Method effective to modulate expression of T-box protein 4 (Tbx4) for reducing progression of lung fibrosis after a lung injury
10973842 · 2021-04-13
Assignee
Inventors
- Paul W. Noble (Beverly Hills, CA, US)
- Dianhua Jiang (Encino, CA, US)
- Ting Xie (Los Angeles, CA, US)
- Carol Jiurong Liang (Encino, CA, US)
Cpc classification
A61K31/713
HUMAN NECESSITIES
A61K48/0058
HUMAN NECESSITIES
A61K48/0016
HUMAN NECESSITIES
A61K48/0066
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
International classification
A61K31/713
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
A61K48/00
HUMAN NECESSITIES
Abstract
The described invention provides a method for reducing progression of lung fibrosis after a lung injury comprising administering a therapeutic amount of a therapeutic agent, wherein the therapeutic amount is effective: (a) to modulate expression of a T-box transcription factor in a population of cells in lung; and (b) to reduce proliferation of the population of cells in lung expressing the T-box transcription factor. According to some embodiments the T-box transcription factor is Tbx4.
Claims
1. A method for reducing progression of lung fibrosis after a lung injury in a subject, comprising administering locally to a lung of the subject a therapeutic amount of a therapeutic agent, wherein the therapeutic agent is a small interfering RNA (siRNA), wherein the therapeutic amount is effective: (a) to inhibit expression of a T-box transcription factor protein T-box protein 4 (Tbx4) in a population of cells in lung expressing Tbx4 and (b) to reduce proliferation of the population of cells in lung expressing Tbx4.
2. The method according to claim 1, wherein the nucleic acid inhibitor is a small interfering RNA (siRNA) of nucleic acid sequence 5′-rGrCrArCrUrGrCrCrArArGrArArArCrArUrGrGrArArArGGT-3′ (SEQ ID NO: 1).
3. The method according to claim 1, wherein the nucleic acid inhibitor is a small interfering RNA (siRNA) of nucleic acid sequence 5′-rUrGrCrArArUrUrArUrCrUrArArGrArArGrUrGrArCrUrUTG-3′ (SEQ ID NO: 2).
4. The method according to claim 1, wherein the population of cells in lung in which Tbx4 is expressed is heterogeneous.
5. The method according to claim 4, wherein the population of cells in lung in which Tbx4 is expressed comprises one or more of a population of pericytes, a population of lipofibroblasts, a population of endothelial cells, or a population of myofibroblasts.
6. The method according to claim 5, wherein the population of cells in lung in which Tbx4 is expressed is further characterized by expression of one or more markers selected from α-smooth muscle actin (αSMA), Col1a1, desmin, vimentin, NG2, and PDGFRβ.
7. The method according to claim 5, wherein the population of myofibroblasts in lung is characterized by expression of Tbx4 and αSMA.
8. The method according to claim 5, wherein the population of cells in lung in which Tbx4 is expressed comprises a population of fibroblasts resident in the lung of the subject.
9. The method according to any one of claims 2 and 3, wherein knockdown of Tbx4 with the siRNA is effective to reduce invasiveness of myofibroblasts.
10. The method according to claim 1, wherein inhibition of expression of Tbx4 is effective to modulate expression of Has-2.
11. The method according to claim any one of claims 2 and 3, wherein the therapeutic amount of the Tbx4 siRNA is effective to decrease TGFβ-induced release of hyaluronic acid (HA).
12. The method according to claim 1, wherein the therapeutic amount of the therapeutic agent is effective to reduce symptoms of pulmonary fibrosis.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DETAILED DESCRIPTION OF THE INVENTION
Glossary
(30) The terms “amino acid residue” or “amino acid” or “residue” are used interchangeably to refer to an amino acid that is incorporated into a protein, a polypeptide, or a peptide, including, but not limited to, a naturally occurring amino acid and known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
(31) The abbreviations used herein for amino acids are those abbreviations which are conventionally used: A=Ala=Alanine; R=Arg=Arginine; N=Asn=Asparagine; D=Asp=Aspartic acid; C=Cys=Cysteine; Q=Gln=Glutamine; E=Glu=Glutamic acid; G=Gly=Glycine; H=His=Histidine; I=Ile=Isoleucine; L=Leu=Leucine; K=Lys=Lysine; M=Met=Methionine; F=Phe=Phenyalanine; P=Pro=Proline; S=Ser=Serine; T=Thr=Threonine; W=Trp=Tryptophan; Y=Tyr=Tyrosine; V=Val=Valine. The amino acids may be L- or D-amino acids. An amino acid may be replaced by a synthetic amino acid which is altered so as to increase the half-life of the peptide or to increase the potency of the peptide, or to increase the bioavailability of the peptide.
(32) The following represent groups of amino acids that are conservative substitutions for one another:
(33) Alanine (A), Serine (S), Threonine (T);
(34) Aspartic Acid (D), Glutamic Acid (E);
(35) Asparagine (N), Glutamine (Q);
(36) Arginine (R), Lysine (K);
(37) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Anatomical Terms
(38) When referring to animals, that typically have one end with a head and mouth, with the opposite end often having the anus and tail, the head end is referred to as the cranial end, while the tail end is referred to as the caudal end. Within the head itself, rostral refers to the direction toward the end of the nose, and caudal is used to refer to the tail direction. The surface or side of an animal's body that is normally oriented upwards, away from the pull of gravity, is the dorsal side; the opposite side, typically the one closest to the ground when walking on all legs, swimming or flying, is the ventral side. On the limbs or other appendages, a point closer to the main body is “proximal”; a point farther away is “distal”. Three basic reference planes are used in zoological anatomy. A “sagittal” plane divides the body into left and right portions. The “midsagittal” plane is in the midline, i.e. it would pass through midline structures such as the spine, and all other sagittal planes are parallel to it. A “coronal” plane divides the body into dorsal and ventral portions. A “transverse” plane divides the body into cranial and caudal portions.
(39) When referring to humans, the body and its parts are always described using the assumption that the body is standing upright. Portions of the body which are closer to the head end are “superior” (corresponding to cranial in animals), while those farther away are “inferior” (corresponding to caudal in animals). Objects near the front of the body are referred to as “anterior” (corresponding to ventral in animals); those near the rear of the body are referred to as “posterior” (corresponding to dorsal in animals). A transverse, axial, or horizontal plane is an X-Y plane, parallel to the ground, which separates the superior/head from the inferior/feet. A coronal or frontal plane is a Y-Z plane, perpendicular to the ground, which separates the anterior from the posterior. A sagittal plane is an X-Z plane, perpendicular to the ground and to the coronal plane, which separates left from right. The midsagittal plane is the specific sagittal plane that is exactly in the middle of the body.
(40) Structures near the midline are called medial and those near the sides of animals are called lateral. Therefore, medial structures are closer to the midsagittal plane, lateral structures are further from the midsagittal plane. Structures in the midline of the body are median. For example, the tip of a human subject's nose is in the median line.
(41) Ipsilateral means on the same side, contralateral means on the other side and bilateral means on both sides. Structures that are close to the center of the body are proximal or central, while ones more distant are distal or peripheral. For example, the hands are at the distal end of the arms, while the shoulders are at the proximal ends.
(42) The term “biomarkers” as used herein refers to are molecules or genes that carry information about the health or disease state of the individual. Generally speaking, biomarkers can be divided into several classes based on the type of the information that they provide. Diagnostic biomarkers allow the distinction of one disease from the other, and can be used in disease classification and diagnosis. Disease susceptibility markers—most often gene mutations and polymorphisms associated with the disease—are often included with diagnostic markers, but in fact differ because in the healthy individual they just indicate an increased risk and their diagnostic value is unclear in complex disease. Prognostic biomarkers are markers that allow the prediction of outcome, usually at the time of presentation. Diagnostic and prognostic markers should be distinguished from disease activity biomarkers that may change during the course of the disease—although in some cases they may overlap. The last group of biomarkers can be broadly defined as treatment efficacy biomarkers—these include markers that a drug is indeed affecting the pathway it is supposed to affect, markers that indicate toxicity and markers that indicate a real beneficial drug effect that could eventually be used as surrogate endpoints in drug studies (Zhang & Kaminski. Curr Opin Pulm Med. 2012 September; 18(5): 441-6).
(43) The term “bronchoalveolar lavage” (BAL) as used herein refers a medical procedure in which a bronchoscope is passed through the mouth or nose into the lungs and fluid is squirted into a small part of the lung and then collected for examination.
(44) The term “5-bromo-2-deoxyuridine” (BrdU) as used herein refers to a thymidine analog, which is readily incorporated in replicating DNA in dividing cells. BrdU is utilized as a tool to measure cell proliferation with anti-BrdU antibodies.
(45) The term “CC10” as used herein refers a 10 kDa protein, which is the major secretory protein of club cells, and is thought to play a protective role in the lung (Bolton et al. Toxicol Pathol. 2008 April; 36(3): 440-8).
(46) The term “cell line” as used herein refers to a population of cultured cells that has undergone a change allowing the cells to grow indefinitely.
(47) The term “cell strain” as used herein refers to a population of cultured cells that has a finite life span.
(48) The term “CFP” as used herein refers to cyan fluorescent protein, which possesses bright fluorescence with excitation/emission maxima at 458 and 480 nm, respectively.
(49) The term “complementary” as used herein refers to two nucleic acid sequences or strands that can form a perfect base-paired double helix with each other.
(50) The term “Confetti” as used herein refers to a reporter construct that functions as a stochastic multicolor Cre recombinase reporter of multiple fluorescent proteins from a single genomic locus. This enables a way to label and distinguish individual/adjacent cells with nuclear localized, membrane-targeted, or cytoplasmic fluorescent proteins in Cre recombined cells.
(51) The term “Col1a1” as used herein refers to a marker for the pro-alpha 1 chain of type I collagen.
(52) The term “Col1a2” as used herein refers to the gene encoding the pro-alpha 2 chain of type I collagen. IPF patients have been found to have increased expression levels of Col1a2 (Konishi et al. Am J Respir Crit Care Med. 2009 Jul. 15; 180(2): 167-75).
(53) The term “chondroitin sulfate proteoglycan 4” (CSPG4) as used herein refers to the human homolog of NG2. NG2 is a proteoglycan marker for macrophages and pericytes.
(54) The term “Cre-lox system” as used herein refers to a sophisticated tool for general knockouts, conditional knockouts, and reporter strains. The Cre-lox mechanism was discovered in P1 bacteriophage (Sauer & Henderson. J Mol Biol. 1988; 85: 5166-70; Sternberg & Hamilton. J Mol Biol. 1981; 150: 467-86), and requires only two components: 1) Cre recombinase, an enzyme that catalyzes recombination between two loxP sites; and 2) LoxP sites, specific 34-base pair (bp) sequences consisting of an 8-bp core sequence, where recombination takes place, and two flanking 13-bp inverted repeats. The term “CreER” refers to a fusion of Cre and a tamoxifen-responsive estrogen receptor, and is commonly used in fate mapping studies.
(55) The term “desmin” as used herein refers to a protein found in intermediate filaments that copolymerizes with vimentin to form constituents of connective tissue, cell wells, filaments etc. It is found in the Z disk of skeletal and cardiac muscle cells. (Paulin & Li. Exp Cell Res. 2004 Nov. 15; 301(1): 1-7))
(56) The term “downstream” with respect to a gene, refers to the direction RNA polymerase moves during transcription, which is toward the end of the template DNA strand with a 3′ hydroxyl group. The term “upstream” is the direction on a DNA opposite to the direction RNA polymerase moves during transcription.
(57) The term “EGFP” as used herein refers to enhanced green fluorescent protein, a protein composed of 238 amino acid residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.
(58) The term “epithelial-mesenchymal transition” (EMT) as used herein refers to a biological process that allows a polarized epithelial cell, which normally interacts with basement membrane via its basal surface, to underdo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype, which includes enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of ECM components (Kalluri & Neilson. J Clin Invest. 2003 December; 112(12): 1776-84). A number of distinct molecular processes are engaged in order to initiate EMT and enable it to reach completion, including activation of transcription factors, expression of specific cell-surface proteins, reorganization and expression of cytoskeletal proteins, production of ECM-degrading enzymes, and changes in the expression of specific microRNAs (Kalluri & Weinberg. J Clin Invest. 2009 June; 119(6): 1420-8).
(59) The term “expression vector” as used herein refers to a modified DNA molecule that carries a gene or DNA which is specially constructed into a suitable host cell and there directs synthesis of a protein product encoded by an inserted sequence.
(60) The term “FACS” as used herein, refers to fluorescence-activated cell sorting, a specialized form of flow cytometry.
