WO2008109794A2 - Rôle de la voie du mk2 dans le traitement des fibroses et de la formation de cicatrices - Google Patents

Rôle de la voie du mk2 dans le traitement des fibroses et de la formation de cicatrices Download PDF

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WO2008109794A2
WO2008109794A2 PCT/US2008/056142 US2008056142W WO2008109794A2 WO 2008109794 A2 WO2008109794 A2 WO 2008109794A2 US 2008056142 W US2008056142 W US 2008056142W WO 2008109794 A2 WO2008109794 A2 WO 2008109794A2
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fibrosis
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mef
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Usamah Kayyali
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Tufts Medical Center Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • Pulmonary fibrosis as a major component of interstitial lung disease is characterized by abnormal fibroblast proliferation and deposition of extracellular matrix proteins that remodel the normal pulmonary tissue structure and compromise its function.
  • interstitial lung disease e.g., idiopathic pulmonary fibrosis (IPF) the mechanism by which fibrosis arises remains poorly understood (1, 2).
  • IPF idiopathic pulmonary fibrosis
  • Pulmonary hypertension is a disease characterized by narrowing and increased resistance of the pulmonary arteries whose etiology is not well understood. While the disease can be primary or secondary to other conditions, a common feature in different types of pulmonary hypertension is vascular remodeling (for review, see Humbert et al., 2004).
  • fibroblasts have been reported to constitute the first type of cells to respond to pulmonary hypertension producing stimuli, e.g., chronic hypoxia in animal models (Stenmark et al., 2002) (Humbert et al., 2004).
  • Fibrosis arises in several tissues in response to inflammation and chemical injury, or idiopathically, leading to accumulation of fibroblasts which deposit extracellular matrix (ECM) proteins. Such processes are part of normal wound healing, and are often reversible by apoptosis of accumulating fibroblasts and resorption of ECM proteins.
  • ECM extracellular matrix
  • the proliferation of fibroblasts and ECM deposits appear excessive and dysregulated, resulting in the gradual displacement of normal tissue by fibrotic foci that disrupt tissue function. Differentiation of fibroblasts into myofibroblasts has long been believed to be an important event in many conditions such as wound repair and fibrosis.
  • myofibroblasts occur in areas of active fibrosis and are responsible for production and deposition of extracellular matrix proteins in pulmonary fibrosis (3).
  • myofibroblast which occurs in areas of active fibrosis and is responsible for production and deposition of extracellular matrix proteins (Vyalov et al., 1993).
  • Myofibroblasts which are believed to play a role in wound contraction, are more contractile than fibroblasts due to increased expression of the smooth muscle-specific actin ⁇ -SMA (for review, see 14).
  • Studies into the mechanism by which myofibroblast differentiation occurs focused on signaling pathways activated by TGF ⁇ in fibroblasts. In the lungs of IPF patients, TGF ⁇ (5), IL- l ⁇ and TNF ⁇ (6, 7) were reported to be elevated. Indeed TGF ⁇ is believed to play a pivotal role in fibrosis and its overepxression in mice is sufficient to cause pulmonary fibrosis independent of injury and inflammation (8, 9).
  • TGF ⁇ is a cytokine that can exert many actions and has been implicated in diseases ranging from cancer to pulmonary hypertension.
  • One action of TGF ⁇ that has been of interest in fibrosis research is its causing fibroblasts to differentiate into myofibroblasts which are known to accumulate in sites of fibrosis (3, 10-13).
  • MAP kinase p38 is a stress-activated kinase that is activated in response to stimuli such as ultraviolet radiation and hyperosmolarity. Once p38 is activated, it phosphorylates a variety of substrates, including the kinase MK2. Upon its phosphorylation by p38, MK2 becomes activated, and in turn, phosphorylates additional substrates, such as the small heat shock protein HSP27. When phosphorylated, HSP27 no longer blocks the polymerization of actin, thus resulting in the stabilization of actin fibers (Benndorf et al., 1994).
  • MK2 smooth muscle ⁇ -actin
  • the invention provides a method for therapeutically treating a vertebrate having fibrosis or scarring as a result of injury or some other condition.
  • the method in this aspect of the invention comprises increasing the expression and/or activity of MK2 in f ⁇ brotic tissue of a mammal.
