Methods of facilitating vascular growth

A method of facilitating vascular growth in a subject in need of such treatment comprises inhibiting EMAP II activity in the subject by an amount effective to stimulate vascular growth in the subject (e.g., in the lungs or heart of the subject). Pharmaceutical formulations useful for carrying out such methods (e.g., an antibody that specifically binds to EMAP II in a pharmaceutically acceptable carrier) and screening techniques useful for identifying additional compounds that can be used for carrying out such methods are also disclosed.

FIELD OF THE INVENTION
 The present invention concerns methods of facilitating vascular growth in a
 subject, such as a subject at risk for ischemic reperfusion injury, or a
 newborn afflicted with bronchopulmonary displaysia. Methods of identifying
 compounds useful for the aforesaid treatments are also disclosed.
 BACKGROUND OF THE INVENTION
 U.S. Pat. No. 5,641,867 to D. Stern et al. (assigned to Columbia
 University) describes purified endoethelial monocyte activating
 polypeptide (EMAP) II, antibodies that specifically bind to EMAP II, and
 methods of treating tumors by administering EMAP II to an afflicted
 subject.
 U. Knies et al., Proc. Natl. Acad. Sci. USA 95, 12322-12327 (October 1998),
 describes the regulation of endothelial monocyte-activating polypepetide
 II release by apoptosis.
 SUMMARY OF THE INVENTION
 A first aspect of the invention is a method of facilitating vascular growth
 in a subject, such as in an organ or tissue of the subject, in need of
 such treatment. The method comprises inhibiting EMAP II activity in the of
 the subject (e.g., in the aforesaid organ or tissue) by an amount
 effective to stimulate vascular growth.
 A second aspect of the present invention is a pharmaceutical formulation
 comprising: an active compound selected from the group consisting of
 compounds that specifically bind to EMAP II, compounds that inhibit the
 expression of EMAP II, and EMAP II receptor antagonists; and a
 pharmaceutically acceptable carrier.
 A third aspect of the present invention is a method of screening for
 compounds useful for facilitating vascular growth in a subject in need
 thereof. The method comprises: contacting a test compound (e.g., a protein
 or peptide)to a probe molecule, the probe molecule selected from the group
 consisting of EMAP II and fragments thereof; and then detecting the
 presence or absence of binding of the test compound to the probe molecule,
 the presence of binding indicating the compound may be useful for
 facilitating vascular growth in a subject.
 A fourth aspect of the present invention is a method of screening for
 compounds useful for facilitating vascular growth in a subject,
 comprising: contacting a test compound (e.g., an oligonucleotide) to probe
 molecule, the probe molecule selected from the group consisting of DNA
 encoding EMAP II, RNA encoding EMAP II, and fragments thereof; and then
 detecting the presence or absence of binding of the test compound to the
 probe molecule, the presence of binding indicating the compound may be
 useful for facilitating vascular growth in the subject.
 A fifth aspect of the present invention is a method of screening for
 compounds useful for facilitating vascular growth in a subject,
 comprising: determining in vitro whether a test compound inhibits
 expression of EMAP II; the inhibition of expression of EMAP II indicating
 the compound may be useful for facilitating vascular growth in a subject.
 The present invention is explained in greater detail in the specification
 set forth below.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
 As noted above, a first aspect of the invention is a method of facilitating
 vascular growth in a subject in need of such treatment. The method
 comprises inhibiting EMAP II activity in the subject by an amount
 effective to stimulate vascular growth.
 Vascular growth may be inhibited in any suitable organ or tissue, including
 but not limited to lung, kidney, heart, aorta, gastrointestinal tract,
 brain, liver, etc. The inhibition may be specific or general, primarily
 influenced by the manner of administration as discussed below. Applicants
 invention is not intended to be limited to any particular theory of
 vascular growth, and hence this term is intended to be construed
 generally, encompassing any type of vascular growth such as
 vasculogenesis, angiogenesis, etc.
 Subjects that may be treated by the present invention include any subject,
 human or adult, for which it is desired to facilitate vascular growth.
 Such subjects include subjects at risk for ischemic reperfusion injury to
 an organ such as those described above (e.g., in the case of transplant,
 low blood pressure, cardiac arrest, etc.), newborn subjects afflicted with
 bronchopulmonary displaysia, subjects afflicted with pulmonary
 hypertension, subjects afflicted with lung hypoplasia, etc.
 While subjects treated by the present invention are primarily human
 subjects, the invention may also be carried out on other animal subjects
 such as dogs, cats, horses, etc. for veterinary purposes.
 The inhibiting step may be carried out by any suitable means. For example,
 it may be carried out by administering a compound that specifically binds
 to EMAP II to the subject in an amount effective to stimulate vascular
 growth. Such compounds may be antibodies (including polyclonal and
 monoclonal antibodies, antibody fragments, humanized or chimeric
 antibodies, etc. that retain the combining region that specifically binds
 to EMAP II). The antibodies may be of any type of immunoglobulin,
 including but not limited to IgG and IgM immunoglobulins. The antibodies
 may be of any suitable origin, such as chicken, goat, rabbit, horse, etc.,
 but are preferably mammalian and most preferably human. The antibody may
 be administered directly or through an intermediate that expresses the
 antibody in the subject. Examples of EMAP II antibodies are provided in
 U.S. Pat. No. 5,641,867 to Stern et al., the disclosure of which is
 incorporated herein by reference. Examples of the different forms of
 therapeutic antibodies are given in U.S. Pat. No. 5,622,700, the
 disclosure of which is incorporated herein by reference.
