means of a transgenic Fas-ligand (FasL) overexpression system. The Fas/FasL receptor-mediated death-signaling pathway is one of the better characterized apoptotic signaling systems.18-20 Stimulation of the Fas receptor (CD95/APO1), a member of the tumor necrosis factor receptor superfamily, by its natural ligand FasL or by Fas-activating antibody ligands results in its trimerization and the recruitment of two key signaling proteins, the adapter protein Fas-associated death domain and the initiator cysteine protease caspase-8. Subsequent activation of the effector caspases through mitochondria-dependent or -independent pathways results in activation of caspase-3, the key effector caspase. Activated caspase-3 cleaves a variety of substrates, including DNA repair enzymes, cellular and nuclear structural proteins, endonucleases, and many other cellular constituents, culminating in effective cell death.18-21
Selection of the Fas/FasL system as inducer of alveolar epithelial apoptosis for this study was a logical choice. First, perinatal murine respiratory epithelial cells were previously demonstrated to be exquisitely sensitive to Fas-mediated apoptosis in vitro and in vivo.10,22 Furthermore, the Fas/FasL signaling pathway lends itself better to experimental manipulation than other, in particular intrinsic (mitochondrial-dependent) pathways. Finally, the Fas/FasL system has been implicated as critical regulator of alveolar type II cell apoptosis in physiological alveolar remodeling10,11 and in various clinical and experimental models of adult lung injury.23-33 It is therefore conceivable that Fas/FasL signaling may play an important role in BPD-associated apoptosis as well.
The in vivo effect of Fas-activation in perinatal lungs was previously tested by systemic administration of a Fas-activating antibody to newborn mice.10 This approach allowed us to demonstrate that perinatal alveolar epithelial cells are susceptible to Fas-mediated apoptosis.10 However, systemic Fas activation resulted in rapid death from liver failure before the effects of exaggerated alveolar apoptosis on alveolar remodeling could be ascertained. To circumvent the deleterious effects of prolonged and systemic FasL exposure, a tetracycline-inducible (tet-on) lung epithelial-specific FasL-overexpressing mouse was generated, adapted from the Tet system of Gossen and colleagues,34 to target apoptosis to respiratory epithelial cells during perinatal lung development.
The results described herein demonstrate that increased apoptosis of respiratory epithelial cells during postcanalicular alveolar remodeling is sufficient to disrupt alveolar development and results in BPD-like alveolar simplification. These findings support our hypothesis that excessive or premature postcanalicular alveolar epithelial apoptosis is a pivotal event in the pathogenesis of BPD. Elucidation of the role and regulation of postcanalicular alveolar epithelial apoptosis may result in important insights into the regulation of alveologenesis and the pathogenesis of BPD. This, in turn, may open new therapeutic opportunities for the prevention and treatment of this disease, as well as other pulmonary conditions associated with dysregulated alveolar epithelial apoptosis, such as acute lung injury, emphysema, and neoplasia.
Materials and Methods
Generation of a Tetracycline-Dependent Respiratory Epithelium-Specific FasL
The tetracycline-inducible system in vivo consists of two independent transgenic mouse lines, an activator line and a responder line. The activator line expresses the reverse tetracycline responsive transactivator (rtTA) in a tissue- or cell-specific manner, whereas the responder line carries a transgene of interest under control of the tet-operator (TetOp). A tet-on tetracycline dependent overexpression system was selected to achieve conditional respiratory epithelium-specific FasL transgene expression. In the tet-on system, transgene expression is induced by binding of the tetracycline analogue, doxycycline (Dox) to rtTA, which in turn activates the (tetOp)7-CMV target promoter, activating transcription of the gene of interest.34,35 Tetracycline-dependent transgene expression was targeted to respiratory epithelial cells by using transgenic CCSP-rtTA activator mice in which the rtTA is placed under control of Clara cell secretory protein (CCSP, CC-10) promoter elements.36,37 The specific CCSP-rtTA transactivator mice used for these studies have been shown previously to be robust activators that effectively drive transgene expression not only in nonciliated bronchial epithelial (Clara) cells, but also in alveolar type II cells.36-42 The 2.3-kb rat CCSP promoter element used in these activator mice is thus expressed differently from the native murine CCSP gene, which is limited to Clara cells.43 Consistent with the endogenous expression pattern of CCSP, which is active from embryonal day 12.5 (E12.5) onward, CCSP-rtTA activator mice have been shown to be particularly suitable for studies of gene function in late gestation and postnatal lungs.37 CCSP-rtTA activator mice, generated in a FVB/N background, were a generous gift from Dr. J. Whitsett37,38 (Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio).