  • the mammal is a human.
  • the fibrotic tissue is lung tissue.
  • the invention provides a method for preventing fibrosis or scarring after injury in a vertebrate.
  • the method in this aspect of the invention comprises increasing the expression and/or activity of MK2 in injured tissue of a mammal.
  • the mammal is a human.
  • the injured tissue is lung tissue.
  • FIG. 1 MK2 ⁇ ⁇ lungs produce more collagen in response to bleomycin treatment than wild type lungs. Bleomycin caused an increase in lung collagen deposition in both wild type and MK2 " " lungs. However, the collagen deposition was significantly higher in
  • MK2 " bleomycin-treated lungs than in wild type bleomycin-treated lungs. * indicates statistically significant difference from saline-treated wild type mean, # indicates statistically significant difference from saline -treated MK2 ⁇ ⁇ mean, ⁇ indicates statistically significant difference from bleomycin-treated wild type mean. P ⁇ 0.05 in ANOVA and Holm-Sidak post-hoc analysis.
  • FIG. 2 MK2 ⁇ ⁇ mice develop more severe fibrosis in response to bleomycin than wild type mice.
  • Treatment of wild type mice with bleomycin (B) cause significant cellularity and collagen deposition (blue staining) relative to saline -treated wild type mice (A).
  • MK2 ⁇ ⁇ mice exhibited more severe cellularity and collagen deposition in response to bleomycin (D), compared to saline-treated MK2 " ⁇ mice (C) and bleomycin-treated wild type mice (B).
  • D saline-treated MK2 " ⁇ mice
  • C saline-treated wild type mice
  • Several slides were blindly ranked for fibrosis by 3 investigators and scored as described in Examples.
  • FIG. 3 Knocking down MK2 also reduces sm ⁇ expression.
  • Transfection with siRNA against MK2 reduced the level of MK2 and ⁇ -SMA relative to mock siRNA transfection in a dose-dependent manner.
  • MK2 siRNA transfection had no effect on total actin expression.
  • FIG. 4 Fibrotic lesions in MK ⁇ ' mice contain few ⁇ -SMA -positive cells compared to wild type fibrotic lesions. Lung sections were stained with an anti-sm ⁇ antibody. Both wild type and MK2 ⁇ ⁇ sections expressed ⁇ -SMA in smooth muscle cells (thick arrows). However, ⁇ -SMA -positive fibroblasts (thin arrows) were much more prominent in fibrotic regions of bleomycin-treated wild type lung sections.
  • A Saline-treated wild type
  • B bleomycin-treated wild type
  • C saline-treated MK2 "7"
  • D bleomycin-treated MK2 ⁇ ⁇ .
  • FIG. 5 Fibrotic lesions in MK ⁇ ' mice contain many vimentin-positive cells compared to wild type fibrotic lesions: Lung sections were stained with an anti-vimentin antibody. Both wild type and MK2 ⁇ ⁇ sections expressed vimentin in fibrotic lesions. However, vimentin-positive fibroblasts were much more prominent in fibrotic regions of bleomycin-treated MK2 ⁇ ⁇ lung sections, reflecting a higher degree of fibrosis.
  • A Saline- treated wild type
  • B bleomycin-treated wild type
  • C saline-treated MK2 "7"
  • D bleomycin-treated MK2 ⁇ ⁇ .
  • FIG. 8 MK2 ⁇ ⁇ MEF are slower than wild type MEF in repopulating a wounded part of the culture dish. Cells were scraped off sub-confluent MEF culture dishes of equal density using a sterile pipette tip. After 24 hours more wild type MEF than MK2 ⁇ ⁇ MEF were observed in the wounded area. Over expressing phosphomimicking pmHSP27 in MK2 ⁇ ⁇ cells did not reverse the defective migratory properties of MK2 " " MEF.
  • FIG. 9 MK2 is not upstream of Smad signaling in response to TGF ⁇ : A: TGF ⁇ causes phosphorylation of both Smad2/3 and p38 in wild type and MK2 ⁇ ⁇ MEF. Smad2/3 phosphorylation is observed before p38 phosphorylation. B: Inhibiting the TGF ⁇ Type I receptor kinase by SB431542 blocked Smad2/3 phosphorylation in both wild type and MIQ 7 MEF. SB431542 also blocked the activation of p38 by TGF ⁇ .