(61) The term “fate mapping” as used herein refers to methods of elucidating how embryonic tissue organization relates to postnatal tissue structure and function, and is used in studying the etiology of human disorders (Jensen & Dymecki. Methods Mol Biol. 2014; 1092: 437-54). When carried out at single-cell resolution, fate mapping is also known as cell lineage tracing. In cell lineage tracing a single cell is marked in such a way that the mark is transmitted to the cell's progeny, resulting in a set of labeled clones, providing information about the number of progeny of the founder cell, their location, and their differentiation status (Kretzschmar & Watt. Cell. 2012 Jan. 20; 148(1-2): 33-45).
(62) The term “fibroblast” as used herein refers to a connective tissue cell that secretes collagen and other components of the extracellular matrix, which migrates and proliferates during normal wound healing.
(63) The term “flow cytometry” as used herein refers to a tool for interrogating the phenotype and characteristics of cells. Flow cytometry is a system for sensing cells or particles as they move in a liquid stream through a laser (light amplification by stimulated emission of radiation)/light beam past a sensing area. The relative light-scattering and color-discriminated fluorescence of the microscopic particles is measured. Analysis and differentiation of the cells is based on size, granularity, and whether the cells are carrying fluorescent molecules in the form of either antibodies or dyes. As the cell passes through the laser beam, light is scattered in all directions, and the light scattered in the forward direction at low angles (0.5-10 degrees) from the axis is proportional to the square of the radius of a sphere and so to the size of the cell or particle. Light may enter the cell; thus, the 90 degree light (right-angled, side) scatter may be labeled with fluorochrome-linked antibodies or stained with fluorescent membrane, cytoplasmic, or nuclear dyes. Thus, the differentiation of cell types, the presence of membrane receptors and antigens, membrane potential, pH, enzyme activity, and DNA content may be facilitated. Flow cytometers are multiparameter, recording several measurements on each cell; therefore, it is possible to identify a homogeneous subpopulation within a heterogeneous population (Marion G. Macey, Flow cytometry: principles and applications, Humana Press, 2007).
(64) The term “FoxD1” as used herein refers to a gene encoding a protein, forkhead box D1, which belongs to the forkhead family of transcription factors, which are characterized by a distinct forkhead domain. The lung contains an extensive population of FoxD1 progenitor-derived pericytes that are an important lung myofibroblast precursor population (Hung C, et al. Am J Respir Crit Care Med. 2013 Oct. 1; 188(7): 820-30).
(65) The term “FoxJ1” as used herein refers to a gene encoding a protein, forkhead box J1, which belongs to the forkhead family of transcription factors, which are characterized by a distinct forkhead domain. FoxJ1 is required for cilia formation and regulates basal body anchoring to the cytoskeleton of ciliated pulmonary epithelial cells (Gomperts B N, et al. J Cell Sci. 2004 Mar. 15; 117(Pt 8): 1329-37).
(66) The term “gene” as used herein is the entire DNA sequence, including exons, introns, and noncoding transcription-control regions necessary for production of a functional protein or RNA.
(67) The terms “gene expression” or “expression” are used interchangeably to refer to the process by which information encoded in a gene is converted into an observable phenotype.
(68) The term “growth” as used herein refers to a process of becoming larger, longer or more numerous, or an increase in size, number, or volume.
(69) The term “interfere” or “to interfere with” as used herein refers to the hampering, impeding, dampening, hindering, obstructing, blocking, reducing or preventing of an action or occurrence.
(70) The term “invasion” or “invasiveness” as used herein refers to a process that includes penetration of and movement through surrounding tissues.
(71) The term “involucrin” as used herein refers to a component of the keratinocyte crosslinked envelope, is found in the cytoplasm and crosslinked to membrane proteins by transglutaminase.
(72) The term “knockdown” or “knockout” are used interchangeably to refer to selectively inactivating a gene.
(73) The term “knock-in” as used herein refers to a genetic engineering method that involves the insertion of a protein coding cDNA sequence at a particular locus in a target organism's chromosome (Gibson, Greg (2009). A Primer of Genome Science 3rd ed. Sunderland, Mass.: Sinauer. pp. 301-302).
(74) The term “lipofibroblast” as used herein refers to a lipid-containing alveolar interstitial fibroblast recognizable by characteristic lipid droplets. Lipofibroblasts participate in the synthesis of ECM proteins, including collagen and elastin, and produce leptin, which binds to AEC2s, stimulating their production of surfactant (Rehan & Torday. PPAR Res. 2012; 2012: 289867).
(75) The term “Masson's trichrome stain” as used herein refers to a stain used in the study of connective tissue, muscle and collagen fibers. It is used primarily for distinguishing collagen from muscle tissue. Typically, it contains nuclear, collagenous, and cytoplasmic dyes in mordants, such as phosphotungstic or phosphomolybdic acid.
(76) The term mCherry” as used herein refers to a 28.8 kDa red fluorescent protein with 256 amino acids.
(77) The term “migration” as used herein refers to movement of a cell from one place or location to another.
(78) The term “modulate” as used herein means to regulate, alter, adapt, or adjust to a certain measure or proportion.
(79) The term “mT/mG” as used herein refers to a double-fluorescent Cre reporter allele that expresses cell membrane-localized red fluorescence in widespread cells and tissues prior to Cre recombinase exposure, and cell membrane-localized green fluorescence in Cre recombinase expressing cells (and future cell lineages derived from these cells).
(80) The term “NG2” as used herein refers to neural/glial antigen. NG2 is found in the plasma membrane of many cell types, and has been reported to be expressed exclusively by pericytes during vascular morphogenesis (Ozerdem et al. Dev Dyn. 2001 October; 222(2): 218-27).
(81) The term “nucleic acid” is used herein to refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
(82) The term “nucleotide” is used herein to refer to a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. In the most common nucleotides, the base is a derivative of purine or pyrimidine, and the sugar is the pentose deoxyribose or ribose. Nucleotides are the monomers of nucleic acids, with three or more bonding together in order to form a nucleic acid. Nucleotides are the structural units of RNA, DNA, and several cofactors, including, but not limited to, CoA, FAD, DMN, NAD, and NADP. Purines include adenine (A), and guanine (G); pyrimidines include cytosine (C), thymine (T), and uracil (U).
(83) The term “oligonucleotide” as used herein refers to relatively short (13-25 nucleotides) unmodified or chemically modified single-stranded DNA molecules.
(84) The following terms are used herein to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.
(85) The term “reference sequence” refers to a sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(86) The term “comparison window” refers to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be at least 30 contiguous nucleotides in length, at least 40 contiguous nucleotides in length, at least 50 contiguous nucleotides in length, at least 100 contiguous nucleotides in length, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence, a gap penalty typically is introduced and is subtracted from the number of matches.
(87) Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS 5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8:155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24:307-331 (1994). The BLAST family of programs, which can be used for database similarity searches, includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
(88) Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters. Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information at ncbi.nlm.nih.gov. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits then are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatching residues; always<0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915).
(89) In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA, 1993, 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. BLAST searches assume that proteins may be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs may be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 1993, 17:149-163) and XNU (Claverie and States, Comput. Chem., 1993, 17:191-201) low-complexity filters may be employed alone or in combination.
(90) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, i.e., where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 1988, 4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
(91) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) relative to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
(92) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity and at least 95% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values may be adjusted appropriately to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, or at least 70%, at least 80%, at least 90%, or at least 95%. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. However, nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide that the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
(93) The terms “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, at least 80%, at least 85%, at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Optionally, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides which are “substantially similar” share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes.
(94) The term “PDGFRβ” as used herein refers to platelet derived growth factor receptor beta.
(95) The term “peptidomimetic” refers to a small protein-like chain designed to mimic or imitate a peptide. A peptidomimetic may comprise non-peptidic structural elements capable of mimicking (meaning imitating) or antagonizing (meaning neutralizing or counteracting) the biological action(s) of a natural parent peptide.
(96) The term “pericytes” as used herein refer to perivascular cells (cells situated or occurring around a blood vessel) that wrap around capillaries. Pericytes are also known as mural cells, Rouget cells, or, because of their contractile fibers, as vascular smooth muscle cells. Pericytes have roles in angiogenesis, and blood vessel maintenance (Bergers & Song. Neuro Oncol. 2005 October; 7(4): 452-64).
(97) The term “phenotype” as used herein refers to the observable characteristics of a cell, for example, expression of a protein.
(98) The term “podoplanin” (PDPN or T1α) as used herein refers to a well-conserved mucin-type transmembrane protein widely distributed in human tissues that plays a critical role in development of the heart, lungs and lymphatic system. It is widely used as a marker for lymphatic endothelial cells and fibroblastic reticular cells of lymphoid organs and for lymphatics in the skin and tumor microenvironment. Tumor cells often upregulate PDPN as they undergo EMT; this upregulation is correlated with increased motility and metastasis (Astarita J L, et al. Front Immunol. 2012 Sep. 12; 3: 283).
(99) The term “polynucleotide” refers to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide may be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides.
(100) The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer formed from the linking together, in a defined order, of amino acid residues. The link between one amino acid residue and the next is known as an amide or peptide bond. The term “polypeptide” as used herein refers to a single chain of amino acids, and a “protein” refers to one or more polypeptides. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms “peptide”, “polypeptide” and “protein” also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms “polypeptide”, “peptide” and “protein” also are inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. It will be appreciated, as is well known and as noted above, that polypeptides may not be entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslational events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well.
(101) The term “primer” refers to a nucleic acid which, when hybridized to a strand of DNA, is capable of initiating the synthesis of an extension product in the presence of a suitable polymerization agent. The primer is sufficiently long to uniquely hybridize to a specific region of the DNA strand. A primer also may be used on RNA, for example, to synthesize the first strand of cDNA.
(102) The term “proliferate” and its other grammatical forms as used herein means multiplying or increasing in number.
(103) The term “progression” as used herein refers to the course of a disease, such as pulmonary fibrosis, as it becomes worse. The term “progression-free survival” as used herein refers to the length of time during and after treatment of a disease that a patient lives with the disease but it does not get worse.
(104) The term “promoter” as used herein refers to a region of DNA upstream, whether downstream, proximal, or distal, from the start of transcription, which is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A given promoter may work in concert with other regulatory regions (enhancers, silencers, boundary elements/insulators) in order to direct the level of transcription of a given gene.
(105) The term “inducible promoter” as used herein refers to a promoter whose downstream transcriptional activity is controlled by the presence of a molecule (i.e., inducer).
(106) The term “constitutive promoter” refers to a promoter whose transcriptional activity is maintained at a relatively constant level in all cells of an organism without regard to cell environmental conditions.
(107) The term “quantitative real-time reverse transcription PCR” or “real-time quantitative reverse transcription PCR” (Real-Time qRT-PCR) refers a PCR technology that enables reliable detection and measurement of products generated during each cycle of the PCR process. RNA is used as the starting material, which is transcribed into complementary DNA (cDNA) by reverse transcriptase; the cDNA is used as the template for the quantitative PCR reaction.
(108) The term “reduce” and its various grammatical forms as used herein refer to a diminution, a decrease, an attenuation or abatement of the degree, intensity, extent, size, amount, density or number of occurrences, events or characteristics.
(109) The term “RFP” as used herein refers to red fluorescent protein, which can be excited by the 488 nm or 532 nm laser line and is optimally detected at 588 nm.
(110) RNA interference (RNAi), or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. It is a natural process by which double-stranded RNAs initiate the degradation of homologous RNA; researchers can take advantage of this process to study gene expression. A simplified model for the RNAi pathway is based on two steps, each involving ribonuclease enzyme. In the first step, the trigger RNA (either dsRNA or miRNA primary transcript) is processed into a short, interfering RNA (siRNA) by the RNase II enzymes Dicer and Drosha. In the second step, siRNAs are loaded into the effector complex RNA-induced silencing complex (RISC). The siRNA is unwound during RISC assembly and the single-stranded RNA hybridizes with a mRNA target. Gene silencing is a result of nucleolytic degradation of the targeted mRNA by the RNase H enzyme Argonaute (Slicer).
(111) Gene silencing, however, can also occur not via siRNA-mediated cleavage of targeted mRNA, but rather, via translational inhibition. If the siRNA/mRNA duplex contains mismatches the mRNA is not cleaved; in these cases, direct translational inhibition may occur, especially when high concentrations of siRNA are present. The mechanism of this translation inhibition is not known.
(112) As a result, siRNA can elicit two distinct modes of post-transcriptional repression. Because the requirement for target complementarity is less stringent for direct translational inhibition than for target mRNA cleavage, siRNAs designed for the latter may inadvertently trigger the former in another gene. Therefore, siRNAs designed against one gene may trigger silencing of an unrelated gene.
