WO2000004929A1 - Utilisation de niveaux reduits d'un produit genique fonctionnel a potentiel de recepteur transitoire - Google Patents

Utilisation de niveaux reduits d'un produit genique fonctionnel a potentiel de recepteur transitoire Download PDF

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Publication number
WO2000004929A1
WO2000004929A1 PCT/US1999/016822 US9916822W WO0004929A1 WO 2000004929 A1 WO2000004929 A1 WO 2000004929A1 US 9916822 W US9916822 W US 9916822W WO 0004929 A1 WO0004929 A1 WO 0004929A1
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cells
transient receptor
receptor potential
potential gene
trp
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PCT/US1999/016822
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Troy Stevens
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South Alabama Medical Science Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the subject invention is directed generally to a method for treating or preventing an inflammatory condition, by decreasing inflammatory gaps in pulmonary endothelial cells, and more particularly to decreasing levels of functional transient receptor potential gene product .
  • Pulmonary endothelial cells are a nonexcitable cell type in which humoral and neural signaling agents increase the free cytosolic Ca 2+ concentration ([Ca 2+ ] 1 ) by inducing Ca + release from intracellular stores and Ca 2+ entry across the cell membrane (4,34) .
  • Increased [Ca 2+ ] has been implicated in many endothelial -directed vascular responses including regulation of vascular tone and permeability (2, 23, 36), angiogenesis (20), and leukocyte trafficking (17) .
  • Activation of Ca 2+ entry appears essential for each of these processes, although many modes of Ca 2+ entry exist and a specific pathway regulating endothelial cell shape has yet to be identified.
  • Non-cardiogenic pulmonary edema represent a significant clinical complication that increases patient morbidity and mortality.
  • ARDS adult respiratory distress syndrome
  • Non-septic ARDS has an estimated mortality rate of 40-60% whereas septic ARDS has an estimated mortality rate exceeding 90%.
  • Clinical management of ARDS patients is supportive, and unfortunately not a single pharmacologic strategy has been utilized successfully to improve patient outcome. The paucity of effective drug therapy and poor prognosis in these patients indicates the mechanisms underlying inception, propagation and resolution of the disease are not well understood.
  • any methods that can decrease these inflammatory gaps could be useful in treating and/or preventing inflammation.
  • the subject invention provides such a method which involves the transient receptor potential (trp) gene. More particularly, the invention provides a method of decreasing inflammatory gaps in pulmonary endothelial cells, the method comprising decreasing levels of functional transient receptor potential gene product (TRP) in the cells. The invention further provides a method of treating or preventing an inflammatory condition in a subject, the method comprising administering to the subject an amount of a compound effective to decrease levels of functional transient receptor potential gene product in the cells of the subject .
  • TRP transient receptor potential gene product
  • Figs. 1A-1C illustrate store-operated Ca 2+ entry in rat (R) pulmonary arterial endothelial cells (PAECs) .
  • 1A individual traces from cells challenged with thapsigargin (TG; l ⁇ M) in presence of 2 mM extracellular Ca 2+ concentration ([Ca 2+ ] 0 ).
  • IB representative trances comparing TG-induced cytosolic Ca 2+ concentration ( [Ca 2+ ] response in presence (solid lineO and absence (dashed line) of [Ca 2+ ] 0 .
  • [Ca 2+ ] 0 was increased from 100 nM to 2 mM at time indicated by Ca 2+ . Arrows, time of addition.
  • 3D readdition of 2 mM [Ca 2+ ] 0 to RPAECs treated as in C;
  • 5A-5C illustrate the ffect of thapsigargin on F-actin organization in presence of 2 mM (5A) , 100 nM (5B) , and 100 nM [Ca 2+ ] 0 followed by restoration of [Ca 2 '] 0 to 2 mM (5C) .
  • Confocal microscopy was performed on 0.3- ⁇ m sections. Three micrographs/ treatment are shown. I: cross section through upper portion of cells. In presence of 2 mM [Ca 2+ ] 0 , diffuse perinuclear staining is evident. In low [Ca 2+ ] 0 , a peripheral actin band with cell-to-cell contact sites is prominent.
  • peripheral band retracted after [Ca + ] 0 was readded, and intercellular actin projections are discernible. II and III: cross sections of middle and lower portions of cells, respectively.
  • peripheral (cortical) actin band is absent, and F-actin appears to align in stress fibers.
  • F-actin appears to align in stress fibers.
  • low [Ca 2+ ] 0 diffuse punctate staining is observed, but cortical actin band is still present.
  • stress fiber formation is obvious.