  • FIG. 10 Consequences of injury in the presence and absence of MK2 are shown. In the presence of MK2 myofibroblast differentiation, resolution or scar formation occurs. In the absence of MK2, fibroblast proliferation and fibrosis or severe scarring occurs.
  • Figure 11 Immunological staining of histological lung sections from GFP-nirus- infected or MK2 -virus infected MK2 ⁇ ⁇ mice are shown.
  • the invention relates to therapeutic approaches to managing conditions related to MK2 activity.
  • the patents and publications cited herein reflect the level of knowledge in the art and are hereby incorporated by reference in their entirety. Any conflict between the teachings of these patents and publications and this specification shall be resolved in favor of the latter.
  • fibroblasts in fibrosis have increasingly focused on a differentiated form of fibroblasts, the myofibroblast. Because of their expression of smooth muscle-specific proteins, such as ⁇ -SMA, their role in wound contraction and cytokine production, as well as their localization near f ⁇ brotic foci, myofibroblasts were suspected of promoting fibrosis. Surprisingly, the present inventors have discovered that mitogen-activated protein kinase-activated protein kinase 2 (MK2) and myofibroblast formation appear to play a role in protection against, rather than in causing fibrosis.
  • MK2 mitogen-activated protein kinase-activated protein kinase 2
  • the invention provides a method for therapeutically treating a mammal having fibrosis or scarring as a result of injury or some other condition.
  • the method in this aspect of the invention comprises increasing the expression and/or activity of MK2 in cells in fibrotic tissue in a mammal.
  • the mammal is a human.
  • the fibrotic tissue is lung tissue.
  • fibrosis and fibrotic refer to the formation or development of excess fibrous connective tissue in an organ or tissue. Fibrosis often occurs in multiple organs including, but is not limited to, the lungs, heart, liver, kiney and skin. Fibrosis occurs in different organs in response to different types of injury, or it can arise without a clear stimulus as in the case of i.e., idiopathic pulmonary fibrosis (IPF). Such stimuli include, but are not limited to, chemotherapeutic agents, sarcoidosis, asbestos, silica dust, and connective tissues diseases such as scleroderma. As used herein, the term “scarring” means the formation or development of scar or fibrous tissue replacing normal tissues destroyed by injury or disease.
  • MK2 expression and/or acttivity can be increased in fibrotic tissue by various means.
  • MK2 expression is increased in cells of fibrotic tisues by transfecting the cells with a virus or other expression vector comprising an MK2 transgene.
  • MK2 activity in cells of fibrotic tissue is increased by inhibiting the activity of an inhibitor of MK2 activity.
  • the therapeutic composition useful for treating fibrosis or scarring includes, but is not limited to, small molecules that inhibit the activity of the autoinhibitory peptide of MK2 preventing it from binding MK2 and thus resulting in MK2 activation.
  • small molecules include the peptide HPRNPARRTPGTRRGAPQEPGAA from human herpes virus 8 and fragments, derivatives, analogs or mimetics thereof. Mutant variants of the autoinhibitory peptide itself may also be used for this purpose.
  • MK2 expression and/or activity in cells of fibrotic tissue is increased by contacting the cells with small molecule that increases the expression and/or activity of MK2, or that decreases the expression or activity of an inhibitor of MK2 expression or activity.
  • administration of therapeutic compositions comprising a virus, expression vector or small molecule can be by any suitable route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, inhalation, intranasal, aerosol, intraocular, intratracheal, intrarectal or vaginal.
  • Administration of the therapeutic compositions of the invention can be carried out using known procedures at dosages and for periods of time effective to treat fibrosis or scarring.
  • the term an "effective amount” or a "sufficient amount” generally refers to an amount sufficient to affect a desired biological effect, such as beneficial results. Thus, an "effective amount” or “sufficient amount” will depend upon the context in which it is being administered. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode.
  • the therapeutic composition can be administered in combination with any other agent useful for treating fibrosis or scarring that does not diminish the effect of the therapeutic composition.
  • the methods could include agents that activate p38 MAP kinase, the upstream activator of MK2, such as hyperosmolar solutions, low oxygen, and sodium arsenite which are expected to cause activation of MK2, as well as agents that inhibit p38, such as SB203580, which are expected to inhibit MK2.
