WO2018196870A1 - 靶向前列腺素e2及其受体的药物与应用 - Google Patents

靶向前列腺素e2及其受体的药物与应用 Download PDF

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WO2018196870A1
WO2018196870A1 PCT/CN2018/085035 CN2018085035W WO2018196870A1 WO 2018196870 A1 WO2018196870 A1 WO 2018196870A1 CN 2018085035 W CN2018085035 W CN 2018085035W WO 2018196870 A1 WO2018196870 A1 WO 2018196870A1
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pge2
vascular
receptor
injury
substance
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PCT/CN2018/085035
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English (en)
French (fr)
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王淼
郝会峰
胡昇
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中国医学科学院阜外医院
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Priority to CN201880028018.9A priority Critical patent/CN110573162A/zh
Publication of WO2018196870A1 publication Critical patent/WO2018196870A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form

Definitions

  • the present invention relates to drugs and methods for modulating vascular remodeling.
  • the present invention relates to drugs, methods and articles for regulating vascular remodeling via the PGE2 signaling pathway and uses thereof.
  • Prostaglandins are combined by a epoxidase grade and inhibited by non-steroidal anti-inflammatory drugs.
  • Prostaglandin E2 (PGE2) is an important cell growth and regulation factor, a metabolite of arachidonic acid cyclooxygenase, a non-carbonic unsaturated fatty acid, and a prostaglandin (PG).
  • PGE2 Prostaglandin E2
  • PG prostaglandin
  • At least four transmembrane G protein-coupled receptors, EP1, EP2, EP3 and EP4 are known to mediate the biological function of PGE2.
  • Similar PGI2 mediates the biological function of PGI2 via receptor IP.
  • the present invention provides a method of modulating vascular remodeling in a subject comprising administering to the subject an effective amount of a substance that up-regulates the signaling activity of the PGE2 receptor EP4 and/or EP2 signaling pathway.
  • the invention also provides the use of a substance that up-regulates the signaling activity of the PGE2 receptor EP4 and/or EP2 signaling pathway for the preparation of a medicament for modulating vascular remodeling in a subject.
  • the invention also provides a substance that up-regulates the signaling activity of the PGE2 receptor EP4 and/or EP2 signaling pathway for use in modulating vascular remodeling in a subject.
  • the invention also provides an article of manufacture for modulating vascular remodeling in a subject comprising a substance that upregulates PGE2 receptor EP4 and/or EP2 signaling pathway signaling activity.
  • the present invention provides a method of modulating vascular remodeling in a subject comprising administering to the subject an effective amount of an agonist of the PGE2 receptor or a drug that increases expression of the PGE2 receptor of the endothelial cell.
  • the invention also provides the use of an agonist of a PGE2 receptor for the preparation of a medicament for modulating vascular remodeling in a subject.
  • the invention also provides agonists of the PGE2 receptor for use in modulating vascular remodeling in a subject.
  • the invention also provides an article of manufacture for modulating vascular remodeling in a subject comprising an agonist of a PGE2 receptor.
  • FIG. 2 Misoprostol (10 ⁇ M) promotes endothelial cell proliferation (A) and inhibits leukocyte adhesion to endothelial cells (B).
  • the EP4 blocker GW627368X (1 ⁇ M) reverses the action of misoprostol, which inhibits endothelial cell proliferation and increases adhesion of leukocytes to endothelial cells. *, p ⁇ 0.05, **, p ⁇ 0.01.
  • FIG. 3 Endothelial repair (A&B) and intimal leukocyte infiltration (C&D) of control mice (Ctl) and endothelial EP4-specific knockout mice (cKO) 7 days after endothelial injury in mice.
  • cKO slows endothelial repair and increases neointimal leukocytes (mostly macrophages, F4/80+) infiltration.
  • n 6 (Ctl)
  • n 7 (cKO) (A&B).
  • n 8 (C&D).
  • EP4 agonist AE1-329 promotes endothelial cell proliferation (A) in a concentration-dependent manner in the presence or absence of inflammatory factor (IL-1 ⁇ ) stimulation, whereas in the presence of IL-1 ⁇ stimulation, EP4 Blocker GW627368X inhibited proliferation of wild mouse endothelial cells in a concentration-dependent manner (B).
  • IL-1 ⁇ inflammatory factor
  • B IL-1 ⁇
  • C endothelial cell proliferation
  • AE1-329 (1 ⁇ M) treated wild endothelial cells for 2 hours to inhibit the adhesion of leukocytes to endothelial cells, while GW627368X (1 ⁇ M) for 2 hours promoted adhesion of leukocytes to endothelial cells.
  • FIG. 7 IP deletion revealed a protective effect of mPGES-1 -derived PGE2 on neointimal formation induced by guidewire injury.
  • DKO Ptgir -/- Ptges -/-
  • littermate IPKO Ptgir -/- mice underwent guidewire injury in the femoral artery and collected blood vessels 28 days after injury and quantified neointimal formation.
  • Figure 8 Deletion of mPGES-1 in IP-deficient mice inhibits re-endothelialization following endothelial damage.
  • the injured femoral artery of DKO and IPKO was collected 7 days after surgery.