(113) The term “Rosa26” as used herein refers to a locus widely used for achieving general expression in mice.
(114) The term “Scgb1a1” as used herein refers to secretoglobin, family 1a, member 1, or uteroglobin, an evolutionary conserved, steroid-inducible secreted protein that has anti-inflammatory and immunomodulatory properties. Mucosal epithelia of virtually all organs that communicate with the external environment express Scgb1a1, and Scgb1a1 is present in blood, urine, and other body fluids (Mukherjee et al. Endocr Rev. 2007 December; 28(7): 707-25).
(115) The term “Sftpc” or “SPC” as used herein refers to pulmonary-associated surfactant protein C, an extremely hydrophobic surfactant protein essential for lung function and homeostasis after birth. It is produced exclusively by AEC2s in the lung. Pulmonary surfactant is a lipid-rich material comprising phosopholipids and other surfactant-associated proteins, and prevents lung collapse by reducing surface tension at the air-liquid interface in the alveoli of the lung (Clark & Clark. Semin Fetal Neonatal Med. 2005 June; 10(3): 271-82).
(116) shRNA (short hairpin RNA) sequences offer the possibility of prolonged gene silencing. shRNAs are usually encoded in a DNA vector that can be introduced into cells via plasmid transfection or viral transduction. There are two main categories of shRNA molecules based on their design: simple stem-loop and microRNA-adapted shRNA. A simple stem-loop shRNA is often transcribed under the control of an RNA Polymerase III (Pol III) promoter [Bartel, DP, MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281-297 (2004), Kim, V. N. MicroRNA biogenesis: coordinated cropping and dicing. Nature Reviews, Molecular Cell Biology 6(5):376-385 (2005)]. The 50-70 nucleotide transcript forms a stem-loop structure consisting of a 19 to 29 bp region of double stranded RNA (the stem) bridged by a region of predominantly single-stranded RNA (the loop) and a dinucleotide 3′ overhang [Brummelkamp, T. R. et al. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296(5567):550-553; Paddison, P. J. et al. (2002) Stable suppression of gene expression by RNAi in mammalian cells. PNAS 99(3):1443-1448; Paul, C. P. et al. (2002) Effective expression of small interfering RNA in human cells. Nature Biotechnology 20(5):505-508]. The simple stem-loop shRNA is transcribed in the nucleus and enters the RNAi pathway similar to a pre-microRNA. The longer (>250 nucleotide) microRNA-adapted shRNA is a design that more closely resembles native pri-microRNA molecules, and consists of a shRNA stem structure which may include microRNA-like mismatches, bridged by a loop and flanked by 5′ and 3′ endogenous microRNA sequences [Silva, J. M. et al. (2005) Second-generation shRNA libraries covering the mouse and human genomes. Nature Genetics 37(11):1281-1288.]. The microRNA-adapted shRNA, like the simple stem-loop hairpin, is also transcribed in the nucleus but is thought to enter the RNAi pathway earlier similar to an endogenous pri-microRNA.
(117) The term “small interfering RNAs,” which comprises both microRNA (miRNA) and small interfering RNA (siRNA), are small noncoding RNA molecules that play a role in RNA interference. siRNAs are synthesized from double-stranded segments of matched mRNA via RNA-dependent RNA polymerase., and siRNAs regulate the degradation of mRNA molecules identical in sequence to that of the corresponding siRNA, resulting in the silencing of the corresponding gene and the shutting down of protein synthesis. The main mechanism of action of siRNA is the mRNA cleavage function. There are no genes that encode for siRNAs. siRNAs can also silence gene expression by triggering promoter gene methylation and chromatin condensation. miRNAs are synthesized from an unmatched segment of RNA precursor featuring a hairpin turn, and miRNAs are encoded by specific miRNA genes as short hairpin pri-miRNAs in the nucleus. miRNAS are also small noncoding RNAs, but they seem to require only a 7- to 8-base-pair “seed” match between the 5′ region of the miRNA and the 3′UTR of the target. While the majority of miRNA targets are translationally repressed, degradation of the target mRNA can also occur. The main mechanism of action of miRNA may be the inhibition of mRNA translation, although the cleavage of mRNA is also an important role (Ross et al. Am J Clin Pathol. 2007; 128(5): 830-36).
(118) The term “αSMA” as used herein refers to alpha-smooth muscle actin, an actin isoform typical of vascular smooth muscle cells, which has been found to play a role in fibroblast contractile activity (Hinz et al. Mol Biol Cell. 2001 September; 12(9): 2730-41).
(119) The term “specifically hybridizes” as used herein refers to the process whereby a nucleic acid distinctively or definitively forms base pairs with complementary regions of at least one strand of the nucleic acid target sequence that was not originally paired to the nucleic acid. A nucleic acid that selectively hybridizes undergoes hybridization, under stringent hybridization conditions, of the nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 100% sequence identity (i.e., complementary) with each other. The term “survival rate” as used herein refers to the percentage of a population that are still alive for a certain period of time after treatment.
(120) The term “T-box” as used herein refers to the family refers to the family of transcription factors that play roles throughout development. T-box genes are expressed in highly specific manners, and are involved in all of the major developmental signaling pathways. Common to all T-box proteins is a 180-200 amino acid DNA-binding motif, or T-box domain, which binds DNA in a sequence-specific manner (Papaioannou V E. Development. 2014 October; 141(20): 3819-33).
(121) There are at least five subfamilies of T-box proteins: 1) the T subfamily, comprising T and Tbx19; 2) the Tbx1 subfamily, comprising Tbx1, Tbx10, Tbx15, Tbx18, Tbx20, and Tbx22; 3) the Tbx2 subfamily, comprising Tbx2, Tbx3, Tbx4, and Tbx5; 4) the Tbx6 subfamily, comprising Tbx6, Drtbx6, Drtbx16, and Mga; and 5) the Tbr1 subfamily, comprising Tbr1, Tbr2, and Tbx21 (Id.).
(122) The term “tdTomato” as used herein refers to the tdTomato fluorescent protein, with an excitation maximum at 554 nm, and an emission maximum at 581 nm.
(123) The term “transcription factor” as used herein refers to a protein that binds to and controls the activity of other genes.
(124) The term “transgenic” as used herein refers to an organism that contains genetic material into which DNA from an unrelated organism has been artificially introduced.
(125) The term “tumor suppressor gene” as used herein refers to a gene whose encoded protein directly or indirectly inhibits progression through the cell cycle and in which a loss-of-function mutation is oncogenic.
(126) A “variant” of a gene or nucleic acid sequence is a sequence having at least 65% identity with the referenced gene or nucleic acid sequence, and can include one or more base deletions, additions, or substitutions with respect to the referenced sequence. The differences in the sequences may by the result of changes, either naturally or by design, in sequence or structure. Natural changes may arise during the course of normal replication or duplication in nature of the particular nucleic acid sequence. Designed changes may be specifically designed and introduced into the sequence for specific purposes. Such specific changes may be made in vitro using a variety of mutagenesis techniques. Such sequence variants generated specifically may be referred to as “mutants” of the original sequence.
(127) A “variant” of a peptide or protein is a peptide or protein sequence that varies at one or more amino acid positions with respect to the reference peptide or protein. A variant can be a naturally-occurring variant or can be the result of spontaneous, induced, or genetically engineered mutation(s) to the nucleic acid molecule encoding the variant peptide or protein. A variant peptide can also be a chemically synthesized variant.
(128) A skilled artisan likewise can produce polypeptide variants having single or multiple amino acid substitutions, deletions, additions or replacements. These variants may include inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids; (b) variants in which one or more amino acids are added; (c) variants in which at least one amino acid includes a substituent group; (d) variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non-conserved positions; and (d) variants in which a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, such as, for example, an epitope for an antibody. The techniques for obtaining such variants, including genetic (suppressions, deletions, mutations, etc), chemical, and enzymatic techniques are known to the skilled artisan. As used herein, the term “mutation” refers to a change of the DNA sequence within a gene or chromosome of an organism resulting in the creation of a new character or trait not found in the parental type, or the process by which such a change occurs in a chromosome, either through an alteration in the nucleotide sequence of the DNA coding for a gene or through a change in the physical arrangement of a chromosome. Three mechanisms of mutation include substitution (exchange of one base pair for another), addition (the insertion of one or more bases into a sequence), and deletion (loss of one or more base pairs).
(129) The term “von Willebrand factor” (vWF) as used herein refers to a glycoprotein produced uniquely by endothelial cells. Among other process, vWF is involved in the coagulation process. This glycoprotein is used as an endothelial cell marker.
(130) The term “XFP” as used herein refers to a general acronym for fluorescent proteins, wherein the “X” is one letter stating the color of the emission.
(131) The term “YFP” as used herein refers to red fluorescent protein, which can be excited by the 514 nm laser line and is optimally detected at 527 nm.
(132) According to one aspect, the described invention provides a method for reducing progression of lung fibrosis after a lung injury comprising administering a therapeutic amount of a therapeutic agent, wherein the therapeutic amount is effective (i) to modulate expression of a T-box transcription factor in a population of cells in lung; and (ii). to reduce proliferation of the population of cells in lung expressing the T-box transcription factor.
(133) According to some embodiments, the T box transcription factor is a Tbox 2 transcription factor.
(134) According to some embodiments, therein the Tbox 2 transcription factor is T-box protein 4 (Tbx4).
(135) According to some embodiments, the therapeutic agent is a small molecule inhibitor, a peptide inhibitor or a nucleic acid inhibitor.
(136) According to some embodiments, the therapeutic agent is a peptide inhibitor. Exemplary peptide inhibitors include peptides that are effective to target one or more domains of Tbx-4. According to some embodiments, the peptide inhibitor comprises a nuclear localization signal peptide.
(137) According to some embodiments, the therapeutic agent is a nucleic acid inhibitor. According to some embodiments, the nucleic acid inhibitor is one or more of a siRNA, a DNAzyme, an antisense oligonucleotide, an aptamer or an oligodeoxynucleotide decoy.
(138) According to some embodiments, the nucleic acid inhibitor is a siRNA. According to some embodiments, the siRNA can be modified to increase stability of the RNA. According to some embodiments, the siRNA is an LNA™-modified siRNA to increase its thermal stability. According to some embodiments, the siRNA is 2′O-methyl modified to improve its stability. According to some embodiments, the therapeutic agent is a siRNA of amino acid sequence 5′-rGrCrArCrUrGrCrCrArArGrArArArCrArUrGrGrArArArGGT-3′(SEQ ID NO: 1). According to some embodiments, the therapeutic agent is a siRNA of amino acid sequence 5′-rUrGrCrArArUrUrArUrCrUrArArGrArArGrUrGrArCrUrUTG-3′ (SEQ ID NO: 2). According to some embodiments, knockdown of Tbx4 with the siRNA is effective to reduce invasiveness of the myofibroblasts.
(139) According to some embodiments, the nucleic acid inhibitor is a DNAzyme that is effective to cleave and inactivate TBX4 mRNA. Generally, DNAzymes are catalytically active DNA molecules (see, e.g., Sterna Biologicals, GmbH & Co. KG, www.sterna-biologicals.com). DNAzymes of the so-called 10-23 family are specifically characterized by their capability to cleave RNA molecules after appropriate binding. Thus, they directly exert RNA endonuclease activity. 10-23 DNAzymes are single-stranded DNA molecules that consist of two binding domains flanking a central catalytic domain. The latter is composed of 15 deoxynucleotides, the sequence of which is conserved throughout all molecules within this specific DNAzyme class. In contrast, the binding domains are variable and are designed to specifically bind the corresponding target mRNA of interest by Watson-Crick base-pairing.
(140) After binding of a DNAzyme to the corresponding sequence in the target mRNA via the binding domains (step 1), the catalytic domain becomes active and directly cleaves the target mRNA molecule (step 2). After successful cleavage of a target mRNA molecule, the DNAzyme-RNA-complex dissociates and the RNA cleavage products are further degraded by endogenous, intracellular enzymes. The DNAzyme molecule is then available for subsequent binding and cleavage of additional mRNA molecules (step 3).
(141) According to some embodiments, the DNAzyme is modified with a 3′-3′ inverted nucleotide at the 3′ terminus to prevent exonuclease degradation.
(142) According to some embodiments, the nucleic acid inhibitor is an antisense oligonucleotide. An antisense oligonucleotide (ASO) is a short strand of deoxyribonucleotide analogue that hybridizes with the complementary mRNA in a sequence-specific manner via Watson-Crick base pairing. Formation of the ASO-mRNA heteroduplex either triggers RNase H activity, leading to mRNA degradation, induces translational arrest by steric hindrance of ribosomal activity, interferes with mRNA maturation by inhibiting splicing, or destabilizes pre-mRNA in the nucleus, resulting in downregulation of target protein expression. Chan, J H, Wong, L S, “Clin. Exp. Pharmacol. Physiol. 2006, 33 (5-6): 533-40. According to some embodiments, the antisense oligonucleotide is a DNA antisense oligonucleotide. According to some embodiments, the antisense oligonucleotide is an RNA antisense oligonucleotide. According to some embodiments, the RNA antisense oligonucleotide is phosphorothioate modified to increase its stability and half-life.
