WO1999024059A1 - Communication microvasculaire a mediation de pdgf et procedes d'utilisation - Google Patents

Communication microvasculaire a mediation de pdgf et procedes d'utilisation Download PDF

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Publication number
WO1999024059A1
WO1999024059A1 PCT/US1998/023892 US9823892W WO9924059A1 WO 1999024059 A1 WO1999024059 A1 WO 1999024059A1 US 9823892 W US9823892 W US 9823892W WO 9924059 A1 WO9924059 A1 WO 9924059A1
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growth factor
derived growth
platelet derived
endothelial cells
factor
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PCT/US1998/023892
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English (en)
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Robert D. Rosenberg
Jay M. Edelberg
William C. Aird
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Massachusetts Institute Of Technology
Beth Israel Deaconess Medical Center
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Priority to AU14541/99A priority Critical patent/AU1454199A/en
Publication of WO1999024059A1 publication Critical patent/WO1999024059A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators

Definitions

  • Endothelial cells of different microvascular beds carry out organ- specific functions, such as regulating angiogensis and modulating hemostasis, by expressing discrete sets of gene products. Endothelial cell diversity is particularly well documented in relation to the hemostatic activity of different vascular beds . For example, previous studies have established that plasminogen activator inhibitor-1 is expressed at high levels in vascular beds of the murine aorta, heart and adipose tissue, and at low levels in the vascular beds of liver, adrenals, and kidney (Sawdey and Loskutoff, J. Clin . Invest . 88:1346-1353 (1991).
  • Heterogenous gene expression can even be observed within different regions of a single vascular bed, as exemplified by the observation that higher levels of thrombomodulin are expressed in abdominal regions of the murine aorta relative to the level expressed in the thoracic region of the same vessel (Weiter-Guettler et al . , Circ . Res . 78:180-187 (1996). Differences in gene expression patterns are also found between microvascular and macrovascular endothelial cells (Speiser et al . , Blood 69 : 964-961 (1987) .
  • endothelial cells derived from arterial, venous and microcirculatory beds are characterized by noticeably different basal mitotic rates, and endothelial cells from macrovascular versus microvascular beds exhibit different growth responses to the addition of exogenous growth factors, including the expression of PDGF isoforms and receptors (Shimada et al . , Artery, 18:268-284 (1991; D'Amore et al . , Growth Fa ctors 8 : 61-75 (1993) .
  • PDGF isoforms and receptors Shiada et al . , Artery, 18:268-284 (1991; D'Amore et al . , Growth Fa ctors 8 : 61-75 (1993) .
  • the present invention is based on the elucidation of a platelet derived growth factor AB (referred to herein as PDGF AB) dependent pathway of microvascular communication between cardiac myocytes and microvascular endothelial cells, which allows for the extravascular regulation of endothelial cell gene expression In vivo, ex vivo and in vi tro . More specifically, the present invention describes a cardiac microvascular communication pathway that is capable of regulating the expression of endothelial cell genes involved in hemostatis and angiogenesis .
  • PDGF AB platelet derived growth factor AB
  • the invention pertains to regulators and methods of regulating the development and function of microvascular endothelial cells comprising regulating the interaction of PDGF AB with the high affinity PDGF receptor (referred to herein as PDGF-R ⁇ ) expressed on the surface of endothelial cells.
  • PDGF-R ⁇ high affinity PDGF receptor
  • substances, or regulators, that regulate the interaction of PDGF AB with endothelial cell PDGF-R ⁇ More specifically, the invention relates to a method wherein the regulated endothelial cell function (also referred to herein as activity) is selected from the group of activities consisting of proliferation, chemotactic migration, angiogenesis, neovascularization and hemostatic activities .
  • the angiogenic activity of microvascular endothelial cells is regulated by inducing the expression of the endothelial cell mitogen vascular endothelial growth factor (referred to herein as VEGF) and its receptor Flk-1.
  • VEGF endothelial cell mitogen vascular endothelial growth factor
  • Flk-1 receptor Flk-1
  • the hemostatic activity of microvascular endothelial cells is regulated by inducing the expression of von Willebrand factor (referred to herein as vWF) .
  • vWF von Willebrand factor
  • the invention also relates to a method of regulating tissue specific microvascular endothelial cell gene expression in PDGF-R ⁇ positive endothelial cells that constitutively express PDGF A comprising contacting the endothelial cells with a soluble factor, produced by cells residing in the local icroenvironment , which induces endothelial cell expression of PDGF B, thereby resulting in the formation of PDGF AB heterodimers .
  • the factor can be derived from cardiac myoctytes, and the factor's ability to mediate the induction of endothelial cell gene expression can be characterized by being susceptible to neutralization by interaction with an anti- epidermal growth factor (referred to herein as EGF) antibody.
  • EGF anti- epidermal growth factor
  • the soluble factor comprises exogenous PDGF AB .
  • the regulated gene is selected from the group consisting of vWF, VEGF, Flk-1 and other genes characterized by encoding a promoter region that is responsive to PDGF AB-mediated PDGF-R ⁇ signal transduction .
  • the invention also pertains to a method of promoting angiogenesis by PDGF-R ⁇ positive microvascular endothelial cells which constitutively express the PDGF A polypeptide chain, comprising contacting endothelial cells with a soluble factor which induces microvascular endothelial cell expression of PDGF B polypeptide chain, thereby resulting in the formation of the AB isoform of PDGF, which upon interaction with its high affinity (e.g. PDGF-R ⁇ ) receptor induces microvascular endothelial cell expression of VEGF and Flk-1.