  • DAPI will dye the nuclei in blue.
  • EC was isolated from the descending aorta of IPKO and DKO mice, and cell proliferation (G) was analyzed in vitro and compared (H). The EC proliferation rate was calculated based on the difference in cell number before and after treatment with 3% FBS for 48 hours.
  • FIG. 9 PGE2 promotes endothelial cell proliferation in vitro via the EP4 or EP2/cAMP/PKA signaling pathway.
  • Mouse arterial endothelial cells MAEC
  • MAEC Mouse arterial endothelial cells
  • Cells from DKO (A) and wild-type (WT) mice B) with PGE2 receptor agonist [AE1-329 (AE1), EP4 agonist; thioprostone (Sul), EP 1/3 agonist; His prostaglandin (Buta), an EP2 agonist, stimulates and shows relative proliferation.
  • WT EC was treated with different concentrations of AE1 with or without IL-1 ⁇ (10 ng/mL) and proliferation was determined (C).
  • GW627368X inhibits IL-1 ⁇ -stimulated MAEC proliferation (D).
  • D The effect of AE1 or GW on the proliferation of Ptges-/-EC stimulated with or without IL-1 ⁇ (10 ng/mL) is shown (E). Under the stimulation of IL-1 ⁇ (10 ng/mL), 1 ⁇ mol/L concentration of GW (EP4 antagonist) inhibits endothelial cell proliferation, but L-798106 (EP3 antagonist), PF-04418948 (EP2 antagonist) or ONO-8130 (EP1 antagonist) No (F).
  • Endothelial proliferation was stimulated with iloprost (IP agonist, 1 ⁇ mol/L) and inhibited by Cay 10441 (IP antagonist, 10 ⁇ mol/L) (J).
  • IP agonist IP agonist
  • Cay 10441 IP antagonist, 10 ⁇ mol/L
  • Db-cAMP treatment reduces leukocyte adhesion EC (L). All results are from at least three separate sets of data. *P ⁇ 0.05.**P ⁇ 0.01; One-way ANOVA was used for data comparison using Bonferroni's (A, G-K), Dunnett's (B) or Turkey's (C-F) post-test. Student's unpaired t-test was used in L.
  • FIG. 10 Induction of deletion of endothelial EP4 promotes neointimal formation.
  • Apoptotic deletion of the endothelial EP4 gene was induced in mice by tamoxifen treatment (A).
  • C Western blotting
  • a representative image EP4, green; vWF, red
  • Figure 12 Pharmacological activation of EP4 promotes endothelial repair and has a protective effect against neointimal formation.
  • the injured blood vessels were stained with H&E, and representative images (A; A1, A2) are shown.
  • the injured blood vessels were stained with H&E, and representative images (A; A3, A4) are shown.
  • EP4 signaling is involved in the proliferation of human endothelial cells.
  • Human microvascular endothelial cells were treated with an agonist (A) or antagonist (B) of 1 ⁇ mol/LPGE2 receptor and relative proliferation was determined.
  • Agonists AE1-329 (EP4), thioprostone (EP1/3) and ketata prostaglandin (EP2).
  • Antagonists GW627368X (EP4), L-798106 (EP3), PF-04418948 (EP2) and ONO-8130 (EP1). Both results come from three sets of independent data. *P ⁇ 0.05, **P ⁇ 0.01; One-way ANOVA with Dunnett's post-test was used for data comparison.
  • FIG. 15 Determination of the proliferative capacity of arterial smooth muscle cells isolated from IPKO and DKO mice.
  • Smooth muscle cells SMC were isolated from the descending aorta of IPKO and DKO mice, and their cell proliferation (A) was analyzed in vitro and compared (B).
  • n 8 wells from 2 independent experiments.
  • FIG. 17 Determination of supernatant PGE2 levels of cultured mouse arterial endothelial cells (MAEC) by HPLC-MS/MS method.
  • MAEC was treated with vehicle (control) or IL-1 ⁇ (10 ng/mL) for 12 hours, showing PGE2 levels (A).
  • n 4, **P ⁇ 0.01; Student’s unpaired t-test.
  • FIG. 19 AE1-329, GW627368X and db-cAMP did not affect EC proliferation in the case of PKA inhibition.
  • PKI PKI inhibitor, 10 ⁇ mol/L
  • EP4 agonist AE1-329, abbreviated as AE1, 1 ⁇ mol/L
  • EP4 antagonist GW627368X, abbreviated as GW, 1 ⁇ mol/L
  • db -cAMP cell permeable cAMP analog, 30 ⁇ mol/L
  • Figure 20 Administration of misoprostol (100 ⁇ g/Kg, i.p., three times a day) reduced the number of white blood cells infiltrating the intima as examined 7 days after vascular injury. **P ⁇ 0.01; Student’s unpaired t-test.
  • Figure 21 Forskolin inhibits vascular remodeling after vascular injury.
  • Figure 21A shows neointimal area, intima and middle in control group (0 mg/Kg/day FSK), FSK1 (2 mg/Kg/day FSK) and FSK2 (4 mg/Kg/day FSK) 7 days after injury. Membrane ratio and media area.