(143) According to some embodiments, the nucleic acid inhibitor is an aptamer. Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides that have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure. Sun, H. et al., Molec. Therapy Nucleic Acids 2014, 3: e182; doi: 10.1038/mbna.2014.32. According to some embodiments the aptamer is modified with polyethylene glycol to increase its half-life.
(144) According to some embodiment, the nucleic acid inhibitor is an oligodeoxynucleotide (ODN) decoy. A decoy oligonucleotide is a synthesized short DNA sequence that has the same sequence as that found on the portion of the promoter region of a gene where a transcription factor lands. Normally when a transcription factor lands on the promoter region of a gene, transcription of the gene is switched on leading to its expression. However, the decoy oligonucleotide acts as the promoter's “lure”, binds with the specific transcription factor in the cell so that the transcription factor cannot land on the genome, and the gene expression is suppressed. According to some embodiments, the gene is TBX4.
(145) According to some embodiments, the lung injury induces proliferation of cells expressing Tbx4 in lung.
(146) According to some embodiments of the method, in (a) the modulating expression of a T-box transcription factor in a population of cells in lung is effective to reduce the proliferation of cells expressing Tbx4 following the injury.
(147) According to some embodiments, the population of cells in lung in which Tbx4 is expressed is heterogeneous.
(148) According to some embodiments, the population of cells in lung in which Tbx4 is expressed comprises one or more of a population of pericytes, a population of lipofibroblasts, a population of endothelial cells, or a population of myofibroblasts. According to some embodiments, the proliferating cells that express cells are characterized by expression of one or more markers selected from α-smooth muscle actin (αSMA), Col1a1, desmin, vimentin, NG2, and PDGFRB.
(149) According to some embodiments, the population of myofibroblasts in lung expressing Tbx4 in lung also express αSMA.
(150) According to some embodiments, the proliferating cells that express Tbx4 comprise a population of fibroblasts resident in the lung of the subject.
(151) According to some embodiments, inhibition of expression of Tbx4 is effective to modulate expression of Has-2.
(152) According to some embodiments, the therapeutic amount of the Tbx4 siRNA is effective to decrease TGFβ-induced release of hyaluronic acid (HA).
(153) According to some embodiments, the therapeutic agent is effective to reduce symptoms of pulmonary fibrosis.
(154) Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
(155) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials have been described. All publications mentioned herein are incorporated herein by reference to disclose and described the methods and/or materials in connection with which the publications are cited.
(156) It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural references unless the context clearly dictates otherwise.
(157) The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application and each is incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
EXAMPLES
(158) The following examples are put forth so as to provide those with ordinary skill in the art with a complete disclosure and description of how to make and use the described invention, and are not intended to limit the scope of what the inventors regards as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.) but some experimental errors and deviation should be accounted for. Unless otherwise indicated, parts are by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
(159) All mouse studies were approved by the Institutional Animal Care and Use Committee at Cedars-Sinai Medical Center. Tbx4 lung enhancer-Cre transgenic mice (Tbx4.sup.LME-Cre or Tbx4-Cre.sup.Tg) and Tbx4.sup.LME-CreER transgenic mice were recently described (Kumar et al. Science. 2014 Nov. 14; 346(6211): 1258810). Tbx4-Cre knockin (Tbx4-Cre.sup.Ki) mice were reported (Naiche et al. Dev Dyn. 2011 October; 240(10): 2290-300). Tbx4-Cre Rosa26-tdTomato mouse lines were obtained by crossing Tbx4-Cre knock-in (Tbx4-Cre.sup.Ki) mice or transgenic mice with lineage reporter Rosa26-tdTomato mice (Madisen et al. Nat Neurosci. 2010 January; 13(1): 133-40).
(160) To obtain Tbx4-CreER Rosa26-tdTomato, Tbx4-CreER mice were crossed to Rosa26-tdTomato mice. Tbx4-CreER Confetti mice were obtained by crossing Tbx4-CreER mice with Rosa26-Confetti mice (Livet et al. Nature. 2007 Nov. 1; 450(7166): 56-62) (Jackson Labs). Triple heterozygous αSMA-GFP Tbx4-Cre Rosa26-tdTomato mice were created by crossing Tbx4-Cre Rosa26-tdTomato mice with αSMA-GFP (a.k.a. Acta2-GFP) mice (Condren et al. PLoS One. 2013; 8(1): e53386) (Robert N. Fariss, National Eye Institute). Triple heterozygous Tbx4-Cre Rosa26-tdTomato mice were bred with Col1a1-GFP mice (Iwaisako et al. Proc Natl Acad Sci USA. 2014 Aug. 12; 111(32): E3297-305) (David A. Brenner, University of California, San Diego) to generate COL1a1-GFP-Tbx4-Cre Rosa26-tdTomato mice. Tbx4-Cre Rosa26-tdTomato mice were bred with NG2-YFP mice (LeBleu et al. Nat Med. 2013 August; 19(8): 1047-53) (Raghu Kalluri, University of Texas MD Anderson Cancer Center) to create the Ng2-YFP Tbx4-Cre Rosa26-tdTomato triple heterozygous mice. Tbx4-CreER mice were bred with Rosa26-DTA mice (Voehringer et al. J Immunol. 2008 Apr. 1; 180(7): 4742-53) (Jackson Labs) to generate Tbx4-CreER Rosa26-DTA mice.
(161) Tbx4.sup.flox/flox mice were described previously (Arora et al. PLoS Genet. 2012; 8(8): e1002866). αSMA-CreER mice (Wendling et al. Genesis. 2009 January; 47(1): 14-8) were crossed with Tbx4.sup.flox/flox mice to create the αSMA-CreER Tbx4.sup.flox/flox mice. Col1a2-CreER Tbx4.sup.flox/flox mice were generated by breeding Col1a2-CreER mice (Zheng et al. Am J Pathol. 2002 May; 160(5): 1609-17) (Jackson Labs) with Tbx4.sup.flox/flox mice. Conditional NG2 cell-specific Tbx4 knockout mice (Ng2-CreER Tbx4.sup.flox/flox) were generated by breeding Ng2-CreER (a.k.a. Cspg4-CreER) mice (Zhu et al. Development. 2011 February; 138(4): 745-53) (Jackson Labs) with Tbx4.sup.flox/flox mice. Tamoxifen (Sigma-Aldrich) dissolved in corn oil was injected intraperitoneally (i.p.) at 1 to 5 doses of 10 μl/g or 20 μg/g body weight to induce Cre-mediated recombination. All mice were on a C57Bl/6 background.
Example 1: Identification of Fibroblast Origin
(162) Murine Lung Fibrosis Model
(163) To determine whether Tbx4+ cells expanded during injury, adult (8 to 16 weeks old) Tbx4-Cre.sup.Tg;Rosa26-tdTomato and Tbx4-Cre.sup.Ki;Rosa26-tdTomato mice (both male and female), were subjected to bleomycin induced lung injury (Li et al. J Exp Med. 2011 Jul. 4; 208(7): 1459-71; Lovgren et al. Sci Transl Med. 2011 Mar. 16; 3(74): 74ra23). Bleomycin at 2.5 U/Kg was injected intratracheally. Mouse lungs were harvested on day 14 or day 21 for examination via histology, hydroxyproline assay, and single cell isolation.
(164) 5-Bromo-2-Deoxyuridine (BrdU) Incorporation Assay
(165) Cell proliferation was quantified by BrdU labeling. BrdU (5 mg/ml, Sigma B-9285) was injected intraperitoneally into bleomycin treated Tbx4-CreER; Rosa26-tdTomato mice at a dose of 10 μl/g body weight at day 21. After 3 h, lungs were collected and fixed by 10% formaldehyde. Lungs were embedded with O.C.T. compound (Sakura Finetek). Frozen sections were processed for antigen retrieval by applying 1.5 N HCl to the section for 30-45 min. The sections were subsequently processed for immunofluorescent staining with anti-BrdU antibody (1:250; Accurate Chemicals), followed by Alexa Fluor 488-conjugated secondary antibody (1:500; Invitrogen). The number of BrdU-positive cells was counted in 5 random fields under 60× magnification and the average number of labeled cells per field was calculated.
(166) Scale Processing and Imaging
(167) Tbx4-CreER Confetti mouse lungs were fixed with 10% formalin for 4-16 hrs and washing with PBS overnight. Tissue was transferred to Scale A2 (an aqueous solution used for making biological tissue samples transparent prepared by dissolving, in pure water, 4M urea, 0.1% (w/v) Triton X-100, and 10% w/v glycerol) and kept on a rocker at 4° C. for 1-2 weeks until maximal tissue clearing occurred. Whole-mount samples immersed in Scale A2 were imaged on the inverted Zeiss 780 confocal microscope in a 35-mm glass-bottom microwell dish with a 20× objective. Z-stack and tiled images were acquired from large areas and volumes of tissue. Images were processed by Zen software.
(168) Histology, Immunofluorescence, and Confocal Imaging
(169) Immunofluorescence staining on frozen sections (5 μm or 12 μm) was performed using primary antibodies to αSMA (A2547, Sigma), Desmin (RB9014-P0, Thermo Scientific), NG2 (AB5320, Millipore), Vimentin (sc-7557, Santa Cruz), Col1a1 (NB600-408, Novus Biologicals), PDGFRB (04-397, Millipore), von Willebrand factor (Ab6994, Abcam), lipid acid (a lipophilic stain used to identify lipofibroblasts; Vybrant® DiO Cell-Labeling Solution, v22886, Life Technology), surfactant protein C (SPC) (AB3786, Millipore), clara cell 10 protein (CC10) (Stripp lab), podoplanin (PDPN) (8.1.1-c, Developmental Studies Hybridoma Bank), secretoglobin family 1A member 1 (Scgb1a1) (Cedars-Sinai Medical Center, Los Angeles, Calif.), bromodeoxyuridine (BrdU) (OBT0030, Accurate Chemical and Scientific), biotinylated recombinant human aggrecan (staining for HA, custom order from R&D Systems Inc., lot PQP051505A) and associated Alexa Fluor 488 conjugated secondary antibodies (Life Technology). Paraffin lung sections were stained with RFP (600-401-379, Rockland). Stained sections were imaged using a Zeiss 780 reverse Laser Scanning Confocal Microscope (Zeiss). The following protocol was implemented:
(170) Preparation of Slides and Samples:
(171) For frozen sections, fresh tissue is snap frozen in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, and stored at −80° C. Sections (4-8 μm thick) are cut and mounted on superfrost or gelatin coated slides. The slide can be stored at −80° C. until needed. Prior to fixation, the slides are warmed to room temperature for 30 minutes.
(172) For paraffin-embedded sections, paraffin is removed from the sections via treatment in xylene for 2×5 minutes. The sections are hydrated with 100% ethanol for 2×3 minutes; with 95% ethanol for 1 minute; and rinsed in distilled water prior to fixation.
(173) The samples are fixed either in ice-cold methanol, acetone (1-10 minutes) or in 3-4% paraformaldehyde in 0.01M phosphate-buffered saline (PBS), pH 7.4 for 15 minutes at room temperature, and washed twice with ice cold PBS.
(174) If the target protein is expressed intracellularly, it is important to permeabilize the cells. Acetone fixed samples do not require permeabilization. The samples are incubated for 10 minutes with PBS containing 0.25% Triton X-100. For analysis of membrane-associated antigens, 100 μM digitonin or 0.5% saponin is used in place of Triton X-100. Cells are washed in PBS three times for 5 mins.
(175) The cells are incubated with 1% bovine serum albumin (BSA) in PBS with 0.05% Tween-20 detergent (PBS-T) for 30 min to block nonspecific binding of the antibodies; alternative blocking solutions are 1% gelatin or 10% serum from the species in which the secondary antibody was raised, or other commercially available blocking solutions.
(176) The cells are incubated with a primary incubation solution comprising one or a mixture of primary antibodies in 1% BSA in PBS-T in a humidified chamber for 1 hour at room temperature or overnight at 4° C. The primary incubation solution is decanted and the cells are washed three times with PBS (5 minutes per wash). The cells are incubated with a secondary incubation solution comprising associated Alexa Fluor 488 conjugated secondary antibodies (for frozen sections) or RFP (for paraffin lung sections) in 1% BSA for 1 hour at room temperature under dark conditions. The secondary incubation solution is decanted and the cells are washed three times with PBS (5 minutes per wash) in the dark.