  • combination with generally means in the course of treating the same condition in the same patient, and includes administering a compound and/or an agent in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart.
  • Such combination treatment may also include more than a single administration of the compound according to the invention, and/or independently an agent.
  • the administration of the compound and/or agent may be by the same or different routes.
  • the invention provides a method for preventing fibrosis or scarring in a mammal.
  • the method in this aspect of the invention comprises increasing the expression and/or activity of MK2 in cells of tissue in a mammal that have been injured, but which is not yet f ⁇ brotic, or only partially f ⁇ brotic.
  • the mammal is a human.
  • the injured tissue is lung tissue.
  • the expression and/or activity of MK2 is achieved by contacting the cells of the injured tissue with a therapeutic composition according to the invention, as described in the first aspect above. Administration of the therapeutic composition, alone or in combination with another agent, is carried out as described for the first aspect of the invention.
  • composition refers to the combination of a virus, expression vector, or small molecule that increases the expression and/or activity of MK2 and a pharmaceutically acceptable carrier.
  • the term “pharmaceutically acceptable” refers to a material that does not interfere with the effectiveness of the compositions of the first, second or third aspects of the invention and is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • the biological system is a living organism, such as a vertebrate.
  • the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application.
  • D-MEM Media and supplements
  • FBS fetal bovine serum
  • penicillin G potassium penicillin G potassium
  • streptomycin penicillin G potassium
  • streptomycin penicillin G potassium
  • streptomycin penicillin G potassium
  • streptomycin penicillin G potassium
  • streptomycin penicillin G potassium
  • streptomycin penicillin G potassium
  • streptomycin penicillin G potassium
  • streptomycin penicillin G potassium
  • streptomycin streptomycin
  • fungizone penicillin G potassium
  • glutamine purchased from Invitrogen (Carlsbad, CA).
  • TGF ⁇ was purchased from R&D Systems (Minneapolis, MN) and activated before use according to manufacturers instructions.
  • SB431542 and all other reagents and drugs were obtained from Sigma (St. Louis, MO). On the day of the experiments, all drugs were diluted in serum-free medium.
  • mice that lack MK2 (MK2 ⁇ ⁇ ) mice were generated as described earlier (42).
  • a neo selection cassette which contains translational stop codons in all three reading frames, was inserted into the Sac I site located in the exon that encodes subdomains V and VI of MK2.
  • MK2 ⁇ ⁇ mice were then back-crossed into the C57BL/6J, and lack of expression of MK2 was verified by PCR and immunob lotting as described earlier (4). Wild type C57BL/6J from Charles River Laboratories (Wilmington, MA) were used as controls for the described experiments. These mice were anesthetized with xylozine-ketamine, and then saline or bleomycin (3 U/kg in 50 ⁇ L) were introduced into their lungs by insufflation.
  • MEF Immortalized mouse embryonic fibroblasts
  • MK2 "7" knockout mice were prepared as described earlier [Kotlyarov et al., 1999].
  • MEF cells were maintained in DMEM containing 10% FBS, penicillin, streptomycin, fungizone and glutamine at 37 0 C in humidified air containing 5 % CO 2 .
  • MEF were passaged in 0.25 % trypsin-0.02 % ethylenediaminetetraacetic acid (EDTA) solution, and one day prior to the experiments, cells were maintained in serum- free or 1% serum-containing media.
  • EDTA ethylenediaminetetraacetic acid
  • pmHSP27 phosphorylated HSP27
  • pMEpuro a selection vector which confers resistance to puromycin to eukaryotic cells
  • Stable transfected wild type MEF cell lines were obtained by selection with geneticin, and resistant colonies were isolated, expanded, and then screened for pmHSP27 by immunob lotting of cell lysates with ant anti HSP27 antibody.
  • MK2 ⁇ ⁇ MEF were already resistant to geneticin, stable transfectants produced by co-transfection with pMEpuro were selected with puromycin, and resistant clones were isolated and expanded as above.
  • proliferation assays cells were seeded in 12-well dishes and cultured for 24, 48 and 72h. Counting was performed after trypsinizing the cells using a Coulter counter apparatus according to manufacturer's instructions.