  • the subject invention is based on the discovery that decreasing levels of functional transient receptor potential gene product (TRP) in a cell (such as by decreasing transient receptor potential (trp) gene expression or by decreasing activity of TRP or by decreasing formation of calcium channels by TRP) can decrease inflammatory gaps in pulmonary endothelial cells. Inflammatory gaps occur between pulmonary endothelial cells due to an increase in intracellular calcium in the cells. The increase in intracellular calcium in the cells occurs via TRP calcium channels. Decreasing levels of functional TRP can therefore be used to decrease intracellular calcium and therefore decrease gap formation and therefore decrease inflammation. TRPs form calcium channels which belong to the family of store-activated calcium channels. One or more isoforms of TRP may be required to form a functional calcium channel. Decreasing "levels" of functional TRP refers to decreasing expression of the trp gene, decreasing activity of the TRP such as by inhibiting one or more TRP isoforms, and/or decreasing the formation of active membrane-spanning calcium channels by the TRP.
  • TRP functional
  • the invention thus provides a method of decreasing inflammatory gaps in pulmonary endothelial cells, the method comprising decreasing levels of TRP in the cells.
  • Levels of TRP in the cells can be decreased by various methods, at the gene and protein and "functional calcium channel” levels.
  • the levels are decreased by decreasing trp gene expression of the TRP in the cells. This can be accomplished by exposing the cells to a compound which decreases trp gene expression of the TRP.
  • the compound could be, for example, an antisense oligonucleotide targeted to the trp gene.
  • the compound which decreases trp gene expression of the TRP could be a ribozyme, which is a special category of antisense RNA molecule having a recognition sequence complementary to the mRNA encoding the TRP.
  • a ribozyme not only complexes with a target sequence via complementary antisense sequences, but also catalyzes the hydrolysis, or cleavage, of the template mRNA molecule. The expression of the TRP protein is therefore prevented.
  • Other methods for decreasing trp gene expression could also involve site-directed mutagenesis of the trp gene to prevent expression of the TRP, or various gene therapy techniques.
  • Levels, in particular activity, of TRP in the cell can also be decreased by exposing the cells to an inhibitor of the TRP.
  • inhibitors of voltage gated calcium channels include, for example, nifedipine, nitrendipine, verapamil, and related compounds.
  • Other inhibitors of the TRP could also readily be identified by various screening methods used in the art (see more detailed discussion below) .
  • peptide inhibitors could also be identified with currently known screening methods (for example, using phage display libraries and other peptide screening methods) .
  • Levels of TRP in the cell can also be decreased by exposing the cells to a compound which interferes with membrane calcium channel formation by the TRP.
  • the method of the subject invention is a method of decreasing inflammatory gaps in pulmonary endothelial cells
  • the cells of interest can be of human or animal origin.
  • the invention further provides a method of treating or preventing an inflammatory condition in a subject, the method comprising administering to the subject an amount of a compound effective to decrease levels of TRP in the cells of the subject.
  • the compound may decrease levels of TRP by decreasing trp gene expression of the TRP, or by inhibiting the TRP, or by interfering with membrane calcium channel formation by the TRP.
  • the method is useful in an inflammatory condition.
  • inflammatory conditions include regional inflammatory disorders, such as asthma (late phase), pancreatitis, inflammatory bowel disease (IBD), peritonitis, rheumatoid arthritis, osteoarthritis , myocardial infarction, ocular inflammatory states, and stroke.
  • inflammatory conditions also include systemic inflammatory disorders, such as systemic inflammatory response syndrome (SIRS) , cardiogenic shock, adult respiratory distress syndrome (ARDS) , multiple- organ dysfunction (MOD) , septic shock, and infant respiratory distress syndrome (IRDS) .
  • SIRS systemic inflammatory response syndrome
  • ARDS adult respiratory distress syndrome
  • MOD multiple- organ dysfunction
  • IRDS infant respiratory distress syndrome
  • the invention employs oligonucleotides targeted to nucleic acids encoding functional transient receptor potential gene product (TRP) .
  • TRP functional transient receptor potential gene product
  • the relationship between an oligonucleotide and its complementary nucleic acid target to which it hybridizes is commonly referred to as "antisense” .
  • "Targeting" an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated.
  • this may be, for example, the cellular gene (or mRNA made from the gene) for trp; i.e., the target is a nucleic acid encoding TRP, the trp gene, or mRNA expressed from the trp gene.
  • the targeting process also includes determination of a site or sites within the nucleic acid sequence for the oligonucleotide interaction to occur such that the desired effect, modulation of gene expression, will result. Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.