  • a method of promoting angiogenesis by PDGF-R ⁇ positive microvascular endothelial cells which constitutively express the PDGF A polypeptide chain comprising contacting endothelial cells with a soluble factor which induces microvascular endothelial cell expression of PDGF B polypeptide chain, thereby resulting in the formation of the AB isoform of PDGF, which upon interaction with its high affinity (e.g.
  • the invention further pertains to a method of inhibiting PDGF AB-mediated PDGF-R ⁇ dependent signal transduction in microvascular endothelial cells comprising contacting endothelial cells with a regulator substance (also referred to as an agent) , that blocks PDGF AB binding to its corresponding receptor.
  • a regulator substance also referred to as an agent
  • the agent is an antibody, polyclonal or monoclonal, antibody fragment or a single chain antibody, which is characterized by an ability to bind to an eptiope that is present in the A or B polypeptide chain of PDGF, or in the ⁇ or ⁇ polypeptide subunit of PDGF receptor chains, or an epitope created by the formation of the dimeric PDGF ligand.
  • the substance is a rationally designed small molecule, or an oligopeptide, or a recombinant PDGF polypeptide chain or dimer which competes with, and thereby antagonizes, the binding of the endogenously produced PDGF to its corresponding receptor expressed on the surface of endothelial cells.
  • the invention also relates to a method of inhibiting PDGF AB-mediated VEGF/Flk-1 dependent neovascularization in microvascular endothelial cells comprising contacting PDGF-R ⁇ positive microvascular endothelial cells expressing PDGF AB heterodimers with a substance, regulator or agent, that inhibits PDGF receptor dimerization, thereby preventing downstream signal transduction events (e.g.
  • the agent is selected from the group consisting of rationally designed small molecules, oligopeptides derived from the sequence of the PDGF A and B polypeptide chains, recombinant polypeptide chains or PDGF heterodimers, and antibodies or functional portions of antibodies.
  • the invention further relates to a method for evaluating a candidate substance for its ability to regulate the interaction of PDGF AB with PDGF ⁇ receptors expressed on microvascular endothelial cells comprising coculturing microvascular endothelial cells and cardiac myocytes in transwell cultures, introducing a candidate substance that is being assayed for its ability to regulate the interaction of PDGF AB with PDGF-R ⁇ into the coculture, and determining the relative level of endothelial cell activity in cocultures maintained in the presence and absence of the candidate substance .
  • FIG. 1 is a schematic of cardiac microvascular communication.
  • PDGF B expression by cardiac microvascular endothelial cells is induced in the presence of cardiac myocytes.
  • PDGF B dimerizes with PDGF A and in turn induces PDGFR ⁇ receptor positive endothelial cells to express endogenous vWF, the vWF acZ-2 transgene, VEGF and Flk- 1.
  • Figures 2A is a graphic representation of ELISA data summarizing the relative percentages of PDGF A polypeptide chain expressed by endothelial cells cultured either alone (control) or in the presence of cardiac myocytes, or samples of recombinant PDGF AA, AB or BB isoforms .
  • Figures 2B is a graphic representation of ELISA data summarizing the relative percentages of PDGF B polypeptide chain expressed by endothelial cells cultured either alone (control) or in the presence of cardiac myocytes, or samples of recombinant PDGF AA, AB or BB isoforms .
  • Figures 3A - 3D are a set of 4 graphic representations of the protein levels of endogenous vWF, vWFlacZ-2 transgene product, VEGF and Flk-1 observed in samples obtained from cardiac microvascular endothelial cells cultured either alone (control) , or in the presence of either cardiac myocytes, PDGF AB, or cardiac myocytes and a neutralizing antibody to PDGF AB .
  • Figures 4A - 4C show the in vivo neutralization of PDGF AB induced expression of vWFlacZ-2 transgene.
  • Figures 4A and 4B illustrate X-Gal staining observed in 12.5 day vWFlacZ-2 embryo hearts injected with either an isotype control antibody (4A) or anti-PDGF antibody (4B) .
  • Figure 4C is a graphic representation of the percentage of hearts in each of the antibody treatment groups exhibiting X-Gal staining.
  • cardiac microvascular communication pathway which is capable of regulating genes involved in angiogenesis and hemostasis represents the first molecularly characterized local pathway by which an organ specifically controls gene expression in endothelial cells.
  • the disclosed model system provides a paradigm for similar tissue specific pathways that regulate the function of endothelial cells residing in alternative vascular beds in response to signals derived from their local icroenvironment .
  • a hallmark of a local communication scheme is the regional control of gene expression in distinct populations of cells.
  • the endothelium exhibits a remarkable diversity of cellular properties that are uniquely adapted to serve the needs of the underlying tissue, making the vascular system an excellent model in which to elucidate mechanisms of cellular diversity.
  • Heterogeneity within the endothelium has been described at the level of cell structure, antigen composition, mRNA expression, and cell function (Aird et al . J. Cell . Biol . 138 : 1117-1124(1997).
  • organ-specific functions of endothelial cells located in different vascular beds require a high level of phenotypic diversity, and subpopulations of microvascular endothelial cells residing in different vascular beds carry out organ- specific tasks by synthesizing discrete sets of gene products .
  • the concept underlying the experimental approach which led to the elucidation of the described communication pathway is a model of gene regulation which predicts that the expression pattern of endothelial cell genes observed within the vascular tree reflect the combined effects of an interplay between multiple signaling pathways, that vary from one microenvironment to another, and intracellular transcription mechanisms.