 The inhibiting step may be carried out by downregulating EMAP II expression
 in the subject by an amount effective to stimulate vascular growth in the
 lungs of the subject. Compounds useful for downregulating EMAP II
 expression are, in general, antisense oligonucleotides that bind to EMAP
 II mRNA and disrupt translation thereof, or oligonucleotides that bind to
 EMAP II DNA and disrupt transcription thereof. Such oligionucleotides may
 be natural or synthetic (such as described in U.S. Pat. No. 5,665,593 to
 Kole, the disclosure of which is incorporated by reference herein in its
 entirety), and are typically at least 4, 6 or 8 nucleotides in length, up
 to the full length of the corresponding DNA or mRNA. Such oligonucleotides
 are selected to bind to the DNA or mRNA by Watson-Crick pairing based on
 the known sequence of the EMAPII DNA as described in U.S. Pat. No.
 5,641,867 to Stern et al., the disclosure of which is incorporated by
 reference herein in its entirety. For example, an antisense
 oligonucleotide of the invention may consist of a 4, 6 or 8 or more
 nucleotide oligonucleotide having a base sequence corresponding to the
 EMAP II DNA sequence disclosed in Stern et al., supra, up to 20, 30, or 40
 nucleotides in length, or even the full length of the DNA sequence. In
 addition, such compounds may be identified in accordance with known
 techniques as described below.
 The inhibiting step may be carried out by administering an EMAP II receptor
 antagonist to the subject in an amount effective to stimulate vascular
 growth in the lungs of the subject. EMAP II receptor antagonists may be
 identified in accordance with known techniques, but are in general analogs
 of EMAP II, such as EMAP II having three to five N-terminal and/or
 C-terminal amino acids deleted.
 Active compounds useful for effecting the aforesaid inhibiting steps may be
 administered by any suitable means, including intraperitoneal,
 subcutaneous, intraarterial intraveneous, intramuscular, and intrathecal
 injection. Injection may be through a syringe, through a canula or
 catheter into a desired vessel or organ, etc. The compounds may be
 administered by inhalation into the airways, and particularly the alveoli,
 of the lungs, such as by the inhalation of respirable aerosol particles
 (e.g., 1 to 5 micron diameter particles) comprising the active compound.
 Pharmaceutical formulations of the invention typically comprise an active
 compound selected from the group consisting of compounds that specifically
 bind to EMAP II (e.g., an antibody as described above), compounds that
 inhibit the expression of EMAP II, and EMAP II receptor antagonists; and a
 pharmaceutically acceptable carrier. Any pharmaceutically acceptable
 carrier may be employed, such as sterile saline solution, sterile water,
 etc. The active compound is included in the pharmaceutically acceptable
 carrier in any suitable amount, such as between about 0.001, 0.005 or 0.01
 percent by weight to about 10, 20 or 50 percent by weight.
 Dosage of the active compound will depend upon the particular active
 compound, the route of administration, the particular disorder being
 treated, the age, weight, and condition of the subject, etc. For example,
 for antisense oligonucleotides, the dosage is preferably one which
 produces intracellular concentrations of the oligonucleotide of from 0.05
 to 50 .mu.M. typically the dosage to a human will be from about 0.01, 0.1
 or 1 mg/Kg up to 50, 100, or 150 mg/Kg. In an additional example, for
 antibodies, the dosage is typically 0.01, 0.05 or 0.1 up to 20, 40 or 60
 mg/Kg.
 Active compounds that are nucleotides or proteins (e.g., antibodies) may be
 administered either directly as described above or through a vector
 intermediate that expresses the same in the subject. Thus vectors used to
 carry out the present invention are, in general, RNA virus or DNA virus
 vectors, such as lentivirus vectors, papovavirus vectors (e.g., SV40
 vectors and polyoma vectors), adenovirus vectors and adeno-associated
 virus vectors. See generally T. Friedmann, Science 244, 1275 16 (June
 1989). Examples of lentivirus vectors that may be used to carry out the
 present invention include Moloney Murine Leukemia Virus vectors, such as
 those described in U.S. Pat. No. 5,707,865 to Kohn. Any adenovirus vector
 can be used to carry out the present invention. See, e.g., U.S. Pat. No.
 5,518,913, U.S. Pat. No. 5,670,488, U.S. Pat. No. 5,589,377; U.S. Pat. No.
 5,616,326; U.S. Pat. No. 5,436,146; and U.S. Pat. No. 5,585,362. The
 adenovirus can be modified to alter or broaden the natural tropism
 thereof, a described in S. Woo, Adenovirus redirected, Nature
 Biotechnology 14, 1538 (November 1996). Any adeno-associated virus vector
 (or AAV vector) can also be used to carry out the present invention. See,
 e.g., U.S. Pat. No. 5,681,731; U.S. Pat. No. 5,677,158; U.S. Pat. No.
 5,658,776; U.S. Pat. No. 5,658,776, U.S. Pat. No. 5,622,856; U.S. Pat. No.
 5,604,090; U.S. Pat. No. 5,589,377; U.S. Pat. No. 5,587,308; U.S. Pat. No.
 5,474,935; U.S. Pat. No. 5,436,146; U.S. Pat. No. 5,354,678; U.S. Pat. No.
 5,252,479; U.S. Pat. No. 5,173,414; U.S. Pat. No. 5,139,941; and U.S. Pat.