  • modulation means either inhibition or stimulation. Inhibition of trp gene expression is presently the preferred form of modulation. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression or Western blot assay of protein expression. Effects on inflammatory gaps between cells can also be measured, as taught in the examples of the instant application.
  • “Hybridization”, in the context of this invention, means hydrogen bonding, also known as Watson- Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them.
  • Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them.
  • “Specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.
  • An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid nonspecific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.
  • oligonucleotides are provided which are targeted to mRNA encoding TRP.
  • mRNA includes not only the coding region which carries the information to encode a gene product using the three letter genetic code, including the translation start and stop codons , but also associated ribonucleotides which form a region known to such persons as the 5 ' - untranslated region, the 3 ' -untranslated region, the 5' cap region, intron regions and intron/exon or splice junction ribonucleotides.
  • oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the coding ribonucleotides.
  • the functions of mRNA to be interfered with include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing or maturation of the RNA and possibly even independent catalytic activity which may be engaged in by the RNA.
  • the overall effect of such interference with the RNA function is to cause interference with trp gene expression.
  • oligonucleotide refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages.
  • oligonucleotide also includes oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.
  • the compounds and/or inhibitors used in the methods of the subject invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound/inhibitor which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds and/or inhibitors used in the subject invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents .
  • the compounds and/or inhibitors for use in the invention may additionally or alternatively be prepared to be delivered in a prodrug form.
  • prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds and/or inhibitors used in the subject invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • oligonucleotides used in the method of the subject invention preferably are from about 8 to about 50 nucleotides in length. In the context of this invention it is understood that this encompasses non-naturally occurring oligomers, preferably having 8 to 50 monomers.
  • oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems . Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the skill of the art. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives.
  • CPG controlled-pore glass
  • to "expose" cells (including the cells of tissues) to a compound and/or inhibitor means to add the compound and/or inhibitor, usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the compounds and/or inhibitor to cells or tissues within an animal (including a human) subject.
  • compositions for therapeutics, methods of decreasing inflammatory gaps in pulmonary endothelial cells and methods of preventing and treating inflammatory conditions are provided.
  • the formulation of therapeutic compositions and their subsequent administration is believed to be within the skill in the art.
  • a patient suspected of needing such therapy is given a compound and/or inhibitor in accordance with the invention, commonly in a pharmaceutically acceptable carrier, in amounts and for periods which will vary depending upon the nature of the particular disease, its severity and the patient's overall condition.
  • the pharmaceutical compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal) , oral or parenteral .
  • Parenteral administration includes intravenous drip or infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation (especially relevant for treatment of asthma) , or intrathecal or intraventricular administration.
  • Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful .
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non- aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
  • cationic lipids may be included in the formulation to facilitate oligonucleotide uptake.
  • One such composition shown to facilitate uptake is Lipofectin (BRL, Bethesda MD) .
  • Dosing is dependent on severity and responsiveness of the condition to be treated, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds and/or inhibitors, and can generally be calculated based on IC 50 's or EC 50 ' s in in vitro and in vivo animal studies.
  • a dose in mg/kg is routinely calculated.
  • the nucleic acid and amino acid sequences of various transient receptor potential genes are known and readily available from GenBank and described in the literature. For example, see GenBank Accession No. NP 003295 which discloses the 759 amino acid sequence of the human transient receptor potential channel 1, GenBank Accession No. AAC16725 which discloses the 123 amino acid sequence of the rat transient receptor potential protein 1, GenBank Accession No.
  • AAD22978 which discloses the 778 amino acid sequence of the African clawed frog cation channel TRP-1, GenBank Accession Nos. NM 003304 and X89066 which each disclose the 4085 bp sequence of the mRNA for human TRPC1 protein, GenBank Accession No. AF061873 which discloses the 369 bp partial sequence of the mRNA for rat trpl, GenBank Accession No. U40980 which discloses the 372 bp partial sequence of the mRNA for house mouse trp-related protein 1, and GenBank Accession No. X90696 which discloses the 304 bp sequence of the mRNA for the African clawed frog trp-like protein.
  • “functional” expression refers to the synthesis and any necessary post-translational processing of a calcium channel molecule in a cell so that the channel is inserted properly in the cell membrane and is capable of conducting calcium ions in accordance with a store- activated channel.
  • a method for screening a chemical agent (compound or inhibitor) for the ability of the chemical agent to modify calcium channel function begins by introducing the nucleic acid molecule encoding the TRP into a host cell, and expressing the TRP encoded by the molecule in the host cell. The expression results in the functional expression of a TRP calcium channel in the membrane of the host cell. The cell is then exposed to a chemical agent and evaluated to determine if the chemical agent modifies the function of the TRP calcium channel. From this evaluation, chemical agents effective in altering the function of the sodium channel can be found and utilized in the methods of the subject invention.