To generate (tetOp)7-FasL responder mice, the 943-bp murine FasL cDNA containing the entire coding region of the protein, a kind gift from Dr. S. Nagata44 (Osaka University Medical School, Osaka, Japan), was subcloned between a CMV minimal promoter and bovine growth hormone intronic and polyadenylation sequences in the pTRE2 vector (BD Biosciences, Franklin Lakes, N.J.) (
Animal Husbandry and Tissue Processing
Transgenic (tetOp)7-FasL progeny derived from founders A through E were crossed with CCSP-rtTA mice to yield a mixed offspring of double-transgenic (CCSP-rtTA+/(tetOp)7-FasL+) and single-transgenic (CCSP-rtTA+/(tetOp)7-FasL−) littermates. For the sake of brevity, double-transgenic mice will be denoted in the text as CCSP+/FasL+ mice, whereas single-transgenic mice will be denoted as CCSP+/FasL− mice.
Dox (1.0 mg/ml) was added to the drinking water of pregnant and nursing dams between E14 and postnatal day 7 (P7). The progeny (CCSP+/FasL+ and CCSP+/FasL−) were sacrificed at E19, P7 (early alveolarization stage45), or P21 (late alveolarization stage) by pentobarbital overdose. The cages were inspected twice daily to record interval postnatal death. Body and wet lung weights were recorded. Pups were genotyped by PCR analysis of tail genomic DNA using the primers described above. For molecular analysis, lungs were snap-frozen in liquid nitrogen and stored at −80° C. For morphological studies, fetal lungs were immersion-fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline, pH 7.4. The lungs of newborn mice were fixed by tracheal instillation of paraformaldehyde at a constant pressure of 20 cm H2O. After overnight fixation, the lungs were dehydrated in graded ethanol solutions, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Controls consisted of Dox-treated single-transgenic CCSP+/FasL− littermates, and age-matched CCSP+/FasL+ and CCSP+/FasL− animals that were not treated with Dox. All animal experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals. Protocols were approved through the Institutional Animal Care and Use Committee.
Alveolar Type II Cell Isolation and Culture
Alveolar type II cells were isolated from fetal mice (E19) by a modification of the methods described by Corti and colleagues46 and Rice and colleagues,47 as described in detail elsewhere.10 Briefly, type II cells were isolated by protease digestion and differential adherence to CD45- and CD32-coated dishes. After isolation and purification, the cells were resuspended in culture medium (HEPES-buffered Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin). Purity was assessed by modified Papanicolaou stain48 and anti-SP-C immunohistochemistry.10 Viability was assessed by trypan blue exclusion. After 24 hours, cells were assayed for FasL mRNA expression as detailed below.
Analysis of FasL Gene Expression
Quantitative Real-Time Polymerase Chain Reaction Analysis FasL mRNA levels were quantified by real-time PCR analysis. Total cellular RNA was extracted from whole lung or cell lysates using Trizol reagent (Invitrogen, Carlsbad, Calif.). Total RNA (2 μg) was DNase-treated (Turbo DNA-free kit; Ambion, Austin, Tex.) and reverse-transcribed using the reverse transcriptase2 first strand kit (SuperArray BioScience) according to the manufacturer's protocols. The cDNA templates were amplified with mouse β-actin (Superarray catalog no. PPM02945A) and FasL (PPM02926A) primer pairs in independent sets of PCR using reverse transcriptase.2 Real-time SYBR Green PCR master mix (Superarray) on an Eppendorf Mastercycler ep realplex (Westbury, N.Y.) according to the manufacturer's protocols. Each sample was run in triplicate, and mRNA levels were analyzed relative to the β-actin housekeeping gene. Relative gene expression ratios were calculated according to the SuperArray-recommended ΔΔCt protocol.49
Immunohistochemical Analysis
The cellular distribution of FasL protein in lung tissues was studied by the streptavidin-biotin immunoperoxidase method using a polyclonal rabbit anti-FasL antibody (Chemicon/Millipore, Billerica, Mass.).10,11 Immunoreactivity was detected with 3,3′-diaminobenzidine tetrachloride. Specificity controls consisted of omission of primary antibody.