 No. 4,797,368. The regulatory sequences, or the transcriptional and
 translational control sequences, in the vectors can be of any suitable
 source, so long as they effect expression of the heterologous nucleic acid
 in the target cells. For example, commonly used promoters are the LacZ
 promoter, and promoters derived from polyoma, Adenovirus 2, and Simian
 virus 40 (SV40). See, e.g., U.S. Pat. No. 4,599,308. The heterologous
 nucleic acid may encode any product that inhibits the expression of the
 EMAP II gene in cells infected by the vector, such as an antisense
 oligonucleotide that specifically binds to the EMAP II mRNA to disrupt or
 inhibit translation thereof, a ribozyme that specifically binds to the
 EMAP II mRNA to disrupt or inhibit translation thereof, or a triplex
 nucleic acid that specifically binds to the EMAP II duplex DNA and
 disrupts or inhibits transcription thereof. All of these may be carried
 out in accordance with known techniques, as (for example) described in
 U.S. Pat. Nos. 5,650,316; 5,176,996, or 5,650,316 for triplex compounds,
 in U.S. Pat. Nos. 5,811,537; 5,801,154; and 5,734,039 for antisense
 compounds, and in U.S. Pat. Nos. 5,817,635; 5,811,300; 5,773,260;
 5,766,942; 5,747,335; and 5,646,020 for ribozymes (the disclosures of
 which are incorporated by reference herein in their entirety). The length
 of the heterologous nucleic acid is not critical so long as the intended
 function is achieved, but the heterologous nucleic acid is typically from
 5, 8, 10 or 20 nucleic acids in length up to 20, 30, 40 or 50 nucleic
 acids in length, up to a length equal the full length of the EMAP II gene.
 Once prepared, the recombinant vector can be reproduced by (a) propagating
 the vector in a cell culture, the cell culture comprising cells that
 permit the growth and reproduction of the vector therein; and then (b)
 collecting the recombinant vector from the cell culture, all in accordance
 with known techniques. The viral vectors collected from the culture may be
 separated from the culture medium in accordance with known techniques, and
 combined with a suitable pharmaceutical carrier for administration to a
 subject. Such pharmaceutical carriers include, but are not limited to,
 sterile pyrogen-free water or sterile pyrogen-free saline solution. If
 desired, the vectors may be packaged in liposomes for administration, in
 accordance with known techniques.
 Any suitable route of administration can be used to carry out the present
 invention, depending upon the particular condition being treated. Suitable
 routes include, but are not limited to, intraveneous, intrarterial,
 intrathecal, intraperitoneal, intramuscular, and intralesional injection.
 Intralesional injection is currently preferred.
 The dosage of the recombinant vector administered will depend upon factors
 such as the particular disorder, the particular vector chosen, the
 formulation of the vector, the condition of the patient, the route of
 administration, etc., and can be optimized for specific situations. In
 general, the dosage is from about 10.sup.7, 10.sup.8, or 10.sup.9 to about
 10.sup.11, 10.sup.12, or 10.sup.13 plaque forming units (pfu).
 In addition to their pharmaceutical or veterinary use, the recombinant
 vectors of the present invention (sometimes also referred to as "active
 agents" herein) are useful in vitro to distinguish cells in culture based
 on their response to the active agents, to induce apoptosis, etc. Such
 techniques are useful for both carrying out cell culture procedures and
 for drug screening purposes.
 In vitro methods of screening compounds for efficacy in carrying out the
 methods of treatment described above are also disclosed herein. In
 general, in one embodiment, such methods comprise determining in vitro
 whether the compound inhibits the expression of EMAP II (preferably the
 mammalian gene, and most preferably the human gene). The inhibition of
 expression of EMAP II indicates the compound is useful in the methods of
 treatment described above. Numerous such screening methods are available.
 The methods can be carried out in a cell or cells, or can be carried out
 in essentially cell free preparation. The method can be carried out by
 screening for compounds that specifically disrupt either transcription or
 translation of EMAP II. The compound to be screened may be a member of a
 library of compounds (the term "compound" as used in this respect
 referring to both small organic compounds and other therapeutic agents
 such as recombinant viral vectors). The method may be carried out as a
 single assay, or may be implemented in the form of a high throughput
 screen in accordance with a variety of known techniques. In another
 embodiment the method of screening compounds comprises determining in
 vitro whether said compound specifically binds to EMAP II (including
 fragments thereof) (preferably the mammalian gene product; most preferably
 the human gene product). The determining step can be carried out by
 screening for binding of a test compound or probe molecule to the entire
 full length EMAP II gene product, or to a peptide fragment thereof (e.g.,
 a fragment of from 5, or 10 amino acids in length up to the full length of
 EMAP II). The binding of the compound to the EMAP II indicates that the
 compound is useful in the methods of treatment described herein. Such
 techniques can be carried out by contacting, a probe compound to EMAP II
 or a fragment thereof in any of the variety of known combinatorial
 chemistry techniques (including but not limited to split pool techniques,
 chip-based techniques and pin-based techniques). Any suitable solid
 support can be used to immobilize the EMAP II or a fragment thereof to
 find specific binding partners thereto (or immobilize the members of the
 library against which the EMAP II or fragment thereof is contacted to find
 specific binding partners thereto), and numerous different solid supports
 are well known to those skilled in the art. Examples of suitable materials
 from which the solid support may be formed include cellulose, pore-glass,
 silica gel, polystyrene, particularly polystyrene cross-linked with
 divinylbenzene, grafted copolymers such as polyethyleneglycol/polystyrene,
 polyacrylamide, latex, dimethylacrylamide, particularly cross-linked with
 N,N'bis-acrylolyl ethylene diamine and comprising
 N-t-butoxycarbonyl-beta-alanyl-N'acrylolyl hexamethylene diamine,
 composites such as glass coated with a hydrophobic polymer such as
 cross-linked polystyrene or a fluorinated ethylene polymer to which is
 grafted linear polystyrene, and the like. Thus the term "solid support"
 includes materials conventionally considered to be semi-solid supports.