  • the envisioned array of local communication pathways provides an effective means of establishing functionally distinct endothelial cell populations which are capable of tailoring their pattern of gene expression to manifest alternative phenotypes in response to the specific needs of the surrounding tissue.
  • the presence of a subpopulation of cells characterized by a unique transcriptional control mechanism which renders them responsive to local signals may constitute a critical population of cells which allow the microvascular endothelium to fulfill its specific functional requirements.
  • Example 2 Such a population is herein exemplified, by the colocalization data of Example 2, which demonstrates that the expression of the transgene and PDGF-R ⁇ were both restricted to a subpopulation of microvascular endothelial cells which constitutively express PDGF A.
  • Previous studies have demonstrated a significant variation in the responsiveness of endothelial cells isolated from different vascular beds to growth factors (Shimada, et al . , Artery 18 : 268-284 (1991); D'A ore et al . , Growth Factors 8 : 61-75 (1993), including the expression of PDGF isofor s and receptors (Linder, V., Pa thobiology 63 : 257-264 (1995) .
  • the present invention is based on the discovery of a PDGF-mediated communication pathway which allows cardiac myoctyes to regulate gene expression in cardiac, and other, microvascular endothelial cells.
  • cardiac myocytes induce the expression of PDGF B polypeptide chains in a subpopulation of PDGF-R ⁇ positive microvascular endothelial cells which constitutively produce PDGF A polypeptide chains.
  • the resulting PDGF AB isoform binds to endothelial cell PDGF receptors resulting in the upregulation in expression of the protein products encoded by the vWF, VEGF and Flk-1 genes .
  • the platelet derived growth factor (PDGF) family includes three disulfide-bonded dimeric ligands, constituted from two related polypeptide chains, PDGF A and PDGF B, which are encoded by different genes (Leveen et al . , Genes & Devel . 8:1875-1887 (1994); Seifert, et al . , J. Biol . Chem . 268 : 4473-4480 (1993) .
  • PDGF expression in the majority of cell types generally accompanies functional or mitogenic activation.
  • PDGF-R ⁇ ⁇ -receptor
  • PDGF-R ⁇ ⁇ -receptor
  • PDGF-R ⁇ ⁇ -receptor
  • the pattern of binding of PDGF isoforms to different cell types is determined by the differential ability of each of the growth factor dimers to bind to their corresponding receptor subunits.
  • PDGF receptor dimerization occurs as a result of ligand binding and appears to be a prerequisite for signal transduction across the plasma membrane (Heldin, et al . , J. Biol . Chem . 264 : 8 905- 8 912 (1989).
  • PDGF encompasses all of the above-described isoforms, as well as biologically active fragments of PDGF. Fragments of PDGF can be obtained by methods well known to those of skill in the art, including digestion and synthesis of PDGF peptides.
  • the PDGF fragments useful in this invention are those which exhibit biological activity comparable to the activity of intact or full length growth factor. Biologically active PDGF, and PDGF fragments, are characterized by an ability to interact with, or bind to, a PDGF receptor expressed on endothelial cells.
  • This interaction of a biologically active PDGF fragment with a PDGF receptor will result in one, or more, of the following endothelial cell activities: dimerization of the PDGF receptor; induction of PDGF-R ⁇ signal transduction; expression of the vWF gene and/or its protein product; expression of the VEGF gene and/or its protein product; expression of the Flk-1 gene and/or its gene product; chemotactic cellular migration; or cellular differentiation. Detection of these activities which reflect the cellular consequences resulting from a PDGF/PDGF-R ⁇ interaction (also referred to herein as binding) , can be accomplished as described in the examples provided herein, or by the use of other methods well known to one of skill in the art.
  • PDGF/PDGF receptor analogs, derivatives and mutants are biologically active analogs, derivatives, and mutants of PDGF polypeptide chains, or PDGF receptor protein subunits.
  • Biological activity of these PDGF/PDGF receptor analogs, derivatives and mutants can be determined as describe above.
  • Previous studies have established that the regulation of endothelial cell gene expression varies between blood vessel type and location of the vascular bed (Aird, et al , J. Cell . Biol . 138 : 1111 - 1124 (1997) .
  • the multimeric glycoprotein von Willebrand factor which is a cofactor for platelet adhesion and a carrier for the antihemophiliac factor (Ruggeri and Ware, FASEB J.
  • vWF is expressed at higher levels on the venous side of the circulation compared with the levels observed to be expressed in arteries and and arterioles .
  • consistently low levels of vWF are present within the sinusoidal endothelial cells of the liver and spleen. Histochemical analysis reveals that the The vWF protein is present in clusters of endothelial cells oriented along the longitudinal axis of blood flow.
  • vWFIacZ-2 transgenic mice described herein have been previously used to demonstrate that endothelial cell expression of vWF transgene under the control of a human promoter sequence is regulated in vivo by a distinct organ- specific transcriptional pathway, which is responsive to signals from the microenviornment .
  • This conclusion was based on the fact that the transgene was observed to have a limited in vivo distribution pattern, relative to the endogenous gene, with the transgene promoter directing a limited pattern of endothelial cell subtype restricted expression in transgenic mice.
  • mice carrying a null mutation in the gene encoding the PDGF B polypeptide chain exhibit a phenotype which includes abnormal kidney glomeruli, heart and blood vessel dilation, anemia, thrombocytopenia and hemorrhages which invariably result in perinatal death ((Leveen, et al., Genes Dev. 8 : 1875-1887 (1994) .
  • a phenotype which includes abnormal kidney glomeruli, heart and blood vessel dilation, anemia, thrombocytopenia and hemorrhages which invariably result in perinatal death ((Leveen, et al., Genes Dev. 8 : 1875-1887 (1994) .