Analysis of Apoptosis
Terminal Deoxynucleotidyl Transferase-Mediated dUTP-FITC Nick-End (TUNEL) Labeling
Pulmonary apoptotic activity was localized and quantified by TUNEL labeling, as previously described.10,11 Negative controls for TUNEL labeling were performed by omission of the transferase enzyme. For quantification of TUNEL signals, a minimum of 25 high-power fields were viewed per sample, and the number of apoptotic nuclei per high-power field [apoptotic index (Al)] was recorded. To assess alveolar type II cell apoptosis, TUNEL labeling was combined with immunohistochemical detection of type II cells using a polyclonal anti-prosurfactant protein C (SP-C) antibody (Abcam Inc., Cambridge, Mass.), as described.11,12 To evaluate Clara cell apoptosis, TUNEL labeling was combined with immunohistochemical detection of Clara cells using a polyclonal anti-CCSP antibody (Upstate Biotechnology, Lake Placid, N.Y.). Negative controls included omission of the primary antibody, which abolished all staining.
Electron Microscopy
For ultrastructural studies, lung samples were fixed with 1.25% glutaraldehyde in 0.15 mol/L sodium cacodylate buffer, postfixed with 1% osmium tetroxide, and dehydrated through a graded ethanol series. Ultrathin sections were stained with uranyl acetate/lead citrate and viewed using a Philips 300 electron microscope (Philips, Research, Eindhoven, The Netherlands).
Western Blot Analysis of Caspase-3 Cleavage Processing and cleavage of the key Fas-dependent executioner caspase, caspase-3, was assayed by Western blot analysis of lung homogenates, as described elsewhere.10,22 Caspase-3 is expressed as an inactive 32-kDa precursor from which the 17-kDa and 20-kDa subunits of the mature caspase-3 are proteolytically generated during apoptosis. The rabbit polyclonal anticaspase-3 antibody used (Cell Signaling, Danvers, Mass.) detects the 17- and 20-kDa cleavage products generated during apoptosis as well as the 32-kDa precursor caspase. Secondary goat antibody was conjugated with horseradish peroxidase and blots developed with an enhanced chemiluminescence detection assay (Amersham Pharmacia Biotech, Piscataway, N.J.). Band intensity was expressed as the combined integrated optical density of the 17- and 20-kDa bands, normalized to the integrated optical density of actin bands (loading control). Specificity controls included preincubation of antibody with blocking peptide.