 General reviews of useful solid supports that include a covalently-linked
 reactive functionality may be found in Atherton et al., Prospectives in
 Peptide Chemistry, Karger, 101-117 (1981); Amamath et al., Chem. Rev. 77:
 183 (1977); and Fridkin, The Peptides, Vol. 2, Chapter 3, Academic Press,
 Inc., pp 333-363 (1979). The solid support may take any suitable form,
 such as a bead or microparticle, a tube, a plate, a microtiter plate well,
 a glass microscope cover slip, etc.
 The present invention can be used with probe molecules, or libraries (where
 groups of different probe molecules are employed), of any type. In
 general, such probe molecules are organic compounds, including but not
 limited to that may be used to carry out the present include oligomers,
 non-oligomers, or combinations thereof. Non-oligomers include a wide
 variety of organic molecules, such as heterocyclics, aromatics,
 alicyclics, aliphatics and combinations thereof, comprising steroids,
 antibiotics, enzyme inhibitors, ligands, hormones, drugs, alkaloids,
 opioids, benzodiazepenes, teipenecs, prophyrins, toxins, catalysts, as
 well as combinations thereof. Oligomers include peptides (that is,
 oligopeptides) and proteins, oligonucleotides (the term oligonucleotide
 also referred to simply as "nucleotide, herein) such as DNA and RNA,
 oligosaccharides, polylipids, polyesters, polyamides, polyurethanes,
 polyureas, polyethers, poly (phosphorus derivatives) such as phosphates,
 phosphonates, phosphoramides, phosphonamides, phosphites, phosphinamides,
 etc., poly (sulfur derivatives) such as sulfones, sulfonates, sulfites,
 sulfonamides, sulfenamides, etc., where for the phosphorous and sulfur
 derivatives the indicated heteroatom for the most part will be bonded to
 C, H, N, O or S, and combinations thereof. Numerous methods of
 synthesizing or applying such probe molecules on solid supports (where the
 probe molecule may be either covalently or non-covalently bound to the
 solid support) are known, and such probe molecules can be made in
 accordance with procedures known to those skilled in the art. See, e.g.,
 U.S. Pat. No. 5,565,324 to Still et al., U.S. Pat. No. 5,284,514 to Ellman
 et al., U.S. Pat. No. 5,445,934 to Fodor et al. (the disclosures of all
 United States patents cited herein are to be incorporated herein by
 reference in their entirety).
 Test compounds used to carry out the present invention may be of any type,
 including both oligomers or non-oligomers of the types described above in
 connection with probe molecules above. Again, such test compounds are
 known and can be prepared in accordance with known techniques.
 Where multiple different probe molecules are desired to be tested, a
 screening substrate useful for the high throughput screening of molecular
 interactions, such as in "chip-based" and "pin-based" combinatorial
 chemistry techniques, can be prepared in accordance with known techniques.
 All can be prepared in accordance with known techniques. See, e.g., U.S.
 Pat. No. 5,445,934 to Fodor et al., U.S. Pat. No. 5,288,514 to Ellman, and
 U.S. Pat. No. 5,624,711 to Sundberg et al.
 In the alternative, screening of libraries of probe molecules may be
 carried out with mixtures of solid supports as used in "split-pool"
 combinatorial chemistry techniques. Such mixtures can be prepared in
 accordance with procedures known in the art, and tag components can be
 added to the discreet solid supports in accordance with procedures known
 in the art. See, e.g., U.S. Pat. No. 5,565,324 to Still et al.
 The present invention is explained in greater detail in the following
 non-limiting examples.
 EXAMPLE 1
 EMAP II Inhibits Lung Neovascularization, Epithelial Morphogenesis and
 Epithelial-Mesenchymal Interactions
 Neovascularization is crucial to lung development and is mediated through a
 variety of angiogenic and anti-angiogenic factors. Herein, it is shown
 that excess Endothelial Monocyte Activating Polypeptide (EMAP) II, an
 anti-angiogenic protein, not only inhibits fetal lung neovascularization,
 but also significantly alters lung epithelial morphogenesis. In a murine
 xenograft model of lung neovascularization and morphogenesis, embryonic
 lungs transplanted under the skin of immunocompromised mice receiving
 intraperitoneal EMAP II, had a 56% reduction in vessel density
 (p&lt;0.0001) compared to control. EMAP II treated lung transplants
 exhibited a marked alteration in lung morphogenesis, including lack of
 type II alveolar cell formation. In contrast, lung implants in animals
 receiving a blocking antibody to EMAP II had an increase in vessel density
 of 50% (p&lt;0.0001) and most distal epithelial cells expressed surfactant
 protein C. Co-cultures of embryonic epithelial and mesenchymal cells
 showed that EMAP II expression is localized to the peri-epithelial cyst
 region. Exposure of these co-cultures to excess EMAP II inhibited
 epithelial cyst formation by 71% (p&lt;0.0001); while, conversely, EMAP II
 antibody increased cyst formation by 54% (p&lt;0.0001). There was a
 time-dependent induction of apoptosis by EMAP II limited to the epithelial
 cells in the co-culture system that was confirmed by apoptosis induction
 in the epithelial cells of the explant model. These studies demonstrate
 that EMAP II modulates vessel growth in the developing lung, inhibition of
 vessel growth, results in altered lung morphogenesis, and effects
 epithelial-mesenchymal interactions where, in the absence of vascular
 growth it induces apoptosis. Therefore, EMAP II, negatively modulates lung
 neovascularization as well as leading to the arrest of lung epithelial
 morphogenesis and apoptosis.