  • perinatal death (Leveen, et al., Genes Dev. 8 : 1875-1887 (1994) .
  • Overall the data from the loss-of-function studies conducted in knockout mice demonstrate that normal development of the cardiac vasculature requires intact PDGF signaling components.
  • PDGF neurotrophic factor 4 : 431 -442 (1993) and allograft rejection (Shaddy, et al . , Am . J. Cardiol . 77:1210-1215 (1996).
  • the putative functions of PDGF generally relate to different responses to pathological conditions.
  • PDGF has been functionally implicated in connective tissue overgrowth resulting from chronic inflammatory processes, atherosclerosis, fibrosis, and tumor stroma formation (Leveen, et al . , Genes and Devel . 8:1875-1887 (1994).
  • PDGF A and B and the PDGF-R ⁇ in cardiac development and disease further suggests that the PDGF pathway may be involved in governing the development and function of microvasculature endothelial cells in general, and the cardiac microvasculature in particular.
  • VEGF vascular endothelial growth factor
  • Flk-1 vascular endothelial growth factor
  • Angiogenesis may be physiologic, as exemplified by endometrial cycling, or pathological, as exemplified by the neovascularization of tumors.
  • VEGF vascular endothelial cell growth factor
  • Flt-1 and Flk-1 The endothelial cell-specific mitogen vascular endothelial cell growth factor (VEGF) is a major mediator of pathological angiogenesis, and the expression of its two receptors, Flt-1 and Flk-1 has been shown to be related to the normal development of the vasculature during embryogenesis (Ferrara et al . , Na ture 380 : 439-442 (1996). ' Mice expressing homozygous mutations for either VEGF receptor die in utero . Recent loss-of-function studies conducted with knockout mice have further confirmed that VEGF and Flk-1 are critical for both normal cardiac vessel development and angiogenesis.
  • mice possessing a single copy of the VEGF gene also exhibit early embryonic lethality, at the same stage of development at which death occurs in the corresponding null animal, providing a unique example of a situation in which a haploid gene dosage is sufficient to cause lethality (Luisa and Dvorak, Thrombosis and Haemostasis 78 : 612 - 611 (1997).
  • Homozygous deletions of Flk-1 die at approximately the same time in their development as the VEGF knockout embryos and exhibit similar defects in cardiac angiogenesis (Shalaby, et al . , Na ture 376: 62- 66 (1995).
  • microvasculature endothelial cells play an essential role in regulating hemostasis and angiogenesis.
  • the described PDGF dependent pathway of cardiac microvascular communication integrates cardiac myocyte produced PDGF-mediated signals into a model of cardiac microvascular hemostatic and angiogenic regulation. Having identified the pathway, the present invention can be used to identify agents, substances, or regulators capable of regulating (or modulating) angiogenesis and hemostasis in both in vi tro and in vivo situations and to identify factors which can induce the expression of microvascular endothelial cell genes.
  • the invention can be used to identify agents which can inhibit the cellular effects of a PDGF/PDGF-R ⁇ interaction either by inhibiting the binding of PDGF AB to its receptor, or alternatively by preventing the downstream signaling events associated with PDGFR ⁇ mediated signal transduction.
  • the regulators identified by the methods described herein can be formulated into pharmaceutical compositions, or medicaments for uses in therapies to regulate the interaction of PDGF AB with the PDGF-R ⁇ .
  • the present invention also encompasses substances (also referred to herein as regulators, factors or agents) that modulate (or regulate) the cellular effects of PDGF/PDGF-R ⁇ interactions.
  • substances also referred to herein as regulators, factors or agents
  • the type of substance useful as an inhibitor of PDGF AB-R ⁇ receptor signal transduction is any molecule which prevents PDGF receptor di erization, or any molecule which inhibits ligand-induced conformational changes in the extracellular domains of the receptor subunits that are necessary to promote receptor-receptor interactions which result in signal transduction.
  • the type of substance useful in the present invention as an enhancer is any molecule which enhances the level of endothelial cell activity that results from a PDGF AB-R ⁇ interaction.
  • the regulatory substance can be a rationally designed small molecule, an oligopeptide derived from the nucleotide sequence of either one of the PDGF polypeptide chains or PDGF receptor protein subunits, recombinant PDGF chain mutants or an antibody.
  • Molecules useful in the present invention include naturally occuring or recombinant proteins, small organic molecules, synthetic peptides, biologically active fragements, antibodies, or functional portions of antibodies.
  • an oligopeptide that mimics the region of PDGF AB that interacts with PDGF-R ⁇ , and which binds to the receptor but does not induce receptor dimerization would exemplify a useful substance.
  • a recombinant PDGF polypeptide chain comprising a mutant amino acid sequence which interacts with a PDGF receptor but fails to induce a conformational change in the receptor subunit which is required for signal transduction, would be a useful subtance in the present invention.
  • a recombinant molecule comprised of multimeric binding sites which efficiently crosslinks PDGF receptors would be a useful substance for inducing or enhancing (also referred to herein as modulating) the cellular effects of a PDGF AB- R ⁇ interaction.
  • antibody includes both polyclonal and monoclonal antibodies or functional portions therof (e.g. an antigen binding portion such as an Fv, Fab, Fab 1 , or F(ab)2 fragment) which binds to an eptiope present in the group consisting of the PGDF A polypeptide chain , PDGF B polypeptide chain , PDGFR ⁇ subunit, PDGFR ⁇ subunit, or an epitope created by the formation of the one of the dimeric isoforms of PDGF.