Analysis of Lung Growth, Alveolarization, and Microvascular Development
Lung growth was assessed by lung weight and lung weight/body weight ratio. In lungs processed for molecular analyses, wet lung weight was recorded. For morphological and morphometric studies, lungs were formalin-fixed by standardized tracheal instillation in situ and lung growth was assessed by determination of inflated lung weight. Morphometric assessment of growth of peripheral air-exchanging lung parenchyma and contribution of the various lung compartments (airspace versus parenchyma) to the total lung volume was performed using standard stereological volumetric techniques, as previously described.50,51 The inflated lung volume, V(lu), was determined according to the Archimedes principle.52 The areal density of air-exchanging parenchyma, AA(ae/lu), was determined by point-counting based on computer assisted image analysis. The number of points falling on air-exchanging parenchyma (peripheral lung parenchyma excluding airspace) in random lung fields was divided by the number of points falling on the entire field (tissue and airspace). AA(ae/lu) represents the tissue fraction of the lung and as such is the complement of the airspace fraction AA(air/lu). The total volume of air-exchanging parenchyma, V(ae), was calculated by multiplying AA(ae/lu) by V(lu). Alveolarization was quantified by computer-assisted morphometric analysis of the mean cord length (MCL) and radial alveolar count (RAC). MCLs were determined by superimposing randomly oriented parallel arrays of lines across randomly selected microscope fields of air-exchanging lung parenchyma (at least 25 random fields per lung) and determining the distance between airspace walls (including alveoli, alveolar sacs, and ducts). The MCL is an indirect estimate of the degree of airspace subdivision by alveolar septa.41 The RAC was determined by counting the number of septa intersected by a perpendicular line drawn from the center of a respiratory bronchiole to the edge of the acinus (connective tissue septum or pleura),53 based on analysis of at least 10 randomly selected lung fields. The MCL was used to calculate mean volume of airspace units using the following formula: (MCL3×π)/3. The internal surface area of the lung available for gas exchange was calculated from the formula [(4×V(lu)]/MCL (adapted from Weibel and Cruz-Orive54) and normalized to body weight to obtain the specific internal surface area. All morphometric assessments were made on coded slides from at least six animals per group by a single observer who was unaware of the genotype or experimental condition of the animal analyzed. For morphometric analysis of vessel density, sections were immunostained for the presence of Factor VIII [von Willebrand factor (vWF)] (DAKO, Carpinteria, Calif.), an endothelium-specific marker. The number of Factor VIII-positive vessels (20 to 80 μm in diameter) per high-power field (×20 objective) was counted in 25 randomly selected fields to assess the vessel density, as described by others.55
Data Analysis
Values are expressed as mean±SD or, where appropriate, as mean±SEM. The significance of differences between groups was determined with the unpaired Student's t-test or analysis of variance with posthoc Scheffé test where indicated. The significance level was set at P<0.05. Statview software (Abacus, Berkeley, Calif.) was used for all statistical work.
Results
Generation of (tetOp)7-FasL Transgenic Mice
Pronuclear microinjection of the (tetOp)7-FasL construct yielded 36 live pups that were screened by PCR. Five transgenic lines (A to E) were established successfully; the transgene copy number of these lines ranged from 2 to 35. The following studies are based on transgenic mouse line D, which has 20 transgene copies and showed intermediate levels of Dox-induced FasL mRNA up-regulation.
Dox Administration Induces Lung-Specific FasL Overexpression in Bitransgenic CCSP+/FasL+ Mice
Pulmonary FasL mRNA Expression
Transgenic (tetOp)7-FasL responder mice (male or female) were crossed with transgenic CCSP-rtTA activator mice of the opposite gender to obtain litters composed of double-transgenic CCSP+/FasL+ and single-transgenic CCSP+/FasL− progeny. Dox was administered to pregnant, and subsequently, nursing dams from E14 to P7. The effect of Dox administration on FasL mRNA expression was studied by quantitative real-time PCR analysis of whole lung homogenates at P7. As shown in
To verify that the CCSP promoter construct is effective in inducing transgene expression in alveolar type II cells, FasL mRNA levels were determined in primary alveolar type II cells isolated from Dox-treated CCSP+/FasL+ and CCSP+/FasL− mice at E19. As seen in
FasL Immunohistochemistry
The cellular distribution of FasL protein was determined by immunohistochemical analysis (
FasL Overexpression in Dox-Treated Bitransgenic CCSP+/FasL+ Mice Is Associated with Increased Postnatal, but Not Fetal Lethality
To assess the effects of FasL transgene overexpression on antenatal and postnatal viability, the proportions of double-transgenic CCSP+/FasL+ and single-transgenic CCSP+/FasL− progeny at E19 and at P7 were determined. CCSP-rtTA mice are homozygous and (TetOp)7-FasL mice hemizygous for their respective transgenes. According to Mendelian laws of inheritance, equal proportions of double-transgenic CCSP+/FasL+ and single-transgenic CCSP+/FasL− progeny would be expected in the absence of a lethal effect.