(177) For counter-staining, cells are incubated with: 0.1-1 μg/ml of counter stain for 1 min, and then rinsed with PBS. A coverslip is mounted with a drop of mounting medium, sealed with nail polish to prevent drying and movement under the microscope, and stored in the dark at −20° C. or 4° C.
(178) The excitation and optimal detection wavelengths of the Alexa Fluor 488, RFP, and Vybrant Dyes are as follows:
(179) TABLE-US-00001 Dye Excitation (nm) Detection (nm) Alexa Fluor 488 488 519 RFP 488 or 532 588 Vybrant DiO 484 501
(180) Hydroxyproline Assay
(181) A hydroxyproline assay for quantitatively measuring hydroxyproline resulting from the hydrolysis of collagen in tissue and protein/peptide hydrolysates was used to measure collagen content in lung tissue from 7-17 mice per group. The ability of the assay to completely hydrolyze and recover hydroxyproline from collagen was confirmed using samples containing known amounts of purified collagen.
(182) An exemplary hydroxyproline assay kit is available from Sigma-Aldrich (Cat. No. MAK008). Vials are briefly centrifuged before opening. To maintain reagent integrity, repeated freeze/thaw cycles are avoided. Oxidation buffer is allowed to come to room temperature before use. DMAB Concentrate is warmed to room temperature prior to use, and stored protected from light and moisture at 2-8° C.
(183) All samples and standards are run in duplicate. Ultrapure water is used for the preparation of standards and samples.
(184) 10 μL of the 1 mg/mL Hydroxyproline Standard Solution is diluted with 90 mL of water to prepare a 0.1 mg/mL standard solution. 0, 2, 4, 6, 8, and 10 μL of the 0.1 mg/mL hydroxyproline standard solution is added into a 96 well plate, generating 0 (blank), 0.2, 0.4, 0.6, 0.8, and 1.0 μg/well standards. Because endogenous compounds may interfere with the reaction, a sample spiked with 0.4 μg of the hydroxyproline standard is included as a spiked sample control to ensure the accurate determination of Hydroxyproline in the test samples,
(185) 10 mg tissue or cells are homogenized in 100 μL of water and transferred to a container, e.g., a pressure-tight vial with PTFE-lined cap or to 2.0 mL polypropylene tube. 100 μL of concentrated hydrochloric acid (HCl, ˜12 M) is added, the container is capped tightly, and hydrolysis allowed to take place at 120° C. for 3 hours. 10-50 μL of supernatant is transferred to a 96 well plate.
(186) All wells are evaporated to dryness under vacuum or the plates are placed in a 60° C. oven to dry the samples.
(187) Chloramine T/Oxidation Buffer Mixture and Diluted DMAB Reagent are stable for 2-3 hours after preparation, and should be prepared after sample preparation, just prior to the start of the assay.
(188) 100 μL Chloramine T/Oxidation Buffer Mixture is required for each reaction well. For each well, 6 μL of Chloramine T Concentrate is added to 94 μL of Oxidation Buffer and mix well.
(189) 100 μL Diluted DMAB Reagent is required for each reaction well. For each well, 50 μL of DMAB Concentrate is added to 50 μL of Perchloric Acid/Isopropanol Solution and mix well.
(190) 100 μL of the Chloramine T/Oxidation Buffer Mixture is added to each sample and standard well and incubated at room temperature for 5 minutes. 100 μL of the Diluted DMAB Reagent is added to each sample and standard well, and incubated for 90 minutes at 60° C. Absorbance at 560 nm (A560) is measured.
(191) The background for the assay is the value obtained for the 0 (blank) hydroxyproline standard and is subtracted from all readings. A standard curve is plotted from the values obtained from the appropriate hydroxyproline standards. The amount of hydroxyproline present in the samples may be determined from the standard curve. The concentration of Hydroxyproline in a sample (C)=S.sub.a/S.sub.v, where S.sub.a=Amount of hydroxyproline in unknown sample (μg) from standard curve; and S.sub.v=Sample volume (μL) added into the wells. For spiked samples, any sample interference is corrected for by subtracting the sample reading from the spiked sample reading according to the following formula:
(192)
(193) Fibroblast Isolation and Culture
(194) Primary fibroblasts were derived from mouse lungs as described previously (Tager A M, et al. Am J Respir Cell Mol Biol. 2004 October; 31(4): 395-404). Briefly, lungs from unchallenged C57Bl/6 mice, and mice 5 and 14 d after bleomycin challenge, were digested for 45 min at 37° C. in Roswell Park Memorial Institute medium (RPMI) with 0.28 U/ml Liberase™ Research Grade (Roche, enzyme blend comprising collagenases I and II and thermolysin, for the dissociation of tissues) and 60 Um′ DNase I, passed through a 70 μm filter, centrifuged at 540×g at 4° C., and plated in tissue culture flasks in Dulbecco's modified Eagle's medium (DMEM) with 15% fetal bovine serum (FBS); cells were passaged when subconfluent after harvest with trypsin-EDTA (Cellgro, Herndon, Va.) (Id.). The cells were used from three to six generations. Human lung fibroblasts were isolated from surgical lung biopsies or lung transplant explants obtained from patients with idiopathic pulmonary fibrosis (IPF) (Meltzer et al. BMC Med Genomics. 2011 Oct. 5; 4:70). Pathological confirmation was obtained for every case. IPF was confirmed by the identification of a usual interstitial pneumonia under the light microscope. Patient demographics were as follows: 2 females and 2 males; ages between 51 and 78 with median age 67; percentage forced vital capacity (FVC %) 51-78, median 57; and percentage diffusing capacity factor of the lung for carbon monoxide (DLCO %) 18-42, median 30.75. Samples of whole lung tissue were obtained at the time during orthotopic lung transplantation surgery. Samples were immediately processed following removal from the body. The specimens were cut into small pieces (˜1 mm in diameter), and were cultured in DMEM supplemented with 15% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml gentamicin, and 0.25 μg/ml amphotericin B. The cells of passages 5-7 were used for invasion assays, siRNA interference assays, and HA amount measurements.
(195) Matrigel Invasion Assay
(196) The invasive behavior of Tbx4 positive and negative fibroblasts isolated from Tbx4-Cre Rosa26-tdTomato mouse lungs was performed essentially as described previously (Li et al. J Exp Med. 2011 Jul. 4; 208(7): 1459-71). Briefly, equal numbers of fibroblasts (5×10.sup.4) in 500 μl of 10% FBS complete medium were plated onto BioCoat Matrigel Invasion Chambers (Corning) containing polyethylene terephthalate (PET) filter-inserts containing 8 μm pores. 750 μl of 10% FBS complete medium with 10 ng/ml PDGF (R & D 120-HD-001) were added to the bottom of wells. After 24 hours of incubation in a CO.sub.2 incubator, media were removed, and the filter inserts with the invaded cells washed once with PBS followed by fixing and staining with a Protocol Hema 3 stain set (Fisher Scientific). Matrigel matrix and non-invading cells on the upper surface of the filter were removed by wiping with a cotton swab; the filters were removed from the insert by a scalpel blade and mounted onto glass slides. The invading cells of each sample were counted in nine randomly selected fields of duplicate filters under a microscope at 400× magnification. Tbx4 siRNA effects on 3T3 cell line and human IPF lung fibroblast invasion were assessed using 30 nM of the siRNA shown in Table 1 below:
(197) TABLE-US-00002 TABLE 1 siRNA Sequences SEQ ID Name Sequence No: mTbx4 5′-rGrCrArCrUrGrCrCrArArGrArAr 1 siRNA ArCrArUrGrGrArArArGGT-3′ hTbx4 5′-rUrGrCrArArUrUrArUrCrUrArAr 2 siRNA GrArArGrUrGrArCrUrUTG-3′
(198) Trilencer-27 universal scrambled negative control siRNA duplex (SR30004, OriGene Technologies, Inc.) was used as a negative control. The cells were transfected with siRNAs for 48 hours before performing the invasion assay.
(199) Mouse Embryonic Fibroblast (MEF) Isolation and Culture
(200) Mouse embryonic fibroblast (MEF) cells were derived from E14 embryos of CMV-Cre Tbx4.sup.f/w or control (CMV-Cre only or Tbx4.sup.f/w only) mice as described previously (Jozefczuk J, Drews K, Adjaye J. Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells. J Vis Exp. 2012; (64):3854).
(201) Briefly, mouse embryonic fibroblasts are isolated as follows. A pregnant mouse is sacrificed at 13 or 14 days post-coitum by cervical dislocation. The uterine horns are dissected out, briefly rinsed in 70% (v/v) ethanol, and placed into a falcon tube containing PBS without Ca.sup.2+Mg.sup.2+ (Gibco, Invitrogen). The uterine horns are placed into a Petri dish and each embryo separated from its placenta and embryonic sac. The head and red organs are dissected, washed in PBS and all embryos placed in a clean Petri dish. The tissue is finely minced using a sterile razor blade until it becomes possible to pipette, and 1 ml of 0.05% trypsin/EDTA (Gibco, Invitrogen), including 100 Kunitz units of DNase I (USB), is added per embryo. The tissue is transferred into a 50 ml falcon tube and incubated for 15 min at 37° C. After each 5 min of incubation, the cells are dissociated by pipetting up and down thoroughly. The trypsin is inactivated by adding about 1 volume of freshly prepared MEF medium containing 450 ml of DMEM, 50 ml of FBS (10% (v/v)), 5 ml of 200 mM L-glutamine (1/100 (v/v)), 5 ml of Penicillin-streptomycin (1/100 (v/v). The cells are centrifuged at low-speed (300×g), 5 min, the supernatant carefully removed, and the cell pellet then resuspended in warm MEF medium.
(202) A number of cells which is approximately equivalent to 3-4 embryos is plated in each T150 (TPP) flask coated with 0.2% gelatine (Gelatine from bovine skin, Type B, Sigma) for 2 hr. The fibroblasts (P0, passage 0) are the only cells that have the ability to attach to the gelatine-coated flasks. Ideally, cells are 80-90% confluent after 24 hr and at this stage a major part of P0 cells is frozen for future usage.
(203) The remaining T150 flask(s) of P0 cells is expanded until passage 3(P3) or passage 4 (P4), inactivated and used as feeders to replate hESCs or to produce conditioned medium (CM) (Jozefczuk et al. J Vis Exp. 2012 Jun. 21; (68): 3854).
(204) The cells were used from three to six generations. MEFs were treated with 5 ng/ml TGF-β1 overnight. RNA was isolated for RT-PCR and supernatant was collected for HA ELISA.
(205) Flow Cytometry
(206) Mouse lung or bone marrow was isolated from Tbx4-Cre Rosa26-tdTomato mice, αSMAGFP Tbx4-Cre Rosa26-tdTomato mice, Col1a1-GFP Tbx4LME-Cre Rosa26-tdTomato mice and NG2-YFP Tbx4LME-Cre Rosa26-tdTomato mice after perfusion. Single mouse lung cells were dissociated by using mouse lung dissociation kit and gentleMACS dissociator (Miltenyi Biotec). Single bone marrow cells from Tbx4-Cre;Rosa26-tdTomato mice were dissociated by using pipette, followed by red blood cell lysis. Cells were analyzed with Fortessa (BD Biosciences). Fluorescence intensity is expressed in arbitrary units on a logarithmic scale or on a linear scale for forward scatter.
(207) Cultured passage 3 fibroblasts from non-treated Tbx4-Cre Rosa26-tdTomato mice were sorted for tdTomato positive and tdTomato negative cells with FACS Aria 3 (BD Biosciences) to 95-98% purity.
(208) Quantitative Real-Time (RT PCR
(209) Real-time RT-PCR was used to quantify the relative mRNA levels of HAS2 in CMV-Cre Tbx4.sup.f/w or control MEF cells with or without human recombinant TGF-β1 treatment using gene-specific primers. In brief, total RNA was purified using RNAqueous-4PCR kit (Life Technology) and was reversed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Life Technology) according to the manufacturer's instructions. HAS2 gene levels in the resultant cDNAs were examined using the ABI 7500 Fast Detection system (Applied Biosystems) with SYBR green as fluorescent dye enabling real-time detection of PCR products according to the manufacturer's protocol (Power SYBR green PCR Master Mix; Applied Biosystems). The relative expression levels of the gene were determined against GAPDH levels in the samples. The RT-PCR primers in listed in Table 2 were used.