  • the type of antibodies that could act as modulators of PDGF AB binding can also be chimeric antibodies or humanized antibodies or a recombinant fusion protein.
  • inhibitor is intended to encompass any qualitative or quantitative reduction in a measured effect or charactertistic, including complete prevention, relative to a control.
  • induce or “enhance” is intended to encompass a qualitative or quantitative increase in the level of expression of a gene, or its encoded protein product, relative to the expression level present in a control sample.
  • modulate and “regulate” are intended to indicate an ability to selectively control a particular activity, it is intended to encompass both the ability to inhibit or to enhance (or induce) the level of a particular cellular activity as described herein.
  • the invention also pertains to an assay for identifying substances which can regulate endothelial cell activity by regulating the interaction of PDGF AB with PDGF ⁇ receptors expressed on microvascular endothelial cells.
  • the assay comprises coculturing microvascular endothelial cells and cardiac myocytes " , which have been demonstrated herein to produce a soluble factor which induces the expression of PDGF B in a subpopulation of PDGF ⁇ receptor positive cardiac microvascular endothelial cells, in transwell cultures in the presence and absences of a candidate substance whose regulatory ability is being determined under conditions suitable for promoting a PDGF AB ⁇ receptor interaction.
  • the regulatory activity of the substance is determined by comparing the level of endothelial cell activity observed in the presence of the substance to the level of activity observed in the absence of the candidate substance.
  • An increase in the level of endothelial cell activity relative to the control indicates that the canditate substance promotes PDGF AB- mediated PDGF ⁇ receptor signal transduction; a decrease in the level or extent of endothelial cell activity relative to the corresponding control level indicates that the candidate substances is an inhibitor of PDGF AB-mediated PDGF ⁇ receptor signal transduction.
  • PDGFs include chemotaxis, differentiation, and regulation of other cell functions, such as contraction or the production of extracellular matrix components, or as shown herein, the regulation of endothelial cell gene expression.
  • Appropriate endothelial cell activities which could provide the basis of a model assay in which to identify substances that are capable of modulating endothelial cell gene expression therefore include proliferation, chemotactic migration, expression of PDGF isoforms, and gene expression. These activities can be monitored according to the protocols used in the examples provided herein or by the used of standard methods that are well known to those skilled in the art.
  • PDGF is produced by numerous cell types in addition to platelets, including monocytes/macrophages, smooth muscle cells, fibroblasts, placenta cytotrophoblasts, neurons, certain glial cells and endothelial cells. Any of these cell types could be utilized in endothelial transwell cocultures as potential sources of a soluble factor or regulator capable of mediating intracellular communication .
  • EGF epidermal growth factor family
  • the EGF family is a large highly redundant group of growth factors, which has been shown to be involved in multiple biological processes, including mediating aspects of vascular physiology and pathophysiology .
  • soluble factors useful in the present invention as potential mediators of intercellular communication include growth factors and cytokines which are known to be produced by cells residing in close proximity to microvascular beds of major organs, which have corresponding receptors that are characteristically expressed on the surface of endothelial cells and which are capable of mediating signal transduction.
  • the method of enhancing the cellular consequences of a of PDGF/PDGF-R ⁇ interaction can be an in vivo method of mediating intracellular communication in a mammal, comprising administering to the mammal an effective amount of an agent which induces or enhances PDGF AB-mediated PDGF-R ⁇ signal transduction.
  • a therapeutically effective amount of one or more substances can be administered to an individual by an appropriate route, either alone or in combination with another " drug in a pharmaceutical composition.
  • routes of administration are possible, including but not limited to, topical, parental (e.g. intravenous, intraarterial, intramuscular, subcutaneous injection) and inhalation (e.g. intrabronchial, intranasal, or oral inhalation) routes of administration, depending on the identity of the organ and vascular bed being targeted.
  • parental e.g. intravenous, intraarterial, intramuscular, subcutaneous injection
  • inhalation e.g. intrabronchial, intranasal, or oral inhalation
  • Formulation of an agent to be administered will vary according to the route of administration selected (e.g. solution, emulsion, capsule, aerosol) .
  • An appropriate composition comprising the agent to be administered can be prepared in a physiologically acceptable vehicle or carrier.
  • suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parental vehicles can include sodium chloride, Ringer's dextrose, , dextrose and sodium chloride, lactated Ringer's or fixed oils, for instance.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replinishers and the like (See, generally, Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., PA, 1985).
  • the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, neublizer or pressurized aerosol dispenser) .
  • a suitable dispenser for administration e.g., an atomizer, neublizer or pressurized aerosol dispenser
  • the actual preferred effective amounts of substance in a specific case will vary according to the specific substance being administered, the particular composition of the formulation, the route of administration, the particular situs of treatment, and the organism being treated. If administered to an individual, dosages for a given recipient will be determined on the basis of the individual characteristics, such as body weight, age, and the type and severity of the condition being treated.
  • microvascular endothelial cell functions can be regulated for the therapeutic benefit of a mammal.
  • vWF von Willebrand factor
  • transgenic mice The generation and identification of transgenic mice as well as the analysis of tissue sections and whole mounts for lacZ activity and vWF immunohistochemistry was carried out as described in Aird, et al . , Proc . Na tl . Acad . Sci . USA 92 : 4567-4571 (1995) .
  • CMEC Cardiac microvascular endothelial cells
  • CMEC were then isolated on a gradient of 25-40% Percoll (Pharmacia) .