  • Figure 21B shows neointimal area, intimal to medial ratio, medial membrane area and body weight of the control (vehicle) and forskolin (2 mg/Kg/day) groups 28 days after injury. *p ⁇ 0.05, **p ⁇ 0.01
  • FIG. 22 Graphical representation of the mechanism of mPGES-1 derived PGE2 in the vascular injury response. Knocking off mPGES-1 reduced PGE2 and increased PGI2. The protective effect of mPGES-1 derived PGE2 was found in IP-deficient mice. PGE2 stimulates endothelial proliferation through the EP4 receptor on endothelial cells, inhibits leukocyte adhesion to endothelial cells, and protects injured blood vessels from neointimal hyperplasia. Arrows indicate stimulation and flat end lines indicate inhibition.
  • Figure 23 FSK reduces atherosclerotic plaque formation.
  • Figure 23A shows oil red O staining of the thoracic aorta
  • Fig. 23B shows oil red O staining of the heart
  • NS control (solvent)
  • FSK 2 dose FSK (4 mg/Kg/day total)
  • lesion area artery Area of atherosclerotic plaque
  • lesion area ratio proportion of atherosclerotic plaque area. *p ⁇ 0.05
  • the present inventors have found for the first time that the prostaglandin E2 (PGE2) signaling pathway has a regulatory effect on vascular remodeling.
  • PGE2 prostaglandin E2
  • upregulation of the PGE2 signaling pathway for example, using a PGE2 receptor agonist (eg, the PGE2 analog misoprostol and the PGE2 receptor agonist AE1-329), can promote endothelial cell proliferation and improve neointimal formation of damaged blood vessels, It inhibits the adhesion of leukocytes to endothelial cells and inhibits vascular remodeling.
  • a PGE2 receptor agonist eg, the PGE2 analog misoprostol and the PGE2 receptor agonist AE1-329
  • the invention provides a method of modulating, for example, vascular remodeling in a subject, comprising administering to the subject an effective amount of a substance that upregulates PGE2 receptor EP4 and/or EP2 signaling pathway signaling activity, and/or increasing PGE2 or A substance whose functional fragment and/or vascular endothelial cell PGE2 receptor EP4 and/or EP2 or a functional fragment thereof.
  • a substance which up-regulates the signaling activity of the PGE2 receptor EP4 or EP2 signaling pathway refers to a PGE2 receptor EP4 or EP2 signaling pathway in a cell treated with the substance under the same conditions as compared with the treatment without the substance.
  • Increased activity results in, for example, increased vascular endothelial cell proliferation, and/or decreased adhesion of leukocytes to vascular endothelial cells, and the like.
  • the substance that up-regulates the signaling activity of the PGE2 receptor EP4 and/or EP2 signaling pathways includes, but is not limited to, PGE2 and functional fragments thereof, PEG2 receptor EP4 or EP2 and functional fragments thereof, EP4 or EP2 A receptor agonist, a molecule that degrades or down-regulates PGE2 and/or EP4 or EP2 receptors, such as an inhibitor of a degradative enzyme, such as an inhibitor of a PGE2 degrading enzyme (eg, 15-PDGH) (eg, SW033291); a functional derivative of PGE2 and Functional analogs, such as misoprostol; receptor EP4 or EP2 agonists, such as phenylprostaglandin, AE1-329 or KMN-80; and substances that up-regulate cAMP-PKA signaling pathway signaling activities, such as cAMP analogs such as Dibutyryl cyclic adenosine monophosphate, cAMP agonist such as
  • the substance which up-regulates the signaling activity of the cAMP-PKA signaling pathway means that the activity of the cAMP-PKA signaling pathway in the cells treated with the substance is increased under the same conditions as compared with the treatment without the substance.
  • the substance that up-regulates cAMP-PKA signaling pathway signaling activity includes, but is not limited to, cAMP, cAMP analog, agonist of cAMP synthase (such as adenylate cyclase (AC)), inhibitor of cAMP degrading enzyme, PKA An agonist, a PGI2 receptor agonist, and the like.
  • a molecule which degrades or down regulates the PGE2 and/or EP4 or EP2 receptor means that the molecule degrades PGE2 or its EP4 or EP2 receptor to lose some or all of its activity, or The activity and/or level of PGE2 or its EP4 or EP2 receptor is reduced such that the binding of PGE2 to its receptor EP4 or EP2 is reduced.
  • An inhibitor that degrades or down-regulates the molecule of the PGE2 and/or EP4 or EP2 receptor prevents the molecule from degrading or down-regulating the PGE2 and/or EP4 or EP2 receptor, under the same conditions, using the inhibitor compared to not using the inhibitor, The binding of PGE2 to its receptor EP4 or EP2 is elevated.
  • the substance capable of increasing the expression of PGE2 or a functional fragment thereof and/or vascular endothelial cell PGE2 receptor EP4 or EP2 or a functional fragment thereof comprises the ability to express PGE2 or a functional fragment thereof and/or PGE2 receptor Expression or gene therapy vector of the body EP4 or EP2 or a functional fragment thereof.