(210) TABLE-US-00003 TABLE 2 RT-PCR Primers SEQ ID Name Sequence No: Mouse HAS2 5′-ACGACGACCTTTACATGA 3 Forward TGGA-3′ (GenBank accession no. NM_008216) Mouse HAS2 5′-GATGTACGTGGCCGATTT 4 Reverse GCT-3′ Mouse Tbx4 5′-TCACTGGATGCGGCAGTT 5 Forward GGTCTCT-3′ (GenBank accession no. NM_011536.2) Mouse Tbx4 5′-CACGTGGGTGCAAAAGGC 6 Reverse TGTGTTT-3′ Mouse 5′-ATCATCTCCGCCCCTTCT 7 GAPDH G-3′ Forward Mouse 5′-GGTCATGAGCCCTTCCAC 8 GAPDH AAC-3′ Reverse
(211) Affymetrix cDNA Microarray
(212) Cultured passage 3 fibroblasts from (untreated) Tbx4-Cre Rosa26-tdTomato mice were sorted for tdTomato positive and tdTomato negative cells with FACS Aria 3 (BD Biosciences) to 95-98% purity. Total RNA was assessed for quality with an Agilent 2100 Bioanalyzer G2939A (Agilent Technologies, Santa Clara, Calif.) and a Nanodrop 8000 spectrophotometer (Thermo Scientific/Nanodrop, Wilmington, Del.). Hybridization targets were prepared with the Ovation Pico WT Amp V2 kit (NuGen, San Carlos, Calif.) and the Encore Biotin Module (NuGen, San Carlos, Calif.) from total RNA, hybridized to GeneChip® Mouse 430 2.0 arrays in Affymetrix GeneChip® hybridization oven 645, washed in Affymetrix GeneChip® Fluidics Station 450 and scanned with Affymetrix GeneChip® Scanner 7G according to standard Affymetrix GeneChip® Hybridization, Wash, and Stain protocols. (Affymetrix, Santa Clara, Calif.).
(213) Table 3 lists the necessary amount of cRNA required for the specific probe array format used.
(214) TABLE-US-00004 TABLE 3 Hybridization Cocktail for Single Probe Arrays 169 Format 49 Format (Mini) Array/ (Standard)/64 100 Format 400 Format Final Component Format Array (Midi Array) (Micro) Array Dilution Fragmented and 15 μg 10 μg 5 μg 0.05 μg/μL Labeled cRNA Control 5 μL 3.3 μL 1.7 μL 50 pM Oligonucleotide B2 (3 nM) 20X Eukaryotic 15 μL 10 μL 5 μL 1.5, 5, 25, and Hybridization 100 pM Controls (bioB, respectively bioC, bioD, cre) 2X 150 μL 100 μL 50 μL 1X Hybridization Mix DMSO 30 μL 20 μL 10 μL 10% Nuclease-free to final volume to final volume to final volume Water of 300 μL of 200 μL of 100 μL Total Volume 300 μL 200 μL 100
(215) For each target, the following are mixed, scaling up volumes if necessary for hybridization to multiple probe arrays. Probe arrays are equilibrated to room temperature immediately before use, the hybridization cocktail is heated to 99° C. for 5 minutes in a heat block, and the array is wet with an appropriate volume of Pre-Hybridization Mix by filling it through one of the septa. The probe array filled with Pre-Hybridization Mix is incubated at 45° C. for 10 minutes with rotation; the hybridization cocktail that has been heated at 99° C., is transferred, to a 45° C. heat block for 5 minutes; and the hybridization cocktail is spun at maximum speed in a microcentrifuge for 5 minutes to collect any insoluble material from the hybridization mixture.
(216) The array is removed from the hybridization oven, vented with a clean pipette tip, and the Pre-Hybridization Mix extracted from the array with a micropipettor. The array is refilled with an appropriate volume of the clarified hybridization cocktail, avoiding any insoluble matter at the bottom of the tube. The probe array is placed into the hybridization oven, set to 45° C., rotated at 60 rpm, and allowed to hybridize for 16 hours.
(217) After 16 hours of hybridization, the array is removed from the hybridization oven, vented by inserting a clean pipette tip into one of the septa, and the hybridization cocktail extracted with a pipettor through the remaining septum. The probe array is refilled completely with the appropriate volume of Wash Buffer A. The stain reagents are prepared, and the probe array washed and stained.
(218) Data were background subtracted and normalized within the array using the LOESS normalization by Genespring GX 11 software.
(219) Hyaluronan (HA) Quantification
(220) The HA content of cultured media of human lung fibroblasts, of 3T3 cell lines, or in the bronchioalveolar lavage fluid from Col-Cre;Tbx4.sup.f/f mice was quantified using a Hyaluronan DuoSet ELISA (DY3614, R&D).
(221) Capture Antibody was diluted to the working concentration in PBS without carrier protein. A 96-well microplate was coated with 100 μL per well of the diluted Capture Antibody, and the plate sealed and incubated overnight at room temperature. Each well was aspirated and washed with Wash Buffer; the process was repeated two times for a total of three washes. Each well was filled with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. After the last wash, any remaining Wash Buffer was removed by aspirating or by inverting the plate and blotting it against clean paper towels. Plates were blocked plates by adding 300 μL of Reagent Diluent to each well, and incubated at room temperature for a minimum of 1 hour, after which each well was aspirated and washed as above.
(222) 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent was added per well; the plate was covered with an adhesive strip and incubated 2 hours at room temperature. Each well was aspirated and washed as above. 100 μL of the Detection Antibody, diluted in Reagent Diluent, was added to each well; the plate was covered with a new adhesive strip and incubated 2 hours at room temperature. Each well was aspirated and washed as above. 100 μL of the working dilution of Streptavidin-HRP was added to each well; the plate was covered and incubated for 20 minutes at room temperature. Each well was aspirated and washed as above. 100 μL of Substrate Solution was added to each well; the plate was incubated for 20 minutes at room temperature. 50 μL of Stop Solution was added to each well, and the plate gently tapped to ensure thorough mixing.
(223) The optical density of each well was determined immediately, using a microplate reader set to 450 nm.
(224) RNA Interference
(225) Tbx4 siRNA effects on 3T3 cell line and human IPF lung fibroblasts HA production were assessed with siRNA specific to Tbx4 (mTbx4 siRNA: 5′-rGrCrArCrUrGrCrCrArArGrArArArCrArUrGrGrArArArGGT-3′ (SEQ ID NO: 1), hTbx4 siRNA: 5′-rUrGrCrArArUrUrArUrCrUrArArGrArArGrUrGrArCrUrUTG-3′ (SEQ ID NO: 2), and Universal scrambled negative control siRNA duplex, SR30004, OriGene Technologies). The cells were transfected with 30 nM siRNAs for 48 hrs, and then treated with 5 mg/ml TGF-β for overnight. The supernatants were collected thereafter for HA measurement. Invasion assays were performed using 48 hr transfected cells.
(226) Promotor Luciferase Assay
(227) A 2880 bp Has2 promoter luciferase reporter plasmid was provided by Dr. Chin Chiang (Liu et al. Dev Biol. 2013 Mar. 15; 375(2): 160-71) (Vanderbilt University Medical Center). The plasmid contains a sequence from −2326 to exon1 of Has2 in pGL3 Basic plasmid (Promega, WI). The 901 bp (Chromosome 15—NC_000081.6:-436 to exon 1) and 664 bp (−112 to exon 1) Has2 promoter luciferase reporter plasmid was generated by PCR and cloned upstream of pGL3 Basic plasmid. Primers listed in Table 4 were used. CMV promoter driven GFP-tagged Tbx4 expression plasmid is purchased from GeneCopoeia (Rockville, Md.).
(228) TABLE-US-00005 TABLE 4 Primers for Luciferase Assay SEQ ID Name Sequence No: Forward 1 5′-CAAGCTCGAGGGAATCCTTGTAACG-3′ 9 Forward 2 5′-CCGCCTCGAGTCCCGCCCAGTCCCT-3′ 10 Reverse 5′-TGCCAAGCTTCTTGTTCAGCTCCTGCTC 11 ATAGA-3′
(229) Luciferase assays were performed essentially as previously (Liu et al., 2013), using the Promega Dual Luciferase Reporter (DLR™) assay system (Promega, WI). In brief, growth media was removed from cultured cells, the cells rinsed in 1×PBS, and all rinse solution removed. The recommended volume of 1×Passive Lysis Buffer (PLB) was dispensed into each culture vessel, which then was gently rocked/shaken for 15 minutes at room temperature. The lysate was transferred to a tube or vial. 100 μl of Luciferase Assay Reagent II (LAR II) was dispensed: into the appropriate number of luminometer tubes to complete the desired number of assays. Up to 20 μl of cell lysate was transferred into a sample tube containing LAR II; the sample was mixed by pipetting 2 or 3 times, and firefly luciferase activity measurement was recorded. 100 μl of Stop & Glo® Reagent was dispensed and the sample tube vortexed briefly to mix. Firefly luciferase activity measurement was recorded.
(230) To determine the effect of Tbx4 on different length of the Has2 promoter activity, 293HEK cells co-transfected with Has2 promoter or pGL3 Basic plasmid and Tbx4 expressing plasmid for 48 hours prior to the luciferase assay. All reporter assays were normalized using Renilla luciferase as an internal control. Each data point represents the mean of triplicate wells with error bar representing the Standard Error of the Mean.
(231) Calculation of the Internal Surface of a Lung
(232) To ensure consistent and reproducible results, all (left and right) lungs were fixed by 10% formalin and with the same pressure, degassed, and embedded in OCT. Step sectioning was performed on each block of lung. For the slides of each step section, more than 5 lung images were sampled randomly under Zeiss AXIO microscopy (×10 objective). Lung morphometric analysis was performed on the images of the lung parenchymal area for uninjured and injured lung without severe fibrotic lesion. Mean linear intercept, also known as mean chord length (Campbell H, Tomkeieff S I. Calculation of the internal surface of a lung. Nature. 1952; 170(4316):116-117), was calculated by point and intersection counting using a grid line system (Foronjy R F, Mercer B A, Maxfield M W, Powell C A, D'Armiento J, Okada Y. Structural emphysema does not correlate with lung compliance: lessons from the mouse smoking model. Exp Lung Res. 2005; 31(6):547-562.).
(233) Western Blot
(234) To detect αSMA and p-actin expression, 25 μg of cell lysate was separated through 10% SDS-PAGE electrophoresis, blotted onto a nitrocellulose membrane (Life Technologies), and incubated with mAbs against αSMA (1:1,000; F3777, Sigma Chemical Co.) and p-actin (1:1,000; 12620, Cell Signaling Technology). The bands were visualized using the ECL system (Bio-Rad).
(235) Collagen 1a1 ELISA
(236) The collagen 1a1 content in cultured media of human lung fibroblasts was quantified using a collagen 1a1 direct ELISA. A standard curve for the assay was generated using collagen type I solution (C3867, Sigma-Aldrich) and collagen 1a1 antibody (ab21286, Abcam). The concentration of the collagen 1a1 in the cell supernatants was determined by extrapolation from the standard curve.
(237) Caspase-3 Activity Assay
(238) IPF fibroblasts were cultured on 96-well plates. Caspase-3 activity assay was performed 48 hours after Tbx4 siRNA transfection. The amount of protein in cell lysates was determined using a BCA protein assay kit (Pierce Chemical Co.). Caspase-3 activity was determined using a Caspase3/CPP32 Fluorometric Assay Kit (BioVision) according to manufacturer's protocol.
(239) Calcein AM Cell Viability Assay
(240) The viability of IPF fibroblasts 48 hours after Tbx4 siRNA transfection was analyzed by Calcein AM Cell Viability Assay Kit (Trevigen) according to manufacturer's protocol.
(241) Statistics
(242) Data are expressed as the mean±SEM. Differences in measured variables between experimental and control groups were assessed by Student's t tests (2-tailed) or Wilcoxon rank-sum test with nonparametric data. One-way ANOVA with Bonferroni test was used for multiple comparisons. The survival curves were compared using the log-rank test. Results were considered statistically significant at P<0.05. GraphPad Prism software was used for statistical analysis.
(243) Results
(244) Tbx4+ Cells Give Rise to Fibroblasts, Smooth Muscle, and Some Endothelial Cells in the Lung
(245) To label Tbx4+ cells in the lung, the Tbx4 lung enhancer-Cre transgenic mice (Tbx4.sup.LME-Cre or Tbx4-Cre.sup.Tg) and Tbx4-Cre knock-in mice (Tbx4-Cre.sup.Ki) were respectively crossed with Rosa26-stop-tdTomato reporter mice to permanently mark any cells that previously or currently express Tbx4. At embryonic (E) day 15.5 and 8 weeks after birth, the fidelity of the genetic system was assessed in the lungs of both Tbx4-Cre.sup.Tg;Rosa26-tdTomato and Tbx4-Cre.sup.Ki Rosa26-tdTomato mice.