At E19 (late gestation), the ratios of double- and single-transgenic Dox-treated fetuses were approximately equal, indicating that the CCSP+/FasL+ double-transgenic status does not confer lethal effects in utero (Table 4). At P7, however, CCSP+/FasL+ pups accounted for only 30% of Dox-treated progeny, suggestive of increased postnatal lethality in Dox-exposed double-transgenic mice (Table 5). The body weight of Dox-treated double-transgenic mice at P7 tended to be less than that of single-transgenic littermates, whereas their lung weight tended to be larger (Table 5). This resulted in significantly larger lung weight/body weight ratios in Doxtreated double-transgenic mice compared with single-transgenic littermates. In the absence of Dox, the ratios of double- and single-transgenic progeny at P7 were equal, indicating that the double-transgenic CCSP+/FasL+ status is not deleterious without Dox-exposure. The body weights of Dox-treated transgenic animals were significantly lower than those of non-Dox-treated animals of the same genotype, suggesting that Dox affects postnatal somatic growth (Table 5). The body weights of Doxtreated or non-Dox-treated pups were not affected by the maternal genotype (CCSP-rtTA versus (tetOp)7-FasL).
3.66 ± 0.27 (8)‡
3.65 ± 0.17 (8)‡
6.15 ± 0.73 (11)†
4.98 ± 0.09 (3)‡
4.77 ± 0.25 (3)‡
†P < 0.01 versus CCSP+/FasL+ littermates;
‡P < 0.01 versus Dox-treated animals of same genotype (Student's t-test).
FasL Overexpression in Dox-Treated Bitransgenic CCSP+/FasL+ Mice
Induces Increased Pulmonary Apoptosis
Lung Morphology and TUNEL Analysis at E19 and P7
Lungs of double-transgenic CCSP+/FasL+ mice treated with Dox from E14 on and examined at E19 and P7 showed abundant cellular debris and detached apoptotic cells within the airspaces (
TUNEL labeling highlighted the dramatic increase in apoptotic activity in lungs of Dox-treated double-transgenic mice (
Interval studies determined that virtually all (>95%) Dox-exposed mice that died between birth and P7 had the double-transgenic CCSP+/FasL+ genotype. Histopathological studies of the lungs of these animals showed that their airspaces were massively occluded by cellular debris admixed with apoptotic nuclear material (not shown). Based on this pathological evidence and the gross appearance of the moribund pups, the disproportionate postnatal lethality of Dox-treated CCSP+/FasL+ mice was attributed to respiratory failure.
TUNEL Analysis Combined with Anti-SP and Anti-CCSP Immunolabeling at E19 and P7
To determine the identity of the apoptotic cells, TUNEL labeling was combined with anti-SP-C or anti-CCSP immunohistochemistry. At E19, lungs of Dox-treated double-transgenic CCSP+/FasL+ mice showed frequent association of TUNEL-positive nuclei with SP-C immunoreactive cellular material within the intra-alveolar debris, suggesting that a large proportion of apoptotic cells were type II cells (
At P7, abundant apoptotic cellular debris remained present within the airspaces of Dox-treated CCSP+/FasL+ mice (
TUNEL labeling combined with anti-CCSP immunostaining at P7 demonstrated increased numbers of TUNEL-positive nuclei in the bronchial epithelium of double-transgenic CCSP+/FasL+ mice, both in CCSP-positive Clara cells and in neighboring CCSP-negative bronchial epithelial cells (
Electron Microscopy
Ultrastructural analysis of the lungs of Dox-treated single-transgenic CCSP+/FasL− mice (E19) revealed frequent, well preserved alveolar type II cells that were readily identified by their cuboidal shape, prominent microvilli, and the presence of cytoplasmic lamellar bodies and glycogen pools (
In contrast, the lungs of Dox-treated double-transgenic CCSP+/FasL+ littermates showed a striking paucity of recognizable alveolar type II cells. Instead, these lungs contained large aggregates of detached cells within the airspaces that were often associated with tubular myelin-like material. These detached intra-alveolar cells showed the characteristic ultrastructural features of apoptosis, including cell shrinkage and peripheral or diffuse chromatin condensation of the nuclei (