  • the expression or gene therapy vector can comprise a nucleotide sequence encoding PGE2 or a functional fragment thereof and/or PGE2 receptor EP4 or EP2 or a functional fragment thereof operably linked to a promoter.
  • the invention provides a method of modulating vascular remodeling in a subject comprising administering to the subject an effective amount of an agonist of a PGE2 receptor or a substance that increases expression of a PGE2 receptor in a vascular endothelial cell. In one embodiment, the method inhibits vascular remodeling in a subject.
  • subject or “individual” or “patient” is used interchangeably herein to mean a mammal, preferably a primate, more preferably a human.
  • the "subject" refers to a non-human mammal, such as a non-human primate, a rodent, etc., such as a cow, a horse, a goat, a sheep, a rat, a mouse, a rabbit, a dog.
  • a non-human mammal such as a non-human primate, a rodent, etc., such as a cow, a horse, a goat, a sheep, a rat, a mouse, a rabbit, a dog.
  • vascular remodeling refers to structural and functional changes that occur in blood vessels to adapt to changes in the internal and external environment, including proliferation, hypertrophy, apoptosis, cell migration, production and degradation of extracellular matrix of blood vessel wall cells, and the like.
  • Vascular remodeling is an important pathological basis for the progression of vascular-related diseases such as atherosclerosis and hypertension or the cause of the development of such diseases.
  • the vascular remodeling of the invention is a vascular remodeling associated with a vascular associated disease or condition.
  • vascular remodeling associated with a blood vessel-related disease or condition means a blood vessel-related disease or condition due to vascular remodeling, or vascular remodeling due to a blood vessel-related disease or condition.
  • the vascular remodeling of the invention is vascular remodeling associated with a vascular endothelial damaging disease or condition.
  • the vascular-related disease or condition of the present invention is selected from the group consisting of a vascular wall injury (such as physical damage caused by an interventional stent, vascular injury caused by atherosclerosis, vascular injury caused by hyperlipidemia, hypertension) Caused by vascular injury, vascular injury caused by diabetes, vascular injury caused by autoimmune disease), vascular stenosis after injury, blood flow dysfunction after injury, thrombosis, vascular restenosis after PCI and Bypass, coronary heart disease, myocardial ischemia, Myocardial infarction, heart failure after myocardial infarction, arrhythmia after myocardial infarction, atherosclerosis, cerebral infarction and any combination thereof.
  • a vascular wall injury such as physical damage caused by an interventional stent, vascular injury caused by atherosclerosis, vascular injury caused by hyperlipidemia, hypertension
  • vascular injury caused by diabetes
  • vascular injury caused by autoimmune disease vascular stenosis after injury
  • blood flow dysfunction after injury
  • an effective amount or “therapeutically effective amount” refers to an amount of a substance, compound, material, drug, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount required to prevent, cure, ameliorate, block or partially arrest the symptoms of a disease or condition.
  • the actual dosage of a composition of the invention administered to a patient can be determined according to the following physical and physiological factors: body weight, sex, severity of symptoms, type of disease being treated, prior or current therapeutic intervention, unknown etiology of the patient, time of administration, The excretion rate of the specific compound and the route of administration. In any event, the concentration of the active ingredient in the composition and the appropriate dosage for the subject will be determined by the medical personnel responsible for administration.
  • PGE2 receptor refers to a molecule present on the surface of a cell, in which PGE2 interacts and produces a corresponding biological action.
  • the PGE2 receptor is known to include EP1, EP2, EP3 and EP4.
  • the PGE2 receptor of the invention is EP2 and/or EP4.
  • PGE2 receptor agonists described herein include molecules capable of mimicking and/or increasing the corresponding biological effects produced by the interaction of PGE2 with its receptor.
  • PGE2 receptor agonists include, but are not limited to, natural ligands of PGE2 receptors and functional fragments thereof, peptidomimetics, agonistic antibodies or antibody fragments, molecules that increase PGE2 expression or activity, increase PGE2 receptor expression, or Activated molecule.
  • the PGE2 receptor agonist includes PGE2 and its derivatives as well as the PGE2 receptor and its derivatives.
  • the PGE2 receptor agonists described herein also include substances that increase or enhance the corresponding biological or biological effects produced by the interaction of PGE2 with its receptor, such as EP4, such as increasing or enhancing the activity of the downstream signaling pathway of PGE2-EP4. substance.
  • the "biological effects” and “biological effects” produced by the interaction of PGE2 with its receptors means that PGE2 or a functional analogue thereof interacts with its receptor, such as EP4 or a functional fragment thereof, for example.
  • Effects or effects upon cell growth conditions such as endothelial cell proliferation, leukocyte and endothelial cell adhesion, etc., include, for example, promoting or inhibiting the rate of growth or proliferation, increasing or decreasing the number of cells, increasing or decreasing cell survival, and the like.
  • a PGE2 receptor agonist such as an EP4 receptor agonist encompasses any substance and molecule capable of promoting endothelial cell proliferation and inhibiting adhesion of leukocytes to endothelial cells.