  • CMEC were cultured in DMEM containing 5% fetal calf serum, 100 ⁇ g/ml heparin, 10 ⁇ g/ml endothelial growth supplement, 1% BME vitamins, 5 ⁇ g/ml insulin, 5 ⁇ g/ml transferrin, 5 ng/ml selenium, and 1% endothelial cell growth factor (Sigma) .
  • the CMEC were grown on flasks coated with 1% gelatin in PBS and passaged up to 5 times prior to use in a coculture assay.
  • the cardiac myocytes were prepared according to the method of Iwaki, K, et al . (1990) . Briefly, ventricular myocytes were prepared from the ventricles of 17.5 day old fetal mice by fragmenting the tissue with a straight-edge razor, followed by digestion with 0.5 mg/ l collagenase II (Worthington Biochemical Corp., Freehold, NJ) and 1.0 mg/ml pancreatin (Sigma Chemical Co., St. Louis, MO) in ADS buffer (116 mM NaCl, 20 mM Hepes, 1 mM NaH 2 P0 4 , 5.5 mM glucose, 5.4 mM KC1, 0.8 mM MgS0 4 , pH 7.4) at 37°C for 10 minutes. The cells were then harvested by centrifugation at 700g at 4°C for 5 minutes .
  • ADS buffer 116 mM NaCl, 20 mM Hepes, 1 mM NaH 2 P0 4 , 5.5 mM glucose,
  • the endothelial cells were cultured at a density of 5 x 10" cell/mm 2 in 12 mm 0.4 ⁇ m pore transwell plates (Costar, Cambridge, MA) either alone or in the presence of the cardiac myocytes. Cocultures were then established by introducing cardiac myocytes into established endothelial cell cultures at a ratio of 1:1. The cells were cultured for 72 hr, with a media change at 48 hr . The cultures were then washed twice with PBS, and the media was changed to DME supplemented with 1%
  • TRANSFECTANTS EXPRESSING DOMINANT NEGATIVE RECEPTORS Transfectants expressing either a dominant negative PDGF-alpha receptor, which preferentially inhibits signaling through the PDGF-R ⁇ pathway (Mercola, et al . , Genes Dev. 4:2333-2341 (1990), or a control with green fluorescent protein were generated using amphotropic retroviruses as described in Hawley, et al . , Gene Ther . 2: 136-138 (1994). Briefly, retrovirus was produced in the PHEONIX packaging cell line, which was plated at 10 6 cells/well (in a 6 well cluster plate), and allowed to adhere overnight.
  • the cells were transfected with the respective MSCV-neo2.1 plasmids (10 ug/plate), by calcium-phosphate coprecipitation and incubated at 37°C for 5 hr .
  • Replication-defective retroviruses were harvested 5 hr later, and flash frozen in liquid nitrogen before use.
  • Cardiac microvascular endothelial cells were plated into 6 well cluster plates at a density of 10 5 cells/well. After growing overnight, DEAE-Dextran (25 ug/ml) and the replication-defective retroviruses were added to the endothelial cells and were incubated at 32°C for 24 hr. Fresh media were exchanged after 24 hr, and the cells were cultured for another 48 hr at 37°C to allow for gene expression before their use in the coculture experiments described above .
  • RNA was isolated from cultures of cardiac microvascular endothelial cells cultured for four days either alone, or in the presence of cardiac myocytes. Cellular lysates were prepared according to the method described in Aird, W.C. et al , J. Cell . Biol . 138 : 1117- 1124 (1997) . In addition, total cellular RNA was also isolated from cardiac microvascular endothelial cells that were cultured either alone or in the presence of PDGF AB (10 ng/mL) for 6, 24 and 96 hr . Samples were assayed for expression of PDGF A and PDGF B message.
  • RNA samples prepared from cardiac myocyte cocultures and samples from the PDGF time course experiment were assayed for the expression of VWF, LacZ, VEGF, Flk-1, G3PDH, and ⁇ -actin.
  • Reverse transcription was performed on equal amount of RNA samples followed by polymerase chain reaction (RT-PCR) of the CDNA.
  • RT-PCR polymerase chain reaction
  • Cellular and secreted protein samples were isolated from cardiac microvascular endothelial cells cultured alone, or in the presence of cardiac myocytes, or in presence of cardiac myocytes and neutralizing PDGF antibody, or alternatively in the presence of recombinant PDGF AA, AB or BB.
  • the relative level of each immunoreactive PDGF polypeptide chain was determined by two-antibody sandwich enzyme-linked immunoassay (ELISA) .
  • the capture antibody was a goat polyclonal antibody to PDGF A (AF- 221-NA, R&D Systems) , coated onto Immunon 2 ELISA strips (Dynatech Laboratories, Chantilly, VA) . Samples (50 ul/well) of culture supernatant harvested from the cardiac endothelial cell cocultures described above were applied to the anti-PDGF coated strips and incubated for 2hrs at room temperature.
  • the assay strips were washed 10 times with PBS, prior to the addition of rabbit polyclonal detection antibodies specific for the PDGF A and B polypeptide chains (anti-PDGF A, sc-128, Santa Cruz, Biotechnology; anti-PDGF B, ZP-215, Genzyme Diagnostics).
  • the chain-specific detection antibodies were used at a dilution of 1:1000.
  • the antigenic levels were determined by adding a peroxidase-labeled donkey anti-rabbit polyclonal (at a 1:1000 dilution), followed by the addition of 1,2 phenyl-enediamine substrate (0.67 g/1), and determination of the absorbance at 490 nm.