 I. EXPERIMENTAL PROCEDURES
 Synthesis of Recombinant (r) EMAP II from E. Coli and generation of a
 peptide antibody. The cDNA of mature human EMAP II was cloned from RT-PCR
 products of U937 cells total RNA based on primers obtained from gene bank
 (accession #10119) into TA vector (Invitrogen). Confirmation of the clones
 was provided by sequence analysis, afterwhich the cDNA was inserted into
 PET28a, 6.times. his-tag containing plasmid. E. coli. (DE.sub.3) underwent
 transformation with the EMAP II/PET28a plasmid and were induced with 1-4
 mM IPTG. After 3-4 hours of induction, the cells were pelleted, lysed and
 the EMAP II protein was purified through the use of a nickel column as per
 protocol (Qiagen) with all procedures performed at 4.degree. C. Briefly,
 pelleted cells were lysed with 50 mM NaH.sub.2 PO.sub.4 pH 8.0, 300 mM
 NaCl, and 10 mM imidazole in the presence of lysozyme of 1 mg/ml.
 Following sonication, cellular debris are removed by centrifugation prior
 to being loaded on the Ni-NTA slurry. Following washing of the column,
 rEMAP II is eluted off with 8M urea, 0.1 M NaH.sub.2 PO.sub.4, and 0.01 M
 Tris.Cl pH 5.9. Purified rEMAP II is dialyzed at 4.degree. C. against PBS
 three times prior to being aliquoted and frozen at -80.degree. C. When an
 aliquot of rEMAP II was thawed, it was used immediately for experiments
 (it was not refrozen and used in future studies). This is essential to
 maintain rEMAP II's activity.
 A peptide sequence of 13 amino acid residues located within a homologous
 region of the human and murine forms of mature EMAP II were used to
 generate an antibody. This peptide was synthesized and the antibody
 produced by Zymed Laboratories Inc. as per protocol and is used for
 immunohistochemistry and western blotting. The antibody is specific to
 EMAP II identified by producing a single band on a western blot that is
 blocked after being incubated with excess EMAP II (data not shown).
 Isolation of epithelial and mesenchymal cells for co-culture. Organotypic
 murine lung cultures were performed following the protocol of Schuger et
 al. [Schuger, Development 110, 1091-9 (1990); Schuger, J. Cell. biol. 139,
 553-62 (1997); Schuger, Int. J. Dev.Biol. 42, 217-220 (1998)]. In brief,
 timed gestation 15d embryos underwent dissection from Swiss-Webster mice
 (Simonsen, Morgan Hill, Calif.), lungs were isolated, underwent digestion
 in PBS containing 0.3% trypsin and 0.1% EDTA for 10 minutes at 37.degree.
 C. prior to being filtered through a 100 .mu.m-pore mesh. The mixed
 epithelial-mesenchymal cells were then resuspended in minimal essential
 medium (MEM:Gibco-BRL) with nonessential amino acids and plated at a
 concentration of 2-2.5.times.10.sup.6 cells/ml in 8 well chamber slides.
 Experiments were performed in the presence of vehicle, rEMAP II (mature
 0.8-3.2 .mu.g/ml), EMAP II peptide antibody (3-6 .mu.g/ml), and rabbit IgG
 (control). Epithelial cyst formation was evaluated by counting the number
 of epithelial cyst per high power field (HPF), we analyzed 10 fields per
 condition and averaged them.
 Xenograft lung transplant model. Timed pregnant Swiss Webster mice at
 gestational day 12 (based on appearance of vaginal plug=day 0) were
 obtained, housed, and handled according to a protocol approved by the
 animal care committee at CHLARI (Childrens Hospital of Los Angeles
 Research Institute). On day 14.5 dams were sacrificed and the embryo
 removed. The lungs and heart were withdrawn as a block microdissection and
 placed in ice cold PBS. The heart was then removed and the lung was placed
 on top of a 0.80 .mu.M Millipore filter disk (Millipore) and implanted
 into a dorsal skinfold chamber of a nude mouse using sterile technique.
 The skin was closed with skin staples. A sibling lung was used for
 histological analysis and comparison to the implanted lung. Nude mice were
 then injected intraperitoneal by (IP) on a daily basis with either vehicle
 (phosphate buffered saline-PBS and albumin), EMAP II (1 .mu.g/day), rabbit
 IgG or EMAP II antibody (25 or 50 .mu.g/every three days).