  • the agonist of the PGE2 receptor comprises PGE2 or a functional fragment thereof; a functional derivative and functional analog of PGE2, such as misoprostol; a PGE2 degrading enzyme (eg, 15-hydroxy prostaglandin dehydrogenase ( An inhibitor of 15-PDGH)), such as SW033291; an agonist of receptor EP1, such as ONO-DI-004; an agonist of receptor EP2, such as butaprost (Butaprost); an agonist of receptor EP3, for example Sulprostone; and an agonist of the receptor EP4, such as AE1-329 or KMN-80.
  • PGE2 degrading enzyme eg, 15-hydroxy prostaglandin dehydrogenase ( An inhibitor of 15-PDGH)
  • SW033291 an agonist of receptor EP1, such as ONO-DI-004
  • an agonist of receptor EP2 such as butaprost (Butaprost)
  • a functional fragment refers to a fragment of a molecule that has or produces the same or similar biological function as the molecule.
  • a functional fragment of PGE2 is a fragment of PGE2 that binds to the PGE2 receptor and results in a biological function that is identical or similar to the biological function produced by PGE2 binding to the PGE2 receptor.
  • an analog refers to another compound that is structurally similar to one compound but slightly different in composition (eg, one atom is replaced by an atom of another element, or a specific functional group is present, or a functional group) Replaced by another functional group).
  • an analog is a compound that is similar or comparable in function and appearance to the control compound, but differs in structure or source.
  • peptidomimetic refers to any peptide-like molecule that specifically binds to the PGE2 receptor and can be used in the present invention.
  • the peptidomimetics can be identified using methods known in the art, such as screening for peptidomimetics, libraries of peptides, DNA or cDNA expression libraries, combinatorial chemistry, and particularly useful phage display libraries. These libraries can be screened for the agonist of the PGE2 receptor of the present invention by contacting the library with a substantially purified PGE2 polypeptide, a PGE2 polypeptide receptor, a fragment thereof or a structural analog.
  • the agonist of the PGE2 receptor comprises PGE2 or a functional fragment thereof, a functional derivative of PGE2, and a functional analog.
  • a functional derivative of PGE2 is a molecule produced from a PGE2 polypeptide that binds to the PGE2 receptor and produces a biological effect identical or similar to that produced by binding of the corresponding receptor to PGE2.
  • a functional analog of PGE2 is a molecule that is structurally similar to PGE2 and that binds to the PGE2 receptor and produces the same or similar biological effects as the PGE2 binds to the corresponding receptor.
  • Functional derivatives and functional analogs of PGE2 include, but are not limited to, for example, truncated PGE2, modified PGE2, or variants thereof, and the like. Such modifications may be those conventionally used in the art to modify proteins to, for example, increase their stability, half-life, solubility, and the like, including but not limited to glycosylation, phosphorylation, albumination, carboxylation, and the like.
  • the agonist of the PGE2 receptor further comprises an inhibitor of a PGE2 degrading enzyme.
  • a PGE2 degrading enzyme refers to an enzyme that degrades PGE2 in the PGE2 metabolic pathway. It is known in the art that 15-hydroxy prostaglandin dehydrogenase (15-PGDH) is a key enzyme for the biodegradation of prostaglandins.
  • an agonist of a PGE2 receptor of the invention comprises an inhibitor of 15-PGDH.
  • inhibitor of PGE2 degrading enzyme refers to any substance capable of reducing or eliminating the activity of an enzyme to degrade PGE2, including but not limited to antagonistic antibodies or antibody fragments, interfering RNA molecules, double-stranded RNA (dsRNA), small molecules For example, SW033291.
  • the inhibitor is a selective or specific inhibitor.
  • selective and specificity are used interchangeably when used in the context of an inhibitor, meaning that the inhibitor has an inhibitory effect only on the target, or the inhibitory effect on the target is relatively It has a higher inhibitory effect on other compounds or molecules, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1000. 10,000 times, etc.
  • Agonists, particularly selective agonists, of the PGE2 receptors known in the art can be used in the present invention.
  • the agonist of the receptor EP1 includes, for example, ONO-DI-004;
  • the agonist of the receptor EP2 includes, for example, phenylprostaglandin;
  • the agonist of the receptor EP3 includes, for example, thioprostone;
  • the agonist of the receptor EP4 includes, for example, AE1- 329 and KMN-80.
  • the agonist of the PGE2 receptor of the invention is an agonist, particularly a selective agonist, of the EP2 and/or EP4 receptor.
  • an agonist of a PGE2 receptor of the invention is selected from the group consisting of PGE2, PGE2 analogs such as misoprostol, tromethamine, KMN-80, and AE1-329.
  • the agonist of the PGE2 receptor also encompasses a substance that increases the downstream molecular (cAMP, PKA) signaling activity of the PGE2-EP4 receptor signaling pathway, thereby enhancing the corresponding biological effects, such as cell growth or proliferation.
  • the agonist of the PGE2 receptor comprises a cAMP-PKA signaling pathway agonist, including, for example, a cAMP analog such as Db-cAMP, a cAMP agonist such as an adenylate cyclase (AC) activator, such as a Fushi Colin, a PGI2 receptor agonist such as iloprost.
  • AE1-329 (PubChem CID: 9468782) is a compound of Formula I.