  • Relative antigenic levels of vWF, VEGF, and Flk-1 were determined by Western dot blotting with the following respective antibodies 082 (Dako, 1:500 dilution), sc-152 (Santa Cruz, 1:1000 dilution), and sc-315 (Santa Cruz, 1:500 dilution).
  • a peroxidase- labeled donkey polyclonal antibody to rabbit IgG was used as the secondary antibody at a dilution of 1:1000.
  • the signal was developed with a chemiluminescence system (ECL; Amersham Corp., Arlington, IL) .
  • Relative ⁇ -galactatosidase activities were determined by cleavage of ONPG (Sigma) . All protein analysis was performed in triplicate sets with a minimum of two independent experiments.
  • vWFLacz-2 hearts were harvested and 8 ⁇ m serial cryosections were obtained. Alternate sections were processed for detection of either the transgene or the PDGF-R ⁇ . Transgene expression (vWFLacZ-2) was determined by X-Gal staining. Immunostaining for PDGF- R ⁇ was performed with the primary and secondary antibodies sc-338 (Santa Cruz) and Cy3-conjugated anti-rabbit IgG (Jackson Immuno Research) respectively. Photomicrographs of adjacent serial sections with X-Gal and immunostaining, respectively, were superimposed. Cardiac microvascular endothelial cells and myocytes were cultured and fixed as previously described.
  • the samples were immunostained for WF or Flk-1, with above antibodies and costained with a goat antibody to PDGFR ⁇ (R&D Systems, AF-307-NA) , and secondary antibody staining with FITC-and Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch) . Duel label photomicrographs were superimposed.
  • EXAMPLE 1 ENDOTHELIAL CELL GENE EXPRESSION IS PROGRAMMED BY SIGNALS FROM THE TISSUE
  • the X-Gal reaction product was detected within blood vessels of the brain, and in a subset of microvessels within the heart and skeletal muscle.
  • cardiac sections stained with X-Gal and processed for immunoperoxidase detection of endogenous vWF the transgene and endogenous gene products colocalized in the endothelial lining of capillary vessels.
  • endothelial cells of the coronary arteries, coronary veins, penetrating arteries, and endocardium of the heart exhibited no detectable B- galactosidase activity but possessed immunoreactive endogenous vWF protein.
  • Transgene expression was similarly absent in the vascular beds of other organs, including the liver, spleen, lung, and kidney as well as in the aorta.
  • LacZ mRNA was detected only in brain, heart and skeletal muscle.
  • mRNA from the endogenous vWF gene and from the endothelial cell restricted thrombomodulin gene was present in all tissues examined.
  • vWF mRNA levels varied from one organ to another and correlated with transcript levels detected by ribonuclease protection assays.
  • the observed vascular-bed specific expression pattern of vWFlacZ-2 suggests that expression of the vWF transgene is regulated in vivo by the interaction of regional transcriptional networks with distinct promoter elements .
  • the colocalization data further suggests that the PDGF-R ⁇ may play a regulatory role in the cardiac microvascular expression of the transgene, thereby implicating a role for PDGF in mediating transcriptional regulation of endothelial cell genes.
  • the expression of the transgene recapitulates the expression of the endogenous vWF gene in the cardiac microcirculation, the expression of PDGF-R ⁇ in these cells may identify them as a distinct subset of microvascular endothelial cells.
  • Cardiac microvascular endothelial cells were cultured alone or in the presence of cardiac myocytes grown in transwells, to assess the potential role of PDGF involvement in the induction of cardiac microvascular gene expression. Samples of secreted protein taken from the transwell cocultures were analyzed by ELISA to determine the chain composition of the PDGF dimers. The data summarized in Figures 2A and 2B demonstrate that cardiac microvascular endothelial cells constitutively express the PDGF A isoform, leading to the formation of PDGF AA homodimers . Cardiac endothelial cells cultured in the presence of cardiac myocytes, were induced to express PDGF B, leading to the formation of PDGF heterodimers.
  • Both the homodimer and heterodimer isoforms of PDGF are ligands for the PDGF-R ⁇ .
  • the in si tu localization data suggests that cardiac myocyte-mediated induction of PDGF-R ⁇ positive endothelial cells gene expression likely involves the induced AB heterodimer, but not the constitutively expressed AA homodimer.
  • This hypothesis was tested by the addition of exogenous recombinant PDGF ligands (e.g. PDGF AA, PDGF AB, or PDGF BB) to endothelial cultures.
  • exogenous recombinant PDGF ligands e.g. PDGF AA, PDGF AB, or PDGF BB
  • EXAMPLE 4 PDGF AB HETERODIMER INDUCTION OF vWF AND vWFLacZ-2 TRANSGENE
  • the PDGF AB induction of vWF and v ⁇ IFLacZ-2 was confirmed by RNA analysis.
  • this result is considered in light of the above reported colocalization data (indicating that vWF transgene expression is restricted to a PDGF ⁇ receptor positive subpopulation of microvascular endothelial cells) it suggests that cardiac myocyte endothelial cell communication is mediated by PDGF AB induction of PDGF-R ⁇ signal transduction.
  • vascular endothehal growth factor VEGF
  • Flk- 1 vascular endothehal growth factor
  • VEGF AB- ⁇ receptor interaction to regulate VEGF and Flk-1 in endothelial cells was therefore examined. Additional studies revealed that myocytes induced VEGF as well as Flk-1 gene expression. Similar to vWF and its transgene, the observed increases in VEGF and Flk-1 were comparably increased by PDGF AB . Moreover, antibody neutralization of PDGF AB also inhibited this induction. The increase of transcript in the presence of PDGF AB confirmed that, like vWF, there is inducible expression of both VEGF and its receptor ( Figures 3C and 3D) . VEGF and Flk-1 expression were observed to be comparably increased by
  • PDGF AB and inhibited by antibody neutralization of PDGF AB .