  • Drugs such as peptide drugs, which inhibit the TRP or which interfere with function TRP calcium channel formation can be made using various methods known in the art. Initially, a monoclonal antibody can be prepared which specifically hybridizes to the TRP, thereby interfering with activity and/or channel formation.
  • the monoclonal antibodies can be produced by hybridomas.
  • a hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
  • the TRP which is used as an immunogen may be modified or administered in an adjuvant in order to increase the TRP ' s antigenicity.
  • Methods of increasing the antigenicity of a protein include, but are not limited to, coupling the antigen with a heterologous protein (such as a globulin or beta- galactosidase) or through the inclusion of an adjuvant during immunization.
  • spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Ag 15 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
  • myeloma cells such as SP2/0-Ag 15 myeloma cells
  • any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz, et al . , Exp Cell Res 175:109-124 (1988)).
  • Hybridomas secreting the desired antibodies are cloned and the class and subclass are determined using procedures known in the art (Campbell, A.M., “Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology", Elsevier Science Publishers, Amsterdam, The Netherlands (1984) ) .
  • antibody containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.
  • the monoclonal (which is itself a compound or inhibitor which can be used in the subject invention) can be used to identify peptides capable of mimicking the inhibitory activity of the monoclonal antibody.
  • One such method utilizes the development of epitope libraries and biopanning of bacteriophage libraries. Briefly, attempts to define the binding sites for various monoclonal antibodies have led to the development of epitope libraries. Parmley and Smith developed a bacteriophage expression vector that could display foreign epitopes on its surface (Parmley, S.F. & Smith, G.P., Gene 73:305-318 (1988)).
  • This vector could be used to construct large collections of bacteriophage which could include virtually all possible sequences of a short (e.g. six-amino-acid) peptide. They also developed biopanning, which is a method for affinity-purifying phage displaying foreign epitopes using a specific antibody (see Parmley, S.F. & Smith, G.P., Gene 73:305-318 (1988); Cwirla, S.E., et al . , Proc Natl Acad Sci USA 87:6378-6382 (1990); Scott, J.K.
  • peptide sequences which mimicked the epitope i.e., sequences which did not identify a continuous linear native sequence or necessarily occur at all within a natural protein sequence. These mimicking peptides are called mimotopes . In this manner, mimotopes of various binding sites/proteins have been found.
  • sequences of these mimotopes do not identify a continuous linear native sequence or necessarily occur in any way in a naturally-occurring molecule, i.e. a naturally occurring protein.
  • sequences of the mimotopes merely form a peptide which functionally mimics a binding site on a naturally- occurring protein.
  • mimotopes are short peptides.
  • the availability of short peptides which can be readily synthesized in large amounts and which can mimic naturally-occurring sequences (i.e. binding sites) offers great potential application.
  • mimotopes to a monoclonal antibody that recognizes TRP can be identified.
  • the sequences of these mimotopes represent short peptides which can then be used in various ways, for example as peptide drugs that bind to TRP and decrease the activity of TRP. Once the sequence of the mimotope is determined, the peptide drugs can be chemically synthesized.
  • the peptides for use in the subject invention can contain any naturally-occurring or non-naturally-occurring amino acids, including the D-form of the amino acids, amino acid derivatives and amino acid mimics, so long as the desired function and activity of the peptide is maintained.
  • the choice of including an (L) - or a (D) -amino acid in the peptide depends, in part, on the desired characteristics of the peptide.
  • the incorporation of one or more (D) -amino acids can confer increased stability on a peptide and can allow a peptide to remain active in the body for an extended period of time.
  • the incorporation of one or more (D) -amino acids can also increase or decrease the pharmacological activity of a peptide.
  • the peptide may also be cyclized, since cyclization may provide the peptide with superior properties over their linear counterparts.
  • Modifications to the peptide backbone and peptide bonds thereof are encompassed within the scope of amino acid mimic or mimetic. Such modifications can be made to the amino acid, derivative thereof, non-amino acid moiety or the peptide either before or after the amino acid, derivative thereof or non-amino acid moiety is incorporated into the peptide. What is critical is that such modifications mimic the peptide backbone and bonds which make up the same and have substantially the same spacial arrangement and distance as is typical for traditional peptide bonds and backbones. An example of one such modification is the reduction of the carbonyl (s) of the amide peptide backbone to an amine.