Western Blot Analysis of Caspase-3 Cleavage
Processing and cleavage of caspase-3, the main executioner of the Fas-dependent apoptotic machinery, was assessed by Western blot analysis using an antibody specific for both procaspase-3 and the active caspase-3 cleavage products. Consistent with Fas-mediated cell death, Dox-exposed CCSP+/FasL+ lungs showed cleavage of procaspase-3 and increased levels of the immunoreactive 17- and 20-kDa active subunits of caspase-3 (
FasL−Induced Alveolar Epithelial Apoptosis Disrupts Alveolar and Microvascular Development in Dox-Treated Bitransgenic CCSP+/FasL+ Mice
To determine the effects of FasL-induced apoptosis on alveolar remodeling, lungs of transgenic mice were studied at P21 after Dox administration from E14 to P7. The ratios of surviving Dox-treated double- and single-transgenic progeny at P21 (29% double transgenic, 71% single transgenic) were similar to those observed at P7, suggesting that no additional mortality occurred after discontinuation of the Dox treatment (Table 6). By P21, the body weights were similar in Dox-treated double- and single-transgenic animals and equivalent to those of animals not exposed to Dox (Table 6). The lung weight/body weight ratio (either wet or inflation-fixed) was significantly larger in Dox-treated CCSP+/FasL+ mice than in CCSP+/FasL− littermates (P<0.01) (Table 6).
0.39 ± 0.03 (3)†
0.35 ± 0.01 (3)†
3.95 ± 0.18 (3)‡
†P < 0.01 versus Dox-treated animals of same genotype;
‡P < 0.05 versus Dox-treated animals of same genotype (Student's t-test).
The total lung volume, V(lu), and the V(lu)/body weight ratio were significantly larger in Dox-treated double-transgenic mice than in single-transgenic littermates (P<0.01) (Table 7). Computer-assisted stereological volumetry was applied to determine the relative contributions of the various lung compartments (specifically, peripheral air-exchanging parenchyma versus airspace) to the observed total lung volume. The areal density of air-exchanging parenchyma, AA(ae/lu), representing the parenchymal tissue fraction, was significantly lower in double-transgenic mice. The total volume of air-exchanging parenchyma, V(ae), which takes into account both AA(ae/lu) and V(lu), was similar in double- and single-transgenic mice, indicating that the increased V(lu) in double-transgenic mice was attributable to distension of the airspaces rather than actual tissue growth.
7.1 ± 1.2 (3)‡
72.0 ± 5.0 (3)†
†P < 0.05 versus Dox-treated animals of same genotype;
‡P < 0.01 versus Dox-treated animals of same genotype (Student's t-test).
The formalin-inflated lungs of Dox-treated double-transgenic CCSP+/FasL+ mice at P21 appeared large and pale compared with those of CCSP+/FasL− littermates (
To estimate the degree of alveolarization in transgenic mice, the MCL and RAC were determined. As shown in
In the absence of Dox, the morphometric assessment of lung growth and alveolarization was similar in double and single-transgenic animals, indicating that the presence of the (TetOp)7-FasL transgene does not have Dox-independent effects on lung development. Compared with non-Dox-treated single-transgenic CCSP+/FasL− mice, Dox-treated single-transgenic animals had significantly larger V(lu), MCL, volume of airspace unit, and specific internal surface area, as well as significantly smaller RAC, indicative of Dox-related effects on alveolarization (
Discussion
In this study, a gain-of-function approach was used to determine the effects of alveolar epithelial apoptosis on postcanalicular alveolar remodeling. A tetracycline-inducible lung epithelial-specific FasL-overexpressing mouse was generated, adapted from the Tet system of Gossen and colleagues,34 to target apoptosis to respiratory epithelial cells during perinatal lung development. Increased alveolar epithelial apoptosis during postcanalicular lung remodeling was determined to be sufficient to disrupt alveolar development and results in a pattern of alveolar simplification that closely mimics the pulmonary pathology of human BPD.