(246)
(247) The labeled Tbx4 lineage cells reside in blood vessels, underneath the bronchioles, and in interstitial mesenchyme at both the embryonic (E15.5) and adult stage (8W) (
(248) To determine whether Tbx4+ cells expanded during injury, adult (8-12 weeks old) Tbx4-Cre.sup.Tg;Rosa26-tdTomato and Tbx4-Cre.sup.Ki;Rosa26-tdTomato mice were intracheally administered with 2.5 U/kg bleomycin solution. The mouse lungs were examined 21 days after bleomycin injury (
(249) In order to address which subpopulations of cell types the Tbx4+ cells represent, immunofluorescence and confocal microscopy were used to localize stromal markers in the lungs of both Tbx4-Cre.sup.Tg;Rosa26-tdTomato and Tbx4-Cre.sup.Ki Rosa26-tdTomato mice.
(250)
(251) Antibody staining of the frozen sections of mouse lung revealed co-staining of the Tbx4 cells with several well-known stromal cell markers, including αSMA (
(252) To further confirm the antibody staining results, reporter mice were used including αSMA-GFP, Col1a1-GFP, and NG2-YFP mice, respectively, to cross with either Tbx4-Cre.sup.Tg Rosa26-tdTomato or Tbx4-Cre.sup.Ki Rosa26-tdTomato mice. In untreated SMA-GFP Tbx4-Cre.sup.Ki;Rosa26-tdTomato triple heterozygous mice, 0.29% of total cells in lung single cell digests were αSMA+Tbx4+ double positive cells by flow cytometric analysis (
(253)
(254) Pericytes have been implicated in tissue fibrosis and suggested to be a source of fibrotic fibroblasts (Humphreys et al., 2010; Hung et al., 2013; Lin et al., 2008). PDGFRB, chondroitin sulfate proteoglycan 4 (CSPG4, or NG2), along with FoxD1 and FoxJ1 have been used as pericyte markers [Hung et al. Am J Respir. Crit Care Med. 2013 Oct. 1; 188(7): 820-30; Rock et al. Proc Natl Acad Sci USA. 2011 Dec. 27; 108(52): E1475-83) REFS]. As shown in
(255) Considering that the majority of αSMA+ cells are Tbx4+ cells, NG2+ cells may represent only a small population of αSMA+ cells, which is consistent with previous studies using NG2-CreER mice, showing that NG2 positive cells do not express high levels of αSMA in fibrotic lungs (Rock et al. Proc Natl Acad Sci USA. 2011 Dec. 27; 108(52): E1475-83). Recent research does not support NG2 as a reliable marker for all pericytes (Duffield. J Clin. Invest. 2014 June; 124(6): 2299-306; Hung et al. Am J Respir. Crit Care Med. 2013 Oct. 1; 188(7): 820-30; LeBleu et al. Nat Med. 2013 August; 19(8): 1047-53). Antibody staining with PDGFRB, another pericyte marker, exhibited much more co-localization than NG2 staining in Tbx4+ cells (
(256) It was recently reported that lipofibroblasts may contribute to a stem cell niche in the murine lung (Barkauskas et al. J Clin Invest. 2013 July; 123(7): 3025-36). To determine whether Tbx4 contributed to lipofibroblasts, Tbx4-Cre.sup.tg;Rosa26-tdTomato lung sections were stained by Vybrant DiO staining. It was found that some Tbx4+ cells were identified as lipofibroblasts.
(257) A minor population of Tbx4+ cells staining positive for von Willebrand factor (vWF) were found in both injured and normal adult mouse lung (
(258)
(259) Tbx4+ lineage cells did not contain any epithelial cells, since Tbx4+ cells had no overlap with any lung epithelial cell markers including AEC2 marker surfactant protein C (SFTPC or SPC), AEC1 marker podoplanin (PDPN; or T1a), and club cell marker CC10 (
(260) Tbx4+ cells, if any, were rare in extrapulmonary locations in both strains of mice (Tbx4-Cre.sup.Tg;Rosa26-tdTomato and Tbx4-Cre.sup.Ki;Rosa26-tdTomato). In bone marrow, a small portion (less than 0.03%) of total live cells showing tdTomato color was found in Tbx4-Cre.sup.Ki;Rosa26-tdTomato mice, while none were observed in Tbx4-Cre.sup.Tg;Rosa26-tdTomato mice (
(261)
(262) Other organs such as hearts, livers, kidneys, spleens, and intestines of normal 8 week old Tbx4cre.sup.Ki;Rosa26-tdTomato mice also were examined, but none had any tdTomato cellular labeling (data not shown).
(263) Together, these data indicate that Tbx4+ cells are heterogeneous cell types including smooth muscle cells, and fibroblasts, as well as some pericytes and endothelial cells. Tbx4+ fibroblasts are expanded during pulmonary fibrosis, and the majority of αSMA+ myofibroblasts are Tbx4+ cells.
(264) Tbx4+ Cells were Proliferative and Induced De Novo Upon Bleomycin Injury in the Lung
(265) To address the question of the source of the expanded Tbx4+ cells, and whether Tbx4+ cells labeled prior to or after injury contribute to the expansion of Tbx4+ cells, tamoxifen induced cell fate mapping experiments were carried out. Tbx4-CreER transgenic mice (Kumar, M E et al, 2014, Science 346: 1258810) were bred with Rosa26-tdTomato mice. In these double transgenic mice, tdTomato labeling of the Tbx4+ cells is controlled in time by the administration of tamoxifen, which activates Cre, allowing for the distinct fate mapping of adult Tbx4+ cells, as well as of injury-induced Tbx4+ cells (
(266)
(267)
(268) One, three, or four doses of tamoxifen were administered one week before harvest. As shown in
(269) One dose or four doses of tamoxifen then were tested one week before bleomycin-induced injury. Fourteen or 21 d after bleomycin treatment, mouse lungs were harvested for frozen section. A significant increase of Tbx4+ cells was detected in mouse lungs 14 and 21 days after bleomycin treatment when compared with untreated lungs (
(270) Tbx4-CreER mice were crossed with confetti mice to determine if Tbx4 lineage cells are clonally expanded (
(271)
(272) These data demonstrated that Tbx4 lineage fibroblasts undergo clonal-like expansion during lung injury and fibrosis.
(273) BrdU labeling also indicated that Tbx4+ cells labeled before injury were proliferating in Tbx4-CreER;Rosa26-tdTomato mice 21 days after bleomycin treatment (
(274)
(275) In order to label Tbx4+ cells after injury, five doses of tamoxifen were administered every other day beginning one week following bleomycin injection in Tbx4-CreER;Rosa26-tdTomato mice and the lungs were harvested on d21 (
(276)
(277) The data indicate that Tbx4+ cells labeled before and after injury both proliferated and contributed to the expansion of Tbx4+ cells.
(278) Ablation of Tbx4 Cells or Signaling Inhibits Pulmonary Fibrosis
(279) To directly demonstrate the functional role of Tbx4+ fibroblasts and their cellular progeny in lung fibrosis, Tbx4-CreER mice were crossed with Rosa26-Stop-DTA mice, which express diphtheria toxin fragment A (DTA) under control of the Tbx4 locus, or Rosa260tdTomato Rosa26-Stop-DTA mice. The tamoxifen-inducible, Cre-mediated combination results in deletion of Tbx4-expressing cells. These mice were given five injections of tamoxifen for 1 week after bleomycin treatment to induce ablation of Tbx4+ cells (
(280)
(281) A significant decrease in collagen content was detected in tamoxifen injected Tbx4-CreER;Rosa26-DTA mouse lungs 21 days after bleomycin treatment (
(282) Injection of tamoxifen induced a marked reduction of progeny of TBX4 cells (tdT+ cells) (
(283)
(284) Thus, the loss of Tbx4+ cells and their progeny attenuated interstitial fibrosis.
(285) To elucidate the role of Tbx4 signaling in lung fibrosis, the impact of deletion of Tbx4 signaling in fibroblasts or myofibroblasts was evaluated. Col1a2-CreER transgenic mice were crossed with Tbx4.sup.flox/flox mice (Arora et al. PLoS Genet. 2012; 8(8): e1002866), resulting in Tbx4 gene deletion in collagen-expressing cells (
(286) In addition, Tbx4.sup.flox/flox mice were crossed with SMA-CreER mice in order to delete the Tbx4 gene in αSMA-expressing myofibroblasts when tamoxifen was administered (
(287) These data indicate that Tbx4-lineage cells play a role in regulating fibrosis in the bleomycin-induced lung injury model and that the loss of TBX4-expressing cells and their progeny attenuated interstitial lung fibrosis.
(288) Tbx4 Regulates Fibroblast Invasion Through HAS2
(289) A fraction of lung fibroblasts are invasive in patients with IPF (Li et al. J Exp Med. 2011 Jul. 4; 208(7): 1459-71; White, E S et al., 2003, J. Pathol. 201: 343-354) and the fibroblast invasion phenotype determines fibrosis progression (Li et al., 2011). It was previously shown that hyaluronan synthase 2 (HAS2), hyaluronan (HA) receptor CD44, and beta-arrestins regulate fibroblast invasion in vitro and in vivo (Li et al. J Exp Med. 2011 Jul. 4; 208(7): 1459-71; Lovgren et al. Sci Transl Med. 2011 Mar. 16; 3(74): 74ra23).
(290) The differences in invasiveness behavior between Tbx4+ fibroblasts and Tbx4− fibroblasts were evaluated. Tbx4− fibroblasts were isolated from Tbx4-Cre.sup.Tg;Rosa26-tdTomato mouse lungs, cultured in vitro for 3 passages, and the tdTomato+ and tdTomato− cells were flow sorted.
(291)
(292) Microarrays were performed by using the RNAs isolated from sorted tdTomato+ and tdTomato− cells, and the tdTomato+ cells were found to have more than a 2 fold increase of Tbx4 transcript expression over the tdTomato− cells.
(293)
(294) Tbx4+ cells were found to be more invasive compared with Tbx4-cells in a Matrigel invasion assay (
(295) To determine if the invasiveness of Tbx4+ fibroblasts was due to expression of HAS2, the expression levels of HAS2 between Tbx4+ fibroblasts and Tbx4-fibroblasts were compared. More Has2 mRNA was in Tbx4+ fibroblasts when compared to Tbx4− cells (
(296) There are three putative T box response elements in Has2 promoter (Arora et al., 2012a). Luciferase promoter assay showed that Tbx4 could significantly induce luciferase activity of luciferase vectors containing the Has2 promoters (
(297) Discussion
(298) This study has identified a TBX4-driven fibrogenic cell population as a heterogeneous mesenchymal population, containing interstitial fibroblasts, vascular and airway smooth muscle cells, some pericytes, and a few endothelial cells in the adult mouse lung in normal and disease states. TBX4-labeled cells did not give rise to any lung cells expressing epithelial cell markers. More importantly, Tbx4-lineage cells expanded, proliferated, and formed organized clonal patches during lung fibrosis in mice in vivo. Almost all of the αSMA+ myofibroblasts and COL1a1+ fibroblasts were Tbx4-derived cells, suggesting a role for this population of cells in lung fibrosis. This is further supported by data showing that lung fibrosis is significantly reduced by ablation of Tbx4-lineage cells. When Tbx4 was deleted in SMA- or COL1a2-expressing fibroblasts, lung fibrosis was also significantly reduced. These data support a non-redundant role for TBX4 in regulating the pathobiology of lung fibrosis and show TBX4 mediated fibroblast invasiveness and HA production by transcriptional regulation of HAS2.