 RT-PCR of lung transplants. Following 14 days, lung xenografts were removed
 from mice that had been treated with rEMAP II, antibody to EMAP II, or
 vehicle, separated from the carrier mouse skin, total RNA was extracted by
 RNA STAT-60 (Tel-Test "B", Inc., Friendswood, Tex.) and the RNA of the
 transplanted lungs were reverse transcribed by superscript II RNase
 H-reverse transcriptase (GIBCO-BRL) using 3 mcg of total RNA template, 4
 .mu.l of 5.times. RT buffer, 2 .mu.l of 0.1 M DTT, 0.5 .mu.g of target
 gene specific 3' primer in a total reaction volume of 18 .mu.l. The
 reaction mix was incubated at 70.degree. C. for 10 minutes followed by
 incubation on ice 2 minutes. One .mu.l of 10 mM dNTP, 1 .mu.l of
 superscript II RNase H-reverse transcriptase were added. The mixture was
 incubated at 49.degree. C. for 1 hour and 30 minutes followed by
 70.degree. C. for 10 minutes. The first strand cDNAs thus synthesized were
 used directly for PCR amplification of the target cDNA. The target cDNA
 primers were: 1) murine PECAM-15' primer-5' GTC ATG GCC ATG GTC GAG TA 3'
 (SEQ ID NO: 1) and the 3' primer-5' CTC CTC GGC ATC TTG CTG AA 3' (SEQ ID
 NO: 2), 2) murine tie-2 5' primer-5'TTG AAG TGA CGA ATG AGA T 3' (SEQ ID
 NO: 3) and the 3' primer-5' ATT TAG AGC TGT CTG GCT T 3' (SEQ ID NO: 4),
 3) murine SP-C 5' primer-5'-CAT ACT GAG ATG GTC CTT GAG-3' (SEQ ID NO: 5),
 and 3' primer-5'-TCT GGA GCC ATC TTC ATG ATG-3' (SEQ ID NO: 6) and 4)
 murine T1-.alpha. 5' primer-5' GAA CAT GAG AGT ACG ACC ACT GTC AAA 3' (SEQ
 ID NO: 7) and the 3' primer-5' TTA GGG CGA GAA CCT TCC AGA AAT CTT 3' (SEQ
 ID NO: 8). .beta. Actin, used as the house keeping gene, was performed on
 all the samples using the primers: 5' primer-5' GTA TGG AAT CCT GTG GCA
 TCC 3' (SEQ ID NO: 9) and the 3' primer-5' TAC GCA GCT CAG TAA CAG TCC 3'
 (SEQ ID NO: 10). In addition, controls were performed on all targeted cDNA
 sequences using primer pairs without the presence of the first-strand cDNA
 template. Target cDNA segments were amplified using 1/10th of the above
 first-strand cDNA template, 10 .mu.l of 10.times. buffer, 0.5 .mu.l of 10
 mM dNTP's, 300 ng of each of 5' and 3' end specific primers, and 1 unit of
 Taq Polymerase (Stratagene) in a 50 .mu.l reaction. The PCR program was
 94.degree. C. 1 min., 62.degree. C. 30 sec., and 72.degree. C. 30 sec. for
 30 cycles. Equal amounts of all amplification cDNA fragments were analyzed
 by agarose gel electrophoresis, photographed, and analyzed.
 In situ hybridization and construction of cDNA probes. Total RNA was
 extracted from 15 day gestation mouse lung tissue by RNA STAT-60 (Tel-Test
 "B", Inc., Friendswood, Tex.). RNA (3 .mu.g) was incubated with oligo(dT)
 primer for 10 minutes at 70.degree. C. First-strand cDNA synthesis was
 performed according to manufacturer's instructions (GIBCO BRL, Grand
 Island, N.Y.). After first-strand synthesis, cDNA was generated by PCR
 amplification with 10 pmol of specific primers for 30 cycles of
 amplification (94.degree. C. 1', 62.degree. C. 1', 72.degree. C. 1'). The
 primers user were as follows: SP-C, sense, 5'-CAT ACT GAG ATG GTC CTT
 GAG-3' (SEQ ID NO: 11), and antisense, 5'-TCT GGA GCC ATC TTC ATG ATG-3'
 (SEQ ID NO: 12). The RNA probe for EMAP II was 456 bp in size and obtained
 from a region that has minimal homology with other known proteins. The
 generated SP-C PCR product was subcloned into TA vector (Invitrogen,
 Carlsbad, Calif.) for the in vitro transcription of RNA.
 Digoxigenin RNA probe labeling by in vitro transcription. DNA of the SP-C
 subclone, in good orientation for in vitro transcription of antisense RNA
 by T7 RNA polymerase, was linearized by Hind III digestion and used as a
 template for probe labeling. Antisense RNA probe labeling with
 digoxigenin-UTP by in vitro transcription with T7 RNA polymerase was
 performed as per manufactures instructions (DIG RNA labeling kit,
 Boehringer Mannheim, Indianapolis, Ind.).
 RNA in situ hybridization (RISH) using DIG-labeled cRNA probes. Murine
 embryo control lung day 14 g.a. and murine transplants, days 14 g.a.+3.5,
 14 g.a.+7, 14 g.a.+10.5, and 14 g.a.+14 were obtained for in situ
 hybridization. The Dig RNA probe anti-sense and sense (control) were made
 using the Dig RNA labeling Kit (SP6/T7) from Boehringer Mannheim
 (Indianapolis, Ind.). RISH was performed on 5-mm paraffin embedded
 material gections according to nonradioactive in site hybridization
 application manual (Boehringer Mannheim, Indianapolis, Ind.). Using DEPC
 treated equipment and solutions, paraffin embedded specimens underwent
 sectioning, rehydration and incubation in a prewarmed 5 .mu.g/ml
 proteinase K solution. Slides were then reimmersed in 4% PFA, treated with
 a 0.25% acetic anhydride and dehydrated. gections were exposed to a
 hybridization solution containing 50% formamide, 10% dextran sulfate, 1
 mg/ml tRNA, 1.times.Denhardt's solution, 4.times.SSC, 50 mM Tris and 5 mM
 EDTA that contained 150-300 ng/ml of dig-labeled RNA probe at 50.degree.
 C. overnight. Slides were washed at 55.degree. C. in 2.times.SSC/50%
 formamide, 1.times.SSC and 0.1 SSC for 30 minutes prior to being incubated
 with RNase A (20 .mu.g/ml) for 30 minutes at 37.degree. C. After being
 rinsed with 2.times.SSC and Dig Nucleic Acid detection was accomplished
 using the Genius 3 kit from Boehringer Mannheim. Briefly, slides were
 incubated in 0.1 M Maleic acid/0.15 M NaCl pH 7.5 for 5 minutes after
 which they underwent blocking in a 1% block reagent. Following blocking,
 slides were incubated with anti-Dig-AP conjugate at 4.degree. C.
 overnight, rinsed, and incubated with a dilute NBT/BCIP solution for 3
 hours a room temperature. glides then underwent counterstaining with a
 0.02% fast green solution for 2 minutes, rinsed in water, air dried and
 mounted. Hybridization with sense probe or without probe was performed as
 negative control and they always showed no signals. All sections were
 examined and photographed under light microscopy.