  • An amino acid mimic is, therefore, an organic molecule that retains the similar amino acid pharmacophore groups as are present in the corresponding amino acid and which exhibits substantially the same spatial arrangement between functional groups.
  • substitution of amino acids by non-naturally occurring amino acids and amino acid mimics as described above can enhance the overall activity or properties of an individual peptide thereof based on the modifications to the backbone or side chain functionalities.
  • these types of alterations can enhance the peptide 's stability to enzymatic breakdown and increase biological activity.
  • Modifications to the peptide backbone similarly can add stability and enhance activity.
  • RPAECs Male Sprague-Dawley rats (CD strain, 350-400 g; Charles River) were euthanized by an intraperitoneal injection of 50 mg of pentobarbital sodium (Nembutal, Abbott Laboratories, Chicago, IL) . After sternotomy, the heart and lungs were removed en bloc, and the pulmonary arterial segment between the heart and lung hili was dissected, split, and fixed onto a 35-mm plastic dish.
  • pentobarbital sodium Nembutal, Abbott Laboratories, Chicago, IL
  • Endothelial cells were obtained by gentle intimal scraping with a plastic cell lifter and were seeded into a 100-mm petri dish containing 10 ml of seeding medium (-1:1 DMEM-Ham's F-12 + 10% fetal bovine serum) (37) . Afer incubation for 1 wk (21% 0 2 -5% C0 2 -74% N 2 at 37°C) , smooth muscle cell contaminants were marked and then removed by pipette aspiration.
  • seeding medium -1:1 DMEM-Ham's F-12 + 10% fetal bovine serum
  • RPAECs were washed with 2 ml of a HEPES (Fisher Scientific, Atlanta, GA) -buffered physiological salt solution (PSS) containing (in g/1) 6.9 NaCl, 0.35 KC1, 0.16 KH 2 P0 4 , 0.141 MgS0 4 , 2.0 D-glucose, and 2.1 NaHC0 3 .
  • the loading solution (1 ml) consisted of PSS plus 3 ⁇ M fura 2 -AM, 3 ⁇ l of a 10% pluronic acid solution, and 2 t ⁇ or 100 nM CaCl 2 .
  • RPAECs were fura loaded for 20 min in a C0 2 incubator at 37°C.
  • Epifluorescence (signal averaged) was measured from three to four endothelial cells in a confluent monolayer, and the changes in [Ca 2+ ] x are expressed as the fluorescence ratio of the Ca + -bound (340-nm) to Ca 2+ -unbound (380-nm) excitation wavelengths emitted at 510 nm.
  • the extracellular and pipette solutions were composed of the following (in mM) : 1) extracellular: 110 tetraethylammonium aspartate, 10 calcium aspartate, 10 HEPES, and 0.5 3 , 4-diaminopyridine ; and 2) pipette: 130 N-mehyl-D-glucamine, 1.15 EGTA, 10 HEPES, 1 Ca(OH) 2 , and 2 Mg + -ATP. Both solutions were adjusted to 290-300 mosM with sucrose and pH 7.4 with methane sulfonic acid, and [Ca 2 *] ! was estimated as 100 nM (10) . The pipette resistance was 2-5 M ⁇ .
  • RPAECs were seeded onto 35-mm plastic culture dishes and grown to confluence. Growth medium was replaced with experimental buffer (same as that used for [Ca 2+ ] x measurements but without fura 2), and the cells were subjected to one of the following protocols: 1) vehicle control (5 min) in 2 mM or 100 nM extracellular Ca 2+ concentration ([Ca 2+ ] 0 ); 2) thapsigargin (1 ⁇ M, 5 min) in 2 mM or 100 nM ([Ca 2+ ] 0 ; or 3) thapsigargin in 100 nM
  • filamentous actin F-actin
  • F-actin filamentous actin
  • RPAECs were seeded onto glass coverslips, and F-actin was stained with Oregon Green-phalloidin (Molecular Probes) with a standard fixation and staining protocol.
  • Cells were analyzed by confocal microscopy (excitation at 496 nm and emmision at 520 nm) .
  • Micrographs were taken at multiple cellular depths (0.3- ⁇ m steps, 13-15 sections) and were used to deduce the microfilamentous cytoskeleton configurations of the cells. Identification of trp gene products in pulmonary endothelial cells .
  • RNA Stat-60 Tel- Test "B” , Friendswood, TX
  • First-strand synthesis was performed with reverse transcriptase and oligo(dT) primer (GIECO BRL) on 1 ⁇ g of DNase I -treated total RNA.