  • the increase of transcript in the presence of PDGF AB confirmed that, like vWF, there is a PDGF-mediated induction of both VEGF and its receptor.
  • Involvement of PDGF-R ⁇ was confirmed by immunostaining which indicated a colocalization of Flk-1 and the PDGF-R ⁇ .
  • the involvement of PDGF-R ⁇ was further confirmed by the observation that the expression of the dominant negative PDGF-R ⁇ in endothelial cells inhibited the induction of gene expression.
  • PDGF AB- ⁇ receptor interaction constitutes a critical pathway in the development and function of cardiac microvessels .
  • the in vivo significance of this interaction was examined by determining the effects of ligand inhibition during cardiac development.
  • PDGF AB was blocked by the injection of a relatively small dose of neutralizing polyclonal antibody (administered at a dose which was chosen because it was known to be insufficient to effect normal cardiac development) into the amniotic fluid surrounding day 12.5 vWFLacZ-2 embryos. Control embryos were injected with non-immune antibody. The following day the hearts were explanted and processed for X-Gal staining.
  • Figures 4A and 4B illustrate the X-Gal staining pattern representative of the majority of samples observed in each treatment group.

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Abstract

L'invention concerne la régulation dépendant du PDGF-AB de l'expression génique des cellules endothéliales, plus particulièrement des cellules endothéliales microvasculaires cardiaques à récepteur PDGF α qui expriment constitutivement le PDGF-A. L'invention concerne aussi des procédés d'utilisation de la voie décrite pour réguler le développement et la fonction des cellules endothéliales.
PCT/US1998/023892 1997-11-07 1998-11-06 Communication microvasculaire a mediation de pdgf et procedes d'utilisation WO1999024059A1 (fr)

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WO2001023604A3 (fr) * 1999-09-28 2002-08-08 Infectio Diagnostic Inc Genes a fort pouvoir de conservation et leur utilisation pour produire des sondes d'acide nucleique specifiques a l'espece, specifiques au gene, specifiques a la famille, specifiques au groupe et universelles et des sondes d'amplification, en vue de detecter et d'identifier rapidement des micro-organismes algaires, archeoba
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US7943346B2 (en) 1994-09-12 2011-05-17 Geneohm Sciences Canada Inc. Probes and primers for detection of bacterial pathogens and antibiotic resistance genes
US8034588B2 (en) 1997-11-04 2011-10-11 Geneohm Sciences Canada Inc. Species-specific, genus-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial and fungal pathogens and associated antibiotic resistance genes from clinical specimens for diagnosis in microbiology laboratories
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J.M. EDELBERG ET AL.: "PDGF MEDIATES CARDIAC MICROVASCULAR COMMUNICATION.", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 102, no. 4, 15 August 1998 (1998-08-15), NEW YORK, N.Y., US, pages 837 - 843, XP002099044 *
R.A. SEIFERT ET AL.: "PDGF-AB REQUIRES PDGF RECEPTOR ALPHA-SUBUNITS FOR HIGH-AFFINITY, BUT NOT FOR LOW-AFFINITY, BINDING AND SIGNAL TRANSDUCTION.", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 6, February 1993 (1993-02-01), BALTIMORE, MD, US, pages 4473 - 4480, XP002099043 *
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7943346B2 (en) 1994-09-12 2011-05-17 Geneohm Sciences Canada Inc. Probes and primers for detection of bacterial pathogens and antibiotic resistance genes
US8426137B2 (en) 1996-11-04 2013-04-23 Genohm Sciences Canada, Inc. Methods and probes for detecting a vancomycin resistance gene
US8034588B2 (en) 1997-11-04 2011-10-11 Geneohm Sciences Canada Inc. Species-specific, genus-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial and fungal pathogens and associated antibiotic resistance genes from clinical specimens for diagnosis in microbiology laboratories
US8067207B2 (en) 1997-11-04 2011-11-29 Geneohm Sciences Canada Inc. Species-specific, genus-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial and fungal pathogens and associated antibiotic resistance genes from clinical specimens for diagnosis in microbiology laboratories
WO2001023604A3 (fr) * 1999-09-28 2002-08-08 Infectio Diagnostic Inc Genes a fort pouvoir de conservation et leur utilisation pour produire des sondes d'acide nucleique specifiques a l'espece, specifiques au gene, specifiques a la famille, specifiques au groupe et universelles et des sondes d'amplification, en vue de detecter et d'identifier rapidement des micro-organismes algaires, archeoba
US8114601B2 (en) 1999-09-28 2012-02-14 Geneohm Sciences Canada Inc. Highly conserved genes and their use to generate probes and primers for detection of microorganisms
US8182996B2 (en) 1999-09-28 2012-05-22 Geneohm Sciences Canada Inc. Compositions and methods for detecting Klebsiella pneumoniae
US10047404B2 (en) 1999-09-28 2018-08-14 Geneohm Sciences Canada, Inc. Highly conserved tuf genes and their use to generate probes and primers for detection of coagulase-negative Staphylococcus
EP1436329A1 (fr) * 2001-09-20 2004-07-14 Alexion Pharmaceuticals, Inc. Anticorps anti-pdgf et procedes de production d'anticorps mis au point
EP1436329A4 (fr) * 2001-09-20 2005-04-27 Alexion Pharma Inc Anticorps anti-pdgf et procedes de production d'anticorps mis au point

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