  • mice were evaluated for the amount of acid-soluble collagen in their lungs using the Sircol Assay (Biocolor Ltd, Newtownabbey, United Kingdom). In brief, lungs were minced into small cubes and incubated overnight in 10 volumes of 0.5 M acetic acid. Next homogenates were incubated with an aliquot of Sircol Dye reagent (Sirus Red in picric acid) for 30 min.
  • Sircol Dye reagent Sirus Red in picric acid
  • the collagen-dye complex was then pelleted by centrifugation at 10,000 x g for 10 minutes.
  • the dye was then extracted with 0.5 M NaOH, and the absorbance of samples along with known collagen standards was measured at 540 nm in a Tecan Spectrafluor plate reader.
  • the same assay was used to assay collagen in conditioned media from MEF cells (without the acid step), and in cell lysates (scraped into 0.5 M acetic acid).
  • lungs from the mice treated as described above were examined histochemically. After perfusion and fixation, lung sections were stained with Eosin/hematoxylin and Gomori's Trichrome stain which stains collagen. In brief, lungs were instilled intratracheally with 4% formaldehyde at 21 cm H 2 O pressure. The tissue blocks were then embedded in paraffin, and 4 ⁇ m sections were cut for staining. Gomori's Trichrome staining was used to highlight collagen deposition.
  • the bleomycin treated animals had significant cellularity in regions that should correspond to airspaces.
  • the Trichrome stain also revealed increased collagen deposition in interstitial spaces of bleomycin-treated mice compared to saline-treated mice ( Figure 2 A-D).
  • the bleomycin-treated MK2 " ⁇ mice developed more severe fibrosis compared to the wild type mice.
  • To quantify the fibrosis in these mice several Trichrome-stained sections from differently treated mice were ranked as follows: 0 for no fibrosis, 2 for significant fibrosis, and 1 for intermediate fibrosis. The slides were blindly scored by three different researches.
  • the table in Figure 2E shows the average and standard deviations of the pooled scores.
  • siRNA against MK2 were used to suppress the expression of MK2 in wild type MEF.
  • 80%-90% confluent cells were transfected with MK2 siRNA (Cat # 16704 Ambion, CA) and mock control siRNA (Cat # 4611 Ambion, CA) with Lipofectamine 2000 (Invitrogen, CA) at indicated concentrations for 30 h according to the manufacturer's instructions.
  • Western blotting was then performed to test the silencing effect of siRNA on the expression levels of target proteins.
  • MK2 siRNA transfection caused a significant reduction in the level of MK2 expressed in wild type MEF compared to mock transfected MEF.
  • ⁇ -SMA When these cells were probed for the expression of s ⁇ -SMA, it was observed that the MK2 siRNA transfection also inhibited the expression of ⁇ -SMA ( Figure 3).
  • ⁇ -SMA The expression of ⁇ -SMA in the lungs of MK2 " " and wild type mice was evaluated.
  • lung sections from mice treated with saline or bleomycin as described above were stained with an antibody against ⁇ -SMA.
  • cell lysates were assayed for protein using the Bradford protein assay [Bradford, 1976] and then diluted with 2x Laemmli loading buffer for SDS-PAGE [Laemmli, 1970]. Equal amounts of protein were then loaded in 4-20% Tris/glycine gels, and electrophoresed for 90 min at 125 V constant voltage. Next, the gel was blotted onto an Immobilon-P membrane by electrophoretic transfer at 25 V constant voltage overnight.
  • the membrane was then washed, blocked with 5% milk, and probed with antibodies against MK2, p38 and phospho-p38 (Cell Signaling, Beverly, MA); ⁇ -SMA (1A4, Sigma, St. Louis, MO); pan- actin (C-2, Santa Cruz, Santa Cruz, CA); smad2/3(18, BD-Transduction Laboratories, San Jose, CA); and phospho-smad2 (138D4, Cell Signaling, Beverly, MA).
  • Appropriate secondary antibodies conjugated to horseradish peroxidase (Pierce, Rockford, IL), and a chemiluminescent substrate according to the manufacturer's instructions (SuperSignal, Pierce, Rockford, IL) were used to visualize immunoreactive bands.