  • Trpl 5'- TCG CCG AAC GAG GTG ATG G-3 ' (sense) and 5 ' -GTT ATG GTA ACA GCA TTT CTC C-3 1 (antisense)
  • Tr 3 5 ' -ACC TCT CAG GCC TAA GGG AG-3 ' (sense) and 5 ' -CCT TCT GAA GTC TTC TCC TGC-3' (antisense)
  • Trp6 5 ' -CT ACA TTG GCG CAA AAC AG-3' (sense) and 5 ' -CAC CAT ACA GAA CGT AGC CG-3' (antisense) .
  • PCR products were ligated into pCR2.1 vectors (TA Cloning Kit, Invitrogen, San Diego, CA) , transformed into competent cells, and screened by PCR for proper inserts. Bacterial cultures were grown for 16-18 h, and the plasmids were purified with Promega Wizard Minipreps (Madison, WI) . Sequencing was performed by an automated fluorescence sequencer (AB1370A) , and deduced amino acid alignments were carried out with the Blast software program.
  • first messengers act as so-called first messengers to induce endothelial cell contraction and decrease cell -cell and cell-matrix tethering, resulting in gap formation between cells that forms a paracellular pathway for transfer of the proteinaceous fluid.
  • the underlying mechanisms linking the host of first messengers to altered cell shape are unknown.
  • Cytosolic Ca 2+ ([Ca 2+ ]i) and adenosine 3 ',5' cyclic monophosphate (cAMP) are two intracellular signals importantly dictating endothelial cell-cell apposition, and thus permissiveness of the endothelial barrier for fluid transudation.
  • Increased cytosolic Ca 2+ ([Ca 2+ ]i) engages the endothelial contractile apparatus to pull cells inwardly.
  • increased [Ca 2+ ] i uncouples cell-cell and cell-matrix tethering so that inward contractions produce focal gaps between cells in the vessel wall .
  • Trp transient receptor potential
  • cAMP elevating agents are commonly used in clinical medicine for the treatment of inflammation.
  • Adenylyl cyclase (the enzyme responsible for synthesis of cAMP) activators and phosphodiesterase (the enzyme responsible for the breakdown of cAMP) inhibitors have both been utilized to increase cellular cAMP content for the treatment of urticaria and asthma among other conditions.
  • phosphodiesterase the enzyme responsible for the breakdown of cAMP
  • cAMP levels decrease in endothelial cells, which permissively increases permeability.
  • endothelial cells express a form of adenylyl cyclase that is inhibited by Ca 2+ entry.
  • cAMP content is reduced.
  • Trp3 and Trp6 are not SOCs (6, 46), Trpl may form SOCs based on the following experimental evidence: 1) Trpl and its splice variant TRPC1A increase store-operated Ca 2+ entry when expressed in COS cells (45, 47) and 2) expression of antisense trp sequences in murine L(tk " ) cells greatly attenuates store-operated Ca 2+ entry evoked by Ins(l,4,5)P 3 (45). Information concerning putative functions for Trp2 , -4, and -5 are lacking in the literature.
  • RPAECs were challenged with thapsigargin, a plant alkaloid that activates store-operated Ca 2+ entry independent of ligand- receptor-G protein-coupled processes (40, 43), and the changes in endothelial cell shape and microfilamentous cytoskeletal arrangement were monitored. It was then determined whether RPAECs express Trpl in order to address the possible molecular basis for the pulmonary endothelial store-operated Ca 2+ entry pathway. Ther data indicate that store-operated Ca + entry promotes cell shape change in rat pulmonary endothelial cells expressing Trpl and further suggest that Ca 2+ entry through SOCs involves site-specific rearrangement of the microfilamentous cytoskeleton.
  • Thapsigargin activates store -operated Ca 2+ entry in RPAECs .
  • Fura 2 epifluorescence was monitored, and as shown in Fig. 1A and summarized in Fig. 1C (open bars), RPAECs incubated in 2 mM [Ca 2+ ] 0 had baseline fluorescence ratios averaging 0.91 + 0.02.
  • Thapsigargin produced a gradual increase in [Ca 2+ ] ⁇ to a peak level followed by a modest decline, producing a new steady state, or plateau, in [Ca 2"1" ].,.
  • Figure 1, B (dashed line) and C solid bars), illustrates that the thapsigargin-induced response was dependent on [Ca 2+ ] 0 .
  • Thapsigargin right shifted the I-V curve and increased the current magnitude (slope conductance 1.64 nS, calculated from -100 to -20 mV without respect to cell capacitance) .
  • Store -operated Ca 2+ entry evokes endothelial cell shape change and F-actin cytoskeletal rearrangement in RPAECs .
  • changes in endothelial cell shape and formation of intercellular gaps in thapsigargin- treated confluent RPAEC monolayers were assessed.