These findings establish a solid causative relationship between alveolar epithelial apoptosis and disrupted alveolarization and support the hypothesis that excessive or premature alveolar epithelial apoptosis is a pivotal event in the pathogenesis of BPD that links the known risk factors of BPD to the final common outcome: impaired alveolar development. Accumulating clinical and experimental evidence shows that the major predisposing factors implicated in BPD, including hyperoxia/oxygen toxicity,22,56,57 mechanical distension (stretch),12,58-61 and proinflammatory factors26,62 are capable of inducing alveolar epithelial apoptosis. The molecular signaling pathways regulating alveolar epithelial apoptosis in early BPD remain undetermined, but likely include both receptor-mediated (extrinsic) and mitochondrial-dependent (intrinsic) death signaling systems.
The precise mechanisms by which exaggerated or premature alveolar epithelial apoptosis induces alveolar disruption remain to be determined. First, the alveolar arrest may simply be attributable to numerical loss of alveolar type II cells during crucial time points of alveolar remodeling. Alveolar type II cells ensure adequate surfactant production around birth and serve as the proliferative source for alveolar type I cells that line most of the alveolar surface.63 It is therefore reasonable to speculate that accumulation of a critical mass of alveolar type II cells is essential for normal postnatal lung remodeling. Interestingly, several lines of evidence in humans and experimental models link apoptosis with lung destruction in emphysematous adult lungs,64-70 suggesting that in fully developed lungs as well, type II cell loss is capable of disrupting the alveolar architecture.
Second, reactive type II cell hyperplasia after initial type II cell apoptosis may contribute, paradoxically, to disrupted alveolar remodeling. Reactive type II cell hyperplasia is a characteristic feature of most forms of acute lung injury, including the early stages of lung injury in newborns. In the present study, FasL overexpression in fetal lungs caused massive apoptosis of alveolar type II cells, resulting in near-total eradication of these cells by E19. By P7, however, the alveoli were repopulated by large numbers of strongly SP-C-immunoreactive alveolar type II cells. This newly emerging population of hyperplastic alveolar type II cells was strikingly refractory to apoptosis, despite continued Dox exposure and pulmonary FasL overexpression. This suggests that, at least with respect to Fas sensitivity, the second-generation type II cells are phenotypically different from the original, naïve type II cells. The cellular ontogeny and phenotypic characteristics of the repopulating type II cells remain to be determined. It is possible, however, that the hyperplastic type II cells may also differ from naïve type II cells in other aspects affecting alveologenesis, such as the epithelial-mesenchymal and epithelial-endothelial interactions required for alveolar septation.
Finally, apoptosis-induced alveolar disruption may be mediated by the action of macrophages and other proinflammatory mediators. Lungs of Dox-treated CCSP+/FasL+ mice at P7 contained large numbers of intra-alveolar macrophages, admixed with apoptotic cellular debris. Similarly, macrophages, neutrophils, and associated proinflammatory mediators are a constant feature in BPD.5 This BPD-associated inflammatory response, attributed to antenatal chorioamnionitis and intrauterine cytokine expression, as well as postnatal lung injury caused by resuscitation, oxygen toxicity, volutrauma, barotraumas, and infection, has been implicated in inhibition of alveolarization in the lungs of preterm infants.71,72 This view of BPD may need to be integrated with the angiocentric paradigm emphasized in the current literature.73 It has been shown that, in addition to impaired alveolar development, there is also a disruption of pulmonary microvascular development in infants with BPD5,6,74 or in BPD-like animal models such as chronically ventilated premature baboons.75,76 Although there is controversy whether angiogenesis is increased6 or decreased,73 there is general agreement that the microvasculature is dysmorphic in BPD.5,6,73,74 In the present study, the pulmonary vessel density was significantly lower in Dox-treated double-transgenic mice compared with single-transgenic littermates, similar to the microvascular anomalies seen in infants with BPD.