  • forskolin (CAS Accession No.: 66575-29-9) is a compound of Formula II.
  • a substance that increases the expression of PGE2 or a functional fragment thereof and/or vascular endothelial cell PGE2 receptor EP4 and/or EP2 or a functional fragment thereof refers to PGE2 or a functional fragment thereof in a subject after administration to a subject.
  • the expression of the PGE2 receptor or its functional fragment on vascular endothelial cells is increased compared to the absence of the substance.
  • Such a substance includes, for example, but not limited to, an expression or gene therapy vector, as described below, comprising a PGE2 or a functional fragment thereof operably linked to a promoter and/or a PGE2 receptor EP4 and/or EP2 or a function thereof.
  • the nucleic acid sequence of a sex fragment refers to PGE2 or a functional fragment thereof in a subject after administration to a subject.
  • the expression of the PGE2 receptor or its functional fragment on vascular endothelial cells is increased compared to the absence of the substance.
  • Such a substance includes, for example
  • the "substance which increases the expression of PGE2 receptor in vascular endothelial cells” means that the expression of the PGE2 receptor on the vascular endothelial cells in the subject is increased after administration to the subject as compared with the case where the substance is not administered.
  • Such materials include, for example but without limitation, expression or gene therapy vectors, as described below, comprising a nucleic acid sequence encoding a PGE2 receptor, such as EP2, EP4, or a functional fragment thereof operably linked to a promoter.
  • the methods of the invention may further comprise administering to the subject other agents, such as drugs that treat or prevent a vascular related disease or condition.
  • the methods of the present invention may further comprise administering to a subject such as, but not limited to, an antibacterial agent, an antifungal agent, an antibiotic, an antiviral agent, an anticoagulant, an antithrombotic agent, an antihyperlipidemic agent, and a strong agent.
  • a subject such as, but not limited to, an antibacterial agent, an antifungal agent, an antibiotic, an antiviral agent, an anticoagulant, an antithrombotic agent, an antihyperlipidemic agent, and a strong agent.
  • Heart medicine antihypertensive agent, cholinergic agent, anticholinergic drug, antispasmodic drug, local anesthetic, analgesic, anesthetic antagonist, antioxidant and vasoactive agent.
  • the methods of the invention may further comprise administering to the subject an inflammatory factor such as IL-1 ⁇ .
  • the PGE2 receptor is administered to a subject by oral, buccal, inhalation, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal or topical administration.
  • An agonist or a substance that increases the expression of PGE2 receptor in vascular endothelial cells is administered to a subject by oral, buccal, inhalation, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal or topical administration.
  • the methods of the invention may administer an agonist of the PGE2 receptor or a substance that increases expression of a vascular endothelial cell PGE2 receptor locally (eg, a site of vascular injury).
  • the methods of the invention comprise or coat a vascular stent or a balloon with an intravascular administration or an agonist of the PGE2 receptor or a substance that increases expression of a vascular endothelial cell PGE2 receptor.
  • the topical application is effected on the balloon of the catheter.
  • the invention provides a substance that up-regulates the signaling activity of the PGE2 receptor EP4 and/or EP2 signaling pathway and/or is capable of increasing PGE2 or a functional fragment thereof and/or vascular endothelial cell PGE2 receptor EP4 and/or EP2 or The use of a substance whose functional fragment is expressed in the preparation of a medicament for inhibiting vascular remodeling in a subject.
  • the invention provides a substance that up-regulates the signaling activity of the PGE2 receptor EP4 and/or EP2 signaling pathway and/or is capable of increasing PGE2 or a functional fragment thereof and/or vascular endothelial cell PGE2 receptor EP4 and/or EP2 or A substance expressed by a functional fragment thereof for inhibiting vascular remodeling in a subject or as a drug for inhibiting vascular remodeling in a subject.
  • the invention also provides the use of an agonist of a PGE2 receptor of the invention in the manufacture of a medicament for modulating vascular remodeling in a subject.
  • the medicament inhibits vascular remodeling in a subject.
  • the agonist of the PGE2 receptor comprises PGE2 or a functional fragment thereof; a functional derivative of PGE2 and a functional analog, such as misoprostol; an inhibitor of a PGE2 degrading enzyme (eg, 15-PDGH) , for example, SW033291; an agonist of receptor EP1, such as ONO-DI-004; an agonist of receptor EP2, such as butaprostaglandin; an agonist of receptor EP3, such as thioprostone; and an agonist of receptor EP4
  • AE1-329 or KMN-80 for example, AE1-329 or KMN-80.
  • the agonist of the PGE2 receptor also encompasses a substance that increases the downstream molecular (cAMP, PKA) signaling activity of the PGE2-EP4 receptor signaling pathway, thereby enhancing the corresponding biological effects, such as cell growth or proliferation.
  • the agonist of the PGE2 receptor comprises a cAMP-PKA signaling pathway agonist, including, for example, a cAMP analog such as Db-cAMP, a cAMP agonist such as an adenylate cyclase (AC) activator, such as a Fushi Kelin, a PGI2 receptor agonist, such as iloprost.
  • the medicament may also comprise other active substances, such as substances that treat or prevent a vascular related disease or condition.