  • FIG. 3 shows scanning electron micrographs of RPAECs after different treatments. Untreated RPAECs (Fig. 3A) in 2 mM [Ca 2+ ] 0 exhibited a characteristic "cobblestone" morphology essentially devoid of intercellular gaps. Thapsigargin produced endothelial cell retraction and intercellular gap formation (Fig. 3B) .
  • FIG. 4A shows F-actin localization in untreated RPAECs incubated with 2 mM [Ca 2+ ] 0 . Under these conditions, cells contained dense peripheral actin bands with apparent focal contact sites between cells. Some transcellular, centrally located filaments were also seen.
  • Figure 4B shows that incubation of RPAECs in low [Ca 2+ ] 0 alone had an effect on F-actin configuration.
  • Trpl Three specific mammalian trp gene products, Trpl, Trp3 , and Trp6, were screened for because all are associated with Ca 2+ influx into nonexcitable cell lines, although only Trpl appears to possess the functional capacity to mediate store-operated Ca 2+ entry.
  • the Trp3 or Trp6 products from confluent RPAECs were not amplified.
  • RT- PCR was performed with HPAECs but likewise detected neither Trp3 nor Trp6 expression. However, both products could be amplified in rat brain, indicating that the primers were capable of amplifying these trp gene products (data not shown) .
  • Trpl In contrast, RT-PCR products for Trpl were identified in both RPAECs and HPAECs.
  • the RPAEC and HPAEC products were 96 and 100% homologous, respectively, to the reported nucleotide sequence for human Trpl (Table A) .
  • the deduced amino acid alignments revealed 100% amino acid homology between both endothelial products and human Trpl over the region studied (Table B) .
  • Trpl is present and may contribute to RPAEC SOC formation, whereas Trp3 and Trp6 likely are not expressed in the pulmonary endothelium.
  • Thapsigargin was utilized to test store-operated Ca 2+ entry pathways because this agent produces intracellular Ca 2+ store depletion without the confounding influences of ligand-receptor-heterotrimeric G protein activation (40, 43, 47) .
  • Fura 2-loaded RPAEC monolayers exhibited an increased [Ca 2+ ] 1 that was dependent on Ca 2+ influx in response to thapsigargin, thereby indicating the presence of store-operated Ca 2+ entry pathways.
  • whole cell patch clamp in single cells was performed.
  • Intracellular and extracellular patch solutions were performed to isolate thapsigargin- induced Ca 2+ currents and determine whether thapsigargin activated a channel (s) responsible for Ca 2" release-activated current (I CRAC ) (16) .
  • I CRAC Ca 2" release-activated current
  • thapsigargin may activate an anion channel capable of conducting large organic anions as previously reported in bovine pulmonary endothelium (27, 29) .
  • anion conductance contribution to the total thapsigargin-stimulated current is then considered, a current analogous to I CRAC is apparent .
  • Activation of SOCs in RPAECs causes the appearance of intercellular gaps and rounding of endothelial cells.
  • One intracellular target affected by SOC activation is plasmalemmal-associated and centrally located F-actin. It is accepted that changes in [Ca 2+ ] ⁇ lead to reconfigurations of the microfilamentous cytoskeleton (21, 22, 30), although it has previously been unclear whether Ca 2 ⁇ release from intracellular stores or Ca 2+ influx is necessary to produce cytoskeletal changes leading to cell shape change. Thapsigargin produced a loss of plasmalemmal F-actin staining concurrent with an increase in central F-actin staining.
  • the findings indicate that at least Trpl, but neither Trp3 nor Trp6, is expressed in pulmonary endothelial cells. It is uncertain how trp gene products may be organized in the membrane to form a functional channel, but it has been proposed that SOCs may be composed of trp homo- and/or heteromultimers (5) . Because the data indicate that neither Trp3 nor Trp6 are present in rat or human pulmonary endothelial cells, the SOC is not composed of Trpl-Trp3 or Trpl-Trp6 heteromultimers.
  • the shape change elicited in response to SOC activation in RPAECs has additional importance for other endothelial -directed physiological processes such as angiogenesis and regulation of leukocyte movement .
  • the angiogenic process requires migration of endothelial cells that, in turn, is dependent on the ability of cells to change shape and decrease their cell-to-cell and cell- to-matrix tethering (3) .
  • Inhibition of non-voltage-gated Ca + channels presumably including SOCs, inhibits angiogenic factor-induced proliferation, migration, and tube formation of human umbilical venous endothelial cells (20), which are endothelial cells derived from conduit vessels.