Several observations require special consideration. First, the clearance mechanisms for apoptotic alveolar type II cells and apoptotic Clara cells were found to be strikingly different. Alveolar type II cell apoptosis resulted in massive detachment of these cells from the alveolar wall and subsequent phagocytosis by intra-alveolar macrophages. The vast majority of apoptotic Clara cells, in contrast, remained attached to the bronchial epithelial wall or underwent phagocytosis by adjacent bronchial epithelial cells. The exact mechanisms underlying differential clearance mechanisms in various apoptotic respiratory epithelial cells remain unclear, but are likely related to the nature and extent of lateral cell-cell interactions.
Second, the apoptotic effects of FasL overexpression were virtually limited to alveolar type II cells and bronchial epithelial Clara cells. FasL up-regulation in Dox-treated CCSP+/FasL+ mice did not induce noticeable apoptosis in non-Clara bronchial epithelial cells, interstitial stromal cells, fibroblasts, or endothelial cells. The resistance of non-Clara bronchial epithelial cells to FasL activation was particularly striking because bronchial epithelial cells strongly express Fas receptor.10,11,77-79 The refractoriness of airway epithelial cells to Fas-induced apoptosis has been reported previously62 and has been ascribed to the expression of prosurvival proteins such as the caspase inhibitors c-IAP1 and c-IAP-2.80,81
Use of a tetracycline-regulated bitransgenic expression system in vivo requires rigorous controls to ensure accurate interpretation of the data.82 Potentially confounding variables that may influence the outcome include integration site effects (such as insertional mutagenesis), copy number effects, the effects of Dox exposure, and potential toxicity of rtTA transgene. Five (TetOp)7-FasL transgenic lines were successfully established. The number of transgene copies in these lines ranged from 2 to 30. When crossed with CCSP-rtTA animals, the up-regulation of pulmonary FasL mRNA ranged from 5-fold to more than 1000-fold. The present study was focused on a transgenic line with intermediate transgene copy numbers (20) and intermediate levels of FasL up-regulation (30-fold). All lines, however, showed similar phenotypical features ascribed to FasL overexpression (i.e., increased pulmonary apoptosis and arrested alveolar development), and the severity of their phenotype correlated with the level of FasL mRNA up-regulation. The occurrence of apoptosis and alveolar disruption in all transgenic lines studied indicates that the pulmonary phenotype described in this study is a specific effect of transgene expression, and not a result of nonspecific integration or copy number effects.
The tetracycline-regulated expression system uses the tetracycline analogue, Dox, for induction of transgene expression. Two distinct Dox-related phenotypic effects were identified in the present study. As previously described by others in postnatal rats,83 Dox treatment during the perinatal period was found to have adverse effects on early postnatal somatic growth. Whether the lower body weight of Dox-treated pups was the result of indirect effects on the nursing dams or direct effects on growth of the pups remains undetermined. In accordance with previous reports,83 Dox was further found to negatively affect alveolar development, a phenomenon that has been attributed to its various nonantibiotic activities that include pan-MMP (matrix metalloproteinase) inhibition83,84 and antiangiogenic and anti-inflammatory effects.85 Importantly, this study was controlled for Dox-related effects by comparing Dox-treated double-transgenic CCSP+/FasL+ animals with Dox-treated CCSP+/FasL− littermates.
In summary, a transgenic mouse that allows external control of FasL expression in the respiratory epithelium was generated. Pulmonary FasL overexpression targeted to the postcanalicular stages of lung development was demonstrated to be sufficient to induce alveolar epithelial apoptosis and arrested alveolar development, mimicking the pulmonary pathology of BPD. These results support the hypothesis that alveolar epithelial apoptosis is a pivotal event in the pathogenesis of BPD. The versatility of the novel tetracycline-inducible CCSP/FasL mouse model should facilitate the analysis of the pathogenesis and new therapeutic approaches for BPD and other perinatal or adult pulmonary diseases characterized by dysregulated alveolar epithelial apoptosis. Furthermore, (TetOp)7-FasL transgenic mice, cross-bred with mice carrying the appropriate cell-specific rtTA or tTA construct, may be invaluable models to study the effects of Fas-mediated apoptosis in a wide range of conditions and organ systems.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
The invention was supported, in whole or in part, by a grant P20-RR18728 from the National Institutes of Health. The Government has certain rights in the invention.