  • the drug includes, for example but is not limited to, an antibacterial agent, an antifungal agent, an antibiotic, an antiviral agent, an anticoagulant, an antithrombotic agent, an antihyperlipidemic agent, a cardiotonic drug, an antihypertensive agent.
  • an antibacterial agent for example but is not limited to, an antibiotic, an antiviral agent, an anticoagulant, an antithrombotic agent, an antihyperlipidemic agent, a cardiotonic drug, an antihypertensive agent.
  • Agents cholinergics, anticholinergics, antispasmodics, local anesthetics, analgesics, narcotic antagonists, antioxidants and vasoactive agents.
  • the medicament may further comprise an inflammatory factor such as IL-1 ⁇ .
  • the medicament preferably comprises at least one pharmaceutically acceptable carrier in addition to an active ingredient such as an agonist of the PGE2 receptor, and may optionally include pharmaceutically acceptable adjuvants, buffers, dispersing agents and the like.
  • the pharmaceutical carrier can be any compatible non-toxic substance suitable for delivery to a patient, such as a polypeptide, antibody or gene therapy vector. Sterile water, alcohols, fats, waxes and inert solids can be used as carriers.
  • the medicament may be prepared or, after reconstitution, a form for administration to a subject by oral, buccal, inhalation, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal or topical administration.
  • the medicament of the invention can be used for treating or preventing blood vessel wall damage (such as physical damage caused by interventional stent, vascular injury caused by atherosclerosis, vascular injury caused by hyperlipemia, vascular injury caused by hypertension, diabetes) Vascular injury, vascular injury caused by autoimmune disease), vascular stenosis after injury, blood flow dysfunction after injury, thrombosis, vascular restenosis after PCI and Bypass, coronary heart disease, myocardial ischemia, myocardial infarction, heart failure after myocardial infarction , arrhythmia after myocardial infarction, atherosclerosis, cerebral infarction and any combination thereof.
  • blood vessel wall damage such as physical damage caused by interventional stent, vascular injury caused by atherosclerosis, vascular injury caused by hyperlipemia, vascular injury caused by hypertension, diabetes
  • Vascular injury, vascular injury caused by autoimmune disease vascular stenosis after injury, blood flow dysfunction after injury, thrombosis, vascular restenosis after
  • the invention also provides an article of manufacture for modulating vascular remodeling in a subject, comprising or coated with an agonist of a PGE2 receptor of the invention or an agonist capable of expressing a PGE2 receptor, and/or Expression of a nucleic acid of a PGE2 receptor or a functional fragment thereof or a gene vector.
  • the article inhibits vascular remodeling in a subject.
  • the agonist of the PGE2 receptor comprises PGE2 or a functional fragment thereof; a functional derivative of PGE2 and a functional analog, such as misoprostol; an inhibitor of a PGE2 degrading enzyme, such as 15-PDGH Inhibitors, such as SW033291; agonists of receptor EP1, such as ONO-DI-004; agonists of receptor EP2, such as butaprostaglandin; agonists of receptor EP3, such as thioprostone; and receptor EP4 An agonist such as AE1-329 or KMN-80.
  • the agonist of the PGE2 receptor also encompasses a substance that increases the downstream molecular (cAMP, PKA) signaling activity of the PGE2-EP4 receptor signaling pathway, thereby enhancing the corresponding biological effects, such as cell growth or proliferation.
  • the agonist of the PGE2 receptor comprises a cAMP-PKA signaling pathway agonist, including, for example, a cAMP analog such as Db-cAMP, a cAMP agonist such as an adenylate cyclase (AC) activator, such as a Fushi Kelin, a PGI2 receptor agonist, such as iloprost.
  • the article of vascular remodeling in a subject of modulation comprises a vascular stent or a balloon with a balloon comprising or coated with an agonist of a PGE2 receptor of the invention or comprising a PGE2 capable of expressing Expression of a nucleic acid of a agonist, and/or a PGE2 receptor or a functional fragment thereof or a gene vector.
  • the PGE2 receptor of the invention is EP2 and/or EP4.
  • the agonist of the PGE2 receptor is an agonist of the EP2 and/or EP4 receptor, for example selected from the group consisting of PGE2, PGE2 analogs such as misoprostol, duprostin, KMN-80 and AE1 -329.
  • the preparation of the invention comprises an agonist of a PGE2 receptor that is released in a sustained release form, such as a PGE2 polypeptide or a functional fragment thereof, a PGE2 analog such as misoprostol, trojana, KMN- 80 and / or AE1-329.
  • a PGE2 receptor that is released in a sustained release form, such as a PGE2 polypeptide or a functional fragment thereof, a PGE2 analog such as misoprostol, trojana, KMN- 80 and / or AE1-329.
  • the vascular stent or the balloon-equipped catheter of the present invention is used for treating or preventing a blood vessel-related disease or condition selected from the group consisting of a blood vessel wall injury (such as physical damage caused by an interventional stent, vascular damage caused by atherosclerosis).
  • a blood vessel wall injury such as physical damage caused by an interventional stent, vascular damage caused by atherosclerosis.