 Histologic and immunohistochemistry analysis of the murine lung
 transplants. Following 14 days, lung xenografts were removed from mice
 that had been treated with rEMAP II, blocking antibody to EMAP II, or
 vehicle, separated from the carrier mouse skin, fixed in 4%
 paraformaldehyde, dehydrated, and paraffin embedded (during all
 procedures, DEPC water and precaution against RNAses were taken). Fixed
 tissue was sectioned at 5 micron intervals. The lung transplants then
 underwent H & E staining for structural analysis. For immunolocalization
 of PECAM-1 antigens (Pharmigen, San Diego, Calif.), a rat anti-murine
 PECAM-1 antibody (4 .mu.g/ml) was employed. Tissues were deparaffinized
 and underwent peroxide quenching. Using a histostain kit from Zymed (San
 Francisco, Calif.), after blocking, the sections were exposed to the
 primary antibody overnight at 4.degree. C. Sections were then incubated
 with secondary biotinylated antibody as per the manufacturer's protocol. A
 brief incubation with the Streptavidin-HRP conjugate system (Zymed) was
 followed by development using the chromogen substrate aminoethylcarbazole.
 Periodic Acid Schiff (PAS) stain was performed, using a kit from Sigma
 (St. Louis, Mo.) according to the manufactures instructions.
 TUNEL analysis of fetal epithelial-mesenchymal cell co-cultures. The
 spatial induction of apoptosis was analyzed in epithelial-mesenchymal cell
 co-culture or lung xenografts using the In Situ Death Detection Kit from
 Boehringer Mannheim. In brief, co-cultured cells were exposed to vehicle,
 EMAP II (3.2 .mu.g/ml), EMAP II antibody (6 .mu.g/ml) or rabbit IgG. Cells
 were evaluated on days one to three for apoptosis. Cells were fixed in 4%
 paraformaldehyde, permeabilized with 0.1% Triton-X and exposed to the
 TUNEL reaction (containing terminal deoxynucleotidyl transferase and a
 nucleotide mixture in a reaction buffer). After which, the cells were
 exposed to a fluorescein antibody, counterstained with propidium iodine
 (0.05 .mu.g/ml), mounted with PBS/glycerol, and observed under a
 floursecent microscope (Olympus). Lung xenografts were fixed in 4%
 paraformaldehyde, dehydrated and paraffin embedded. 5 micron sections were
 cut, rehydrated and prior to exposure to the TUNEL reaction. Apoptotic
 cells were revealed using alkaline phosphatase and observed under light
 microscopy.
 Statistics: Statistical analysis was performed using student t-test on the
 computer program Statview.
 II. RESULTS
 Purification of recombinant EMAP II. In order to determine the function of
 EMAP II in the developing lung, it was important to develop an easy and
 reproducible production system for recombinant EMAP II. We used a PET28a
 6.times. His-tag system to quickly and efficiently isolate mature rEMAP II
 under native conditions. Recombinant (r) EMAp II was expressed in E. coli
 (shown in the Coomassie blue gel, 1st column FIG. 1), induced with 1-4 mM
 IPTG and the E. coli pelleted after 3-4 hours of induction (2nd column
 FIG. 1). The purified, recombinant mature form of EMAP II (column 3, FIG.
 1) had MR 23 kDa (with the 6.times. His-tag) on both reduced and
 nonreduced SDS-PAGE. Activity of rEMAP II, measured by induction of
 TNF-.alpha. and monocyte migration [Kao, 1994 #44], was found to be
 closely analogous to that previously observed with meth A-derived EMAP II.
 LPS levels were &lt;15 pg/ml as measured with a LAL kit (Biowhittaker
 QCL-1000). Heat-treated EMAP II was inactive in these assays. The peptide
 antibody generated in a rabbit, is specific to EMAP II, identified by
 producing a single band on Western analysis that is blocked after being
 incubated with excess EMAP II (data not shown).
 EMAP II inhibition of fetal lung vascular development. To better define
 EMAP II's role in embryonic lung neovascularization, murine lungs obtained
 at gestational age 14.5 days, were implanted subcutaneously into nude
 mice. Mice then received either vehicle or rEMAP II (1 .mu.g/day) IP every
 day for 14 days. A separate group of mice were treated with either EMAP II
 blocking antibody (25 or 50 .mu.g) or rabbit IgG every 3 days for 14 days.
 Lung transplants were then excised and evaluated for vascular and
 structural development using PECAM-1 and hematoxylin and eosin staining
 respectively. Compared to lung xenografts implanted in mice treated with
 vehicle alone, implants in mice receiving the anti-angiogenic protein EMAP
 II exhibited a striking 56% reduction in lung vessel formation.
 Differences between lung vessel formation (assessed by counting the number
 of vessels identified per high powered field (HPF) with PECAM-1 antibody)
 in control (FIG. 2A) and EMAP II treated (FIGS. 2B, D) animals were highly
 statistically significant by student t-test (p&lt;0.0001). In contrast,
 animals receiving blocking antibody to EMAP II had a significant dose
 dependent increase of 50% (p&lt;0.0001) in vessel counts per HPF (FIGS.
 2C, E) (n=10/group, performed on 3 separate occasions). Consistent with
 these histologic findings, mRNA harvested from lung xenografts of animals
 treated with rEMAP II demonstrated a reduction in PECAM-1 and Tie-2 by
 RT-PCR compared to control. Converse results, an increase in PECAM-1 and
 Tie-2 PCR products, were obtained from xenografts in animals treated with
 the blocking EMAP II antibody (FIG. 2F). Negative controls for PCR
 amplification of the PECAM-1 and Tie-2 transcripts, without RT,
 demonstrated no specific PCR product in each rxn (data not shown).