  • Embryonic fibroblasts from MK2 'A mice exhibit a higher proliferative rate and secrete more collagen than wild type fibroblasts:
  • MK A fibroblasts are defective in migratory activity-Reduced migratory behavior in MK ' fibroblasts is not due to lack of phosphorylated HSP27:
  • MK2 ⁇ ⁇ MEF have been described to migrate more slowly than their wild type counterparts (23).
  • wild type MEF repopulated the wounded area much faster than the MK2 ⁇ ⁇ cells.
  • MK2 is known to alter the actin cytoskeleton through its direct action on the small heat shock protein HSP27, stably transfected phosphomicmiking (pm) HSP27 MK2 ⁇ ⁇ MEF were generated.
  • Fibroblast proliferation and ⁇ -SMA expression have been linked to growth factors such as TGF ⁇ , which are known to exert many of its effects through Smad signaling.
  • TGF ⁇ growth factors
  • Smad2 phosphorylation was assayed.
  • Smad2 phosphorylation is activated by TGF ⁇ similarly in wild type or MK2 ⁇ ⁇ MEF, indicating that MK2 is not upstream of Smad in TGF ⁇ -activated signaling.
  • mice were intubated and the viral supernatant was instilled with a syringe through the tube, followed by brief ventilation to insure delivery of the virus to the lung.
  • Some mice received the control pMMP-IRES-GFP (GFP-virus) and others received the pMMP-IRES-GFP-MK2 (MK2 virus).
  • lungs were immunostained with either an antibody against MK2 or control IgG.
  • the Vector (Vector, Burlingame,CA) avidin biotin system was used for secondary antibody labeling, followed by the NovaRed horse radish peroxidase substrate. Slides were then counterstained with hematoxylin. As shown in Figure 11, lungs of mice infected with MK2-encoding virus exhibited stronger antiMK2 labeling than either IgG controls or GFP -virus-infected mouse lungs. The staining appeared strongest in epithelial cells which would be the first to encounter the virus, but was also visible in the parenchyma. These results demonstrate that these viral vectors can be used successfully to overexpress MK2 in mouse lungs.
  • mice are transfected with pMMP-IRES-GFP MK2 as described in Example 9. After 48 hours, transfected mice are challenged with bleomycin as described in Example 2, and histological lung samples are analyxed for fibrosis, also as described in Example 2. It is expected that mice transfected with pMMP-IRES-GFP MK2 will develop less fibrosis than mice transfected with pMMP-IRES-GFP or untransfected mice.

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Abstract

L'invention concerne des procédés permettant de traiter ou de prévenir une fibrose ou une cicatrisation dans un tissu de mammifère en augmentant l'expression et/ou l'activité de la MK2 chez les cellules d'un tissu fibrogène ou lésé du mammifère.
PCT/US2008/056142 2007-03-08 2008-03-07 Rôle de la voie du mk2 dans le traitement des fibroses et de la formation de cicatrices Ceased WO2008109794A2 (fr)

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Publication number Priority date Publication date Assignee Title
EP2349310A4 (fr) * 2008-10-20 2012-11-21 Moerae Matrix Inc Polypeptide pour traiter ou prévenir les adhésions
KR20150100615A (ko) * 2012-09-10 2015-09-02 모레 매트릭스 인코포레이티드 피부 흉터형성을 치료하기 위한 조성물 및 방법

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KAYALLI ET AL.: 'Cytoskeletal changes in hypoxic pulmonary endothelial cells are dependent on MAPK-activated protein kinase MK2' JOUMAL OF BIOLOGICAL CHEMISTRY vol. 277, no. 45, 2002, pages 42596 - 42602 *
MUCHANETA-KUBARA ET AL.: 'Myofibroblast phenotypes expression in experimental renal scarring' NEPHROL DIAL TRASPLANT vol. 12, 1997, pages 904 - 915 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2349310A4 (fr) * 2008-10-20 2012-11-21 Moerae Matrix Inc Polypeptide pour traiter ou prévenir les adhésions
KR20150100615A (ko) * 2012-09-10 2015-09-02 모레 매트릭스 인코포레이티드 피부 흉터형성을 치료하기 위한 조성물 및 방법
KR102040710B1 (ko) 2012-09-10 2019-11-05 모레 매트릭스 인코포레이티드 피부 흉터형성을 치료하기 위한 조성물 및 방법

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