  • a study (17) has shown that human umbilical venous endothelial cell -directed regulation of leukocyte trafficking is [Ca 2+ ] x dependent.
  • RPAECs possess thapsigargin-activated SOCs that conduct current similar to I CRAC .
  • RPAECs respond to this mode of Ca 2+ entry with changes in cell shape, interendothelial gap formation, and rearrangement of the F-actin cytoskeleton. Cytoskeletal rearrangement may be differentially regulated by the extracellular and intracellular Ca 2+ pools, with Ca 2+ influx being necessary to produce a cytoskeleton configured for cell shape change.
  • pulmonary endothelial cells from rats (and humans) express Trpl, which may be integral for forming native SOCs in these cell types.
  • pulmonary conduit vessel -derived endothelial SOC activation leading to interendothelial gap formation may be the basis for some forms of pulmonary edema and/or a component of angiogenesis and regulation of leukocyte trafficking to and from the vasculature.
  • Table A sequence comparison (nucleotides 1-195) between human Trpl (hTrpl) and RT-PCR products from human PAECs (HPAECs) and RPAECs. *Differences between RPAEC and hTrpl sequences .
  • 1 hTrpl TCG CCG AAC GAG GTG ATG GCG CTG AAG GAT HPAEC TCG CCG AAC GAG GTG ATG GCG CTG AAG GAT
  • RPAEC TCG CCG AAC GAG GTG ATG GCG CTG AAG GAT
  • RPAEC GCT GTT ACC ATA ACA Table B deduced amino acid sequence (amino acids 27-91] of RPAEC and HPAEC RT-PCR products with sequence alignment .

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Abstract

L'invention concerne un procédé pour réduire des brèches inflammatoires dans des cellules endothéliales pulmonaires. Le procédé consiste à réduire dans les cellules les niveaux d'un produit génique fonctionnel à potentiel de récepteur transitoire. L'invention concerne en outre un procédé de traitement ou de prévention d'une maladie inflammatoire chez un sujet. Le procédé consiste à administrer au sujet une quantité effective d'un composé pouvant réduire dans les cellules du sujet les niveaux du produit génique fonctionnel à potentiel de récepteur transitoire.
PCT/US1999/016822 1998-07-24 1999-07-23 Utilisation de niveaux reduits d'un produit genique fonctionnel a potentiel de recepteur transitoire WO2000004929A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002087608A1 (fr) * 2001-04-27 2002-11-07 Boehringer Ingelheim Pharma Gmbh & Co. Kg Nouveaux analgesiques qui sont des inhibiteurs de canaux trp
WO2003087158A2 (fr) * 2002-04-16 2003-10-23 Bayer Healthcare Ag Regulation de canal potentiel recepteur transitoire humain
WO2004013629A2 (fr) * 2002-07-29 2004-02-12 Novartis Ag Composes organiques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998008979A1 (fr) * 1996-08-30 1998-03-05 The Regents Of The University Of California Procede et composes de regulation de l'entree capacitive d'ions calcium dans des cellules de mammifere
US5932417A (en) * 1996-10-15 1999-08-03 The Regents Of The University Of California Method of screening compounds for controlling capacitative calcium ion entry into mammalian cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998008979A1 (fr) * 1996-08-30 1998-03-05 The Regents Of The University Of California Procede et composes de regulation de l'entree capacitive d'ions calcium dans des cellules de mammifere
US5932417A (en) * 1996-10-15 1999-08-03 The Regents Of The University Of California Method of screening compounds for controlling capacitative calcium ion entry into mammalian cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TOMITA ET AL: "Intracellular Ca2+ Store-Operated Influx of Ca2+ Through TRP-R, a Rat Homolog of TRP, Expressed in Xenopus Oocytes", NEUROSCIENCE LETTERS, vol. 248, no. 3, 5 June 1998 (1998-06-05), pages 195 - 198, XP002919942 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002087608A1 (fr) * 2001-04-27 2002-11-07 Boehringer Ingelheim Pharma Gmbh & Co. Kg Nouveaux analgesiques qui sont des inhibiteurs de canaux trp
WO2003087158A2 (fr) * 2002-04-16 2003-10-23 Bayer Healthcare Ag Regulation de canal potentiel recepteur transitoire humain
WO2003087158A3 (fr) * 2002-04-16 2004-06-10 Bayer Healthcare Ag Regulation de canal potentiel recepteur transitoire humain
WO2004013629A2 (fr) * 2002-07-29 2004-02-12 Novartis Ag Composes organiques
WO2004013629A3 (fr) * 2002-07-29 2004-05-06 Novartis Ag Composes organiques

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