(299) These data also show that virtually all αSMA-expressing myofibroblasts as well as COL1a1-expressing fibroblasts were derived from TBX4-expressing cells. Without being bound by theory, these data suggest that within the lung, a local lineage of interstitial resident fibroblasts promotes fibrogenesis, which is consistent with recent studies in skin fibrosis (Dulauroy S, Di Carlo S E, Langa F, Eberl G, Peduto L. Lineage tracing and genetic ablation of ADAM12(+) perivascular cells identify a major source of profibrotic cells during acute tissue injury. Nat Med. 2012; 18(8):1262-1270; 38. Rinkevich Y, et al. Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential. Science. 2015; 348(6232):aaa2151), lung fibrosis (Hung C, et al. Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis. Am J Respir Crit Care Med. 2013; 188(7):820-830), and liver fibrosis (Iwaisako K, et al. Origin of myofibroblasts in the fibrotic liver in mice. Proc Natl Acad Sci USA. 2014; 111(32):E3297-E3305). The demonstration that Tbx4-lineage cells were devoid of all epithelial cell markers also is in line with previous work (Rock J R, et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc Natl Acad Sci USA. 2011; 108(52):E1475-E1483) suggesting that cell lineage commitment does not cross between epithelial and stromal compartments in lung fibrosis. Epithelial-mesenchymal transition (EMT) has been suggested in the pathogenesis of cardiac fibrosis (Kalluri R, Neilson E G. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest. 2003; 112(12):1776-1784), lung fibrosis (Kim K K, et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci USA. 2006; 103(35):13180-13185; Tanjore H, et al. Contribution of epithelial-derived fibroblasts to bleomycin-induced lung fibrosis. Am J Respir Crit Care Med. 2009; 180(7):657-665; Chapman H A. Epithelial-mesenchymal interactions in pulmonary fibrosis. Annu Rev Physiol. 2011; 73:413-435; Willis B C, et al. Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-p1: potential role in idiopathic pulmonary fibrosis. Am J Pathol. 2005; 166(5):1321-1332), and kidney fibrosis (Zeisberg M, et al. BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med. 2003; 9(7):964-968; Iwano M, Plieth D, Danoff T M, Xue C, Okada H, Neilson E G. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest. 2002; 110(3):341-350.). Conversely, the data did not support a major role for EMT in lung fibrosis. In addition, extrapulmonary contribution of fibroblasts and myofibroblasts is not regulated by Tbx4-derived cells. Bone marrow-derived stromal progenitor cells such as fibrocytes (Bucala R, Spiegel L A, Chesney J, Hogan M, Cerami A. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med. 1994; 1(1):71-81) have been suggested in lung fibrosis (Hashimoto N, Jin H, Liu T, Chensue S W, Phan S H. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest. 2004; 113(2):243-252; Moore B B, et al. CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am J Pathol. 2005; 166(3):675-684). A very small fraction of Tbx4-derived cells were observed in the bone marrow. Collectively, these data indicate that the majority of myofibroblasts and fibroblasts that accumulate during fibrosis are Tbx4 lineage-derived mesenchymal intrapulmonary cells regardless of the particular cell surface expression marker.
(300) Antibody staining data indicated that Tbx4-lineage cells constitutively labeled from the embryonic stage or inducibly labeled post-natally can differentiate into heterogeneous stromal cell types including αSMA+ and COL1a1+ cells. To confirm these antibody staining results, several transgenic reporter lines were used, including αSMA-GFP and COL1a1-GFP mice, to detect the co-localization of αSMA- and Col1a1-expressing cells within the Tbx4-lineage cells by means of confocal microscopy and FACS analysis. The data showed that αSMA-expressing cells are significantly increased in the fibrotic lung. Almost all of the αSMA-expressing cells (over 90%) were Tbx4-lineage cells. A large portion of Tbx4-lineage-only (TBX4+αSMA−) cells also were detected, indicating that not only αSMA+ cells but other non-αSMA mesenchymal cells accumulate during fibrosis. These data are in line with a previous study showing that multiple stromal populations contribute to pulmonary fibrosis, including αSMA, vimentin, desmin, PDGFRp, and NG2, by using antibody staining (Rock J R, et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc Natl Acad Sci USA. 2011; 108(52):E1475-E1483.). In addition, the data showed that the vast majority of COL1a1+ cells are Tbx4-lineage cells. COL1a1+ cells are considered resident fibroblasts (Hung C, et al. Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis. Am J Respir Crit Care Med. 2013; 188(7):820-830; Kramann R, et al. Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis. Cell Stem Cell. 2015; 16(1):51-66; Iwaisako K, et al. Origin of myofibroblasts in the fibrotic liver in mice. Proc Natl Acad Sci USA. 2014; 111(32):E3297-E3305), while some data suggest that circulating fibrocytes can express COL1a1 (Bucala R, Spiegel L A, Chesney J, Hogan M, Cerami A. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med. 1994; 1(1):71-81; van Deventer H W, Palmieri D A, Wu Q P, McCook E C, Serody J S. Circulating fibrocytes prepare the lung for cancer metastasis by recruiting Ly-6C+ monocytes via CCL2. J Immunol. 2013; 190(9):4861-4867.). Without being bound by theory, the data presented in this study suggest that the majority of COL1a1+ cells are resident fibroblasts, although a small proportion of COL1a1 single-positive cells, the circulating fibrocytes, were observed.
(301) Pericytes are defined as mesenchymal cells that share a common basement membrane with endothelial cells (Bergers G, Song S. The role of pericytes in blood-vessel formation and maintenance. Neurooncology. 2005; 7(4):452-464). They have been suggested as a source of fibroblasts during tissue fibrosis (Kramann R, et al. Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis. Cell Stem Cell. 2015; 16(1):51-66; Humphreys B D, et al. Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol. 2010; 176(1):85-97). The lineage labeling data demonstrated that a maximum of 20% of NG2+ cells are Tbx4-lineage cells. Considering that the majority of αSMA+ cells are Tbx4-lineage cells, our results indicate that NG2+ cells represent a minor population of αSMA+ cells. These data are consistent with a previous study using Ng2-CreER mice, which demonstrated that NG2+ pericytes do not express high levels of αSMA in fibrotic lungs (Rock J R, et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc Natl Acad Sci USA. 2011; 108(52):E1475-E1483). These data also support recent reports that NG2 may not be a reliable marker for all pericytes (Hung C, et al. Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis. Am J Respir Crit Care Med. 2013; 188(7):820-830; LeBleu V S, et al. Origin and function of myofibroblasts in kidney fibrosis. Nat Med. 2013; 19(8):1047-1053; Duffield J S. Cellular and molecular mechanisms in kidney fibrosis. J Clin Invest. 2014; 124(6):2299-2306). Antibody staining with another pericyte marker, PDGFRβ, showed much more co-localization with Tbx4-lineage cells than NG2 staining, which suggests that PDGFRβ may be a better marker for pericytes than NG2. Collectively, these data show that NG2+ pericytes are not the major contributor to myofibroblasts in lung fibrosis.
(302) The antibody staining data also showed that Tbx4-lineage progenitor cells not only contain αSMA+, COL1a1+, and NG2+ cells, but also include endothelial cells. The data indicated that this population does not increase during fibrosis, consistent with a report that PECAM-1 co-stained with Tbx4-lineage cells in embryonic mouse lung (Zhang W, et al. Spatial-temporal targeting of lung-specific mesenchyme by a Tbx4 enhancer. BMC Biol. 2013; 11:111).
(303) Mesenchyme-specific clonal analysis was used to examine expansion of TBX4-expressing cells during fibrosis. The data showed that TBX4-expressing cells expanded clonally. Within 3 weeks after bleomycin injury, clone size was at least 6 cells. These patches seemed to be monoclonal, indicating that regeneration foci are derived from a single Tbx4 progenitor. These data showed that the mesenchymal progenitors during fibrogenesis are similar to the embryonic-stage mesenchymal cells, which are highly proliferative and progressively expand (Kumar M E, Bogard P E, Espinoza F H, Menke D B, Kingsley D M, Krasnow M A. Mesenchymal cells. Defining a mesenchymal progenitor niche at single-cell resolution. Science. 2014; 346(6211):1258810). These findings indicate that mesenchymal progenitors proliferate and self-renew during lung fibrogenesis, which supports the previously suggested concept that during lung fibrosis, embryonic gene programs are reactivated (Selman M, Pardo A, Kaminski N. Idiopathic pulmonary fibrosis: aberrant recapitulation of developmental programs?. PLoS Med. 2008; 5(3):e62).
(304) Without being bound by theory, TBX4, acting as a mesenchymal transcription factor, may control downstream gene expression and impact key fibroblast effector functions. In order to gain insights into possible target genes, Tbx4-lineage and non-Tbx4-lineage cell gene expression was analyzed and identified mesenchymal genes that could be regulated by TBX4. Among them, Has2 was upregulated in TBX4-expressing cells. Has2 is 1 of the 3 HA synthase genes that have been identified. αSMA-Has2 mice showed increased fibrosis after bleomycin injury, and mice with conditional deletion of Has2 in mesenchymal cells exhibited less fibrosis (Li Y, et al. Severe lung fibrosis requires an invasive fibroblast phenotype regulated by hyaluronan and CD44. J Exp Med. 2011; 208(7):1459-1471). TBX4 regulation of Has2 and its impact on the fibroblast invasive phenotype was analyzed. These data showed that TBX4 regulated both fibroblast invasiveness and HA production by binding to the Has2 promoter and triggering Has2 gene expression. These data establish TBX4 as a proximal regulator of Has2 expression, HA production, and fibroblast invasiveness.
(305) Without being bound by theory, the array data suggested that TBX4 may regulate additional fibroblast and ECM remodeling genes, including Fgf Pdgfr, Mmp, and Adam family genes. It has been shown that TBX4 and TBX5 are linked to the activity of FGF, BMP, and WNT signaling pathways that are required for limb outgrowth and patterning (Rodriguez-Esteban C, Tsukui T, Yonei S, Magallon J, Tamura K, Izpisua Belmonte J C. The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity. Nature. 1999; 398(6730):814-818). TBX4 and TBX5 are considered critical for expression of mesenchymal FGF10 in embryonic mouse lung (Cebra-Thomas J A, Bromer J, Gardner R, Lam G K, Sheipe H, Gilbert S F. T-box gene products are required for mesenchymal induction of epithelial branching in the embryonic mouse lung. Dev Dyn. 2003; 226(1):82-90). It has been reported that TBX4 and TBX5 trigger limb formation by the induction of both Wnt and Fgf (55. Takeuchi J K, Koshiba-Takeuchi K, Suzuki T, Kamimura M, Ogura K, Ogura T. Tbx5 and Tbx4 trigger limb initiation through activation of the Wnt/Fgf signaling cascade. Development. 2003; 130(12):2729-2739). There are no reports to date directly linking Tbx4 and Mmp gene expression, although inhibition of the T-box transcription factor Brachyury showed downregulation of Mmp2 and Mmp24 in the context of EMT in cancer (Fernando R I, Litzinger M, Trono P, Hamilton D H, Schlom J, Palena C. The T-box transcription factor Brachyury promotes epithelial-mesenchymal transition in human tumor cells. J Clin Invest. 2010; 120(2):533-544.). Moreover, T-BET, another T-box transcription factor, was found increased together with MMP3 when TGF-p was blocked in human gut (Di Sabatino A, et al. Blockade of transforming growth factor beta upregulates T-box transcription factor T-bet, and increases T helper cell type 1 cytokine and matrix metalloproteinase-3 production in the human gut mucosa. Gut. 2008; 57(5):605-612).
(306) Without being bound by theory, the data suggest a hierarchical relationship of TBX4-lineage fibroblasts with other mesenchymal populations, such as FGF10+, GLI1+, ADAM12+, PDGFRa+, and FOXD1+ cells. TBX4 is a potential upstream regulator of these genes and is detected as early as E9.25 exclusively in lung mesenchyme without epithelial expression (Takeuchi J K, et al. Tbx5 and Tbx4 genes determine the wing/leg identity of limb buds. Nature. 1999; 398(6730):810-814; Arora R, Metzger R J, Papaioannou V E. Multiple roles and interactions of Tbx4 and Tbx5 in development of the respiratory system. PLoS Genet. 2012; 8(8):e1002866; Kumar M E, Bogard P E, Espinoza F H, Menke D B, Kingsley D M, Krasnow M A. Mesenchymal cells. Defining a mesenchymal progenitor niche at single-cell resolution. Science. 2014; 346(6211):1258810; Naiche L A, Arora R, Kania A, Lewandoski M, Papaioannou V E. Identity and fate of Tbx4-expressing cells reveal developmental cell fate decisions in the allantois, limb, and external genitalia. DevDyn. 2011; 240(10):2290-2300; Naiche L A, Papaioannou V E. Loss of Tbx4 blocks hindlimb development and affects vascularization and fusion of the allantois. Development. 2003; 130(12):2681-2693; Sakiyama J, Yamagishi A, Kuroiwa A. Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Development. 2003; 130(7):1225-1234). TBX4 induces mesenchymal genes and the pluripotent stem cell-related transcription factors (Gata2 and Gata3; Smad1, Smad2, Smad3, and Smad5; and Snail) in adult mice, suggesting that TBX4 can be a critical driver of fibroblast differentiation.
(307) Collectively, this study has demonstrated that Tbx4-lineage cells (i) represent most of the resident mesenchymal progenitors; (ii) expand during fibrosis; and (iii) form clonal patches. These data also demonstrate that TBX4 drives fibroblast matrix production and invasiveness through the regulation of Has2.
(308) While the present invention has been described with reference to the specific embodiments thereof it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adopt a particular situation, material, composition of matter, process, process step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.