 EMAP II inhibits epithelial maturation. It was postulated that pulmonary
 vascularization might influence epithelial cell differentiation. After
 administration of rEMAP II, histologic analysis of lung xenografts in
 these mice showed a marked inhibition of structural maturation (FIGS.
 3D-F) compared to vehicle treated animals (FIGS. 3A-C). This was
 demonstrated by a lack of well-defined bronchi with characteristic
 epithelium (FIG. 3A), or of distal airways with attenuated epithelium
 consistent with alveolar epithelium, as compared to those xenografts where
 the mouse received vehicle alone (FIG. 3B,C). In addition, lung xenografts
 in mice treated with EMAP II had alveolar epithelial cells that appeared
 dysplastic (FIG. 3E,F) and an apparent stasis in respiratory duct
 formation (FIG. 3D, arrows) as compared to those transplants in mice
 receiving vehicle alone (3A). To discern whether morphologic progression
 actually occurred, we assessed the xenografts for markers of distal lung
 morphogenesis. Lung xenografts in mice receiving vehicle alone underwent
 type II alveolar cell differentiation as marked by SP-C expression (FIG.
 4A,B). In contrast, lung xenografts in animals receiving EMAP II had a
 marked reduction in SP-C expression throughout the entire transplanted
 lung, even in the most peripheral airways (FIG. 4C,D). Further supporting
 our findings, animals receiving blocking EMAP II antibody had a strikingly
 increased number of type II cells, with essentially every distal
 epithelial cell expressing SP-C (FIG. 4E,F). Therefore, it appeared that
 excess EMAP II lead to profound inhibition of peripheral lung epithelial
 morphogenesis and differentiation.
 Consistent with the in situ hybridization findings, mRNA harvested from
 lung xenografts of animals treated with rEMAP II demonstrated a reduction
 in SP-C by RT-PCR compared to controls. In contrast, animals treated with
 the blocking EMAP II antibody exhibited an increase in the SP-C amplicon
 confirming the in situ results (FIG. 4G). Interestingly, T1-.alpha. a type
 I alveolar epithelial cell marker,was slightly elevated in xenografts
 treated with rEMAP II, whereas a marked reduction in T1-.alpha. was found
 in the blocking EMAP II antibody treated lungs, the inverse of the high
 level of SP-C expression, a type II cell marker (FIG. 4G). Negative
 controls for PCR amplification of the SP-C and T1-.alpha. transcripts,
 without RT, demonstrated no specific PCR product in each rxn (data not
 shown).
 We also evaluated glycogen production in the lung xenografts. Xenografts
 obtained from mice treated with rEMAP II demonstrated excess glycogen
 production (denoted by the magenta color) (FIGS. 5D-F) compared to vehicle
 alone (FIGS. 5A-C), further supporting the concept that EMAP II inhibited
 epithelial differentiation.
 EMAP II disruption of the epithelial-mesenchymal interface. To further
 examine the role of the anti-angiogenic protein EMAP II in lung
 morphogenesis, the localization of EMAP II in epithelial-megenchymal
 co-cultures was defined. Evaluation of lung epithelial-mesenchymal
 co-cultures after 3 days of incubation revealed EMAP II expression to be
 predominately in the peri-epithelial cyst region by both in situ
 hybridization (FIG. 6A) as well as immunohistochemistry (FIG. 6B)
 consistent with those results seen in fetal lung tissue [Schwarz, Am. J.
 Physiol. 276, L365-75 (1999)]. Interestingly, while EMAP II is expressed
 in epithelial and mesenchymal cells, its strongest expression is noted to
 be at the epithelial-mesenchymal junction as noted by the arrows in FIG.
 6A.
 To determine the effect of EMAP II on epithelial cyst formation,
 epithelial-mesenchymal co-cultures were exposed to increasing
 concentrations of rEMAP II, EMAP II blocking antibody, or vehicle (PBS or
 rabbit IgG respectively). Epithelial cyst formation was analyzed as the
 total number of cyst formed per high power field (HPF). There was a
 dose-dependent, 71% inhibition (p&lt;0.0001) of epithelial cyst formation
 and an alteration in structure in co-cultures exposed to EMAP II (FIG. 7B,
 D) as compared to control (FIG. 7A, arrows indicate normal epithelial cyst
 formation with the epithelial cells being surrounded by flattened laminin
 positive cells). Conversely, in the presence of the EMAP II blocking
 antibody (FIGS. 7C, E) there was a 54% increase (p&lt;0.01) in cyst
 formation that was also dose-dependent. Because we recently observed that
 EMAP II induces apoptosis in growing and dividing endothelial cells
 [Schwarz, Journal. of Experimental. Medicine 290 (1999)], we employed the
 TUNEL assay to determine whether induction of apoptosis due by EMAP II was
 responsible for the decrease in numbers of epithelial cyst. We found a
 time-dependent induction of apoptosis, starting in the peri-epithelial
 cyst region and progressing to include the entire epithelial cyst in
 co-cultures treated with rEMAP II as compared to control (data not shown).
 Apoptosis was also markedly decreased in those cultures exposed to the
 EMAP II blocking antibody as compared to control (data not shown).
 Congigtent with our findings in vitro, lung xenografts in animals treated
 with EMAP II had a marked increase in apoptosis localizing to the
 epithelial cells (data not shown).
 The foregoing is illustrative of the present invention, and is not to be
 construed as limiting thereof. The invention is defined by the following
 claims, with equivalents of the claims to be included therein.
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