  • vascular injury caused by hyperlipidemia vascular injury caused by hypertension
  • vascular injury caused by diabetes vascular injury caused by autoimmune disease
  • vascular stenosis after injury blood flow dysfunction after injury, thrombosis, PCI and Bypass postoperative blood vessels Restenosis, coronary heart disease, myocardial ischemia, myocardial infarction, heart failure after myocardial infarction, arrhythmia after myocardial infarction, atherosclerosis, cerebral infarction, and any combination thereof.
  • the invention also provides an expression or gene therapy vector (eg, a viral vector, such as an adenoviral vector, an adeno-associated viral vector, etc.), wherein the nucleotide sequence is capable of driving a nucleotide sequence in an endothelial cell, preferably at Under the control of promoters expressed in vascular endothelial cells:
  • an expression or gene therapy vector eg, a viral vector, such as an adenoviral vector, an adeno-associated viral vector, etc.
  • an expression or gene therapy vector wherein a nucleotide sequence encoding a PGE2 receptor such as EP1, EP2, EP3, EP4, in particular EP2 and/or EP4, or a functional fragment thereof, is operably linked to a promoter ;
  • the gene therapy vector can optionally comprise one or more additional nucleotide sequences encoding additional proteins.
  • the additional protein may be another therapeutic molecule, or (optional) a marker protein, such that cells containing the expression construct can be identified, selected and/or screened, as described in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rd edition)", Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York.
  • the article includes, for example but is not limited to, an antibacterial agent, an antifungal agent, an antibiotic, an antiviral agent, an anticoagulant, an antithrombotic agent, an antihyperlipidemic agent, a cardiotonic agent, an antihypertensive agent.
  • an antibacterial agent for example but is not limited to, an antibiotic, an antiviral agent, an anticoagulant, an antithrombotic agent, an antihyperlipidemic agent, a cardiotonic agent, an antihypertensive agent.
  • Agents cholinergics, anticholinergics, antispasmodics, local anesthetics, analgesics, narcotic antagonists, antioxidants and vasoactive agents.
  • the preparation can include an inflammatory factor such as IL-1 ⁇ .
  • Range and amount may be expressed as “about” a particular value or range. It also includes the exact amount. Thus “about 5%” means “about 5%” and “5%”.
  • a pharmaceutically acceptable carrier is meant to include or not include the pharmaceutically acceptable carrier.
  • IP knockout mice The preparation of PGI receptor (IP) knockout mice is described in the literature (Nature. 1997 Aug 14; 388 (6643): 678-82). Using C57BL/6 mice, embryonic stem cells disrupting the IP-encoding gene were obtained and injected into the blastocyst of the pregnant mouse to obtain chimeric mice. The obtained mice were backcrossed with C57BL/6 mice to obtain IP +/- mice, and IP knockout (IP -/- ) mice were obtained by IP +/- mouse selfing.
  • DKO mice (IP -/- mPGES -/- ) were obtained by hybridization of IP knockout C57BL/6 mice (IP -/- mPGES +/- ) with mPGES knockout C57BL/6 mice (mPGES -/- ).
  • the experimental mPGES -/- C57BL/6 mice and their control mice were derived from mPGES +/- C57BL/6 mouse self-crossing (Circulation. 2011 Feb 15; 123(6): 631-9).
  • EP4 deficient mice were constructed using the tamoxifen-CreERT2 strategy. Briefly, as previously described (Circulation.2017 Mar 28; 135(13): 1253-1264), C57BL/6 mice (EP4f/f) containing loxP-sites on both sides of the EP4 gene were used by Ralf Adams Cdh5-promoter-driven CreERT2 (Cdh5(PAC)-CreERT2+) C57BL/6 mice (Nature. 2010; 465:483-486) were hybridized.
  • C57BL/6 mice were purchased from the China Food and Drug Administration and used to assess the effect of EP4 agonist AE1-329 on vascular remodeling.
  • the vascular injury model experiments were performed using IPKO and DKO mice prepared in Example 1.
  • mice The mouse femoral artery intimal injury model was performed as described in the literature (Circulation. 2011 Feb 15; 123(6): 631-9). The mice were first anesthetized with pentobarbital at a dose of 90 mg/kg. The left femoral artery was then exposed by blunt dissection under strictly sterile conditions. A small arterial branch between the rectus femoris and the femoral muscle is isolated separately, and a lateral arteriotomy is performed on this branch, and a flexible angioplasty guide wire (0.35 mm in diameter) is placed along the artery. The direction is inserted into the femoral artery with an insertion depth of more than 5 mm.
  • the guide wire was left in place for 3 minutes to damage and expand the femoral artery.
  • the blood flow of the femoral artery was restored by removing the circumflex line at the proximal and distal ends of the femoral artery, and then the skin incision was closed with a suture thread No. 5.
  • the left and right femoral arteries were collected. Each femoral artery was embedded in paraffin, and the sections were taken from the branch of the femoral artery with a thickness of 4.5 ⁇ m, separated by 30 ⁇ m, and cut into the proximal end by 400 ⁇ m. The cut sections were subjected to conventional hematoxylin and eosin (HE) staining. Immunofluorescence detection of biomarkers was performed on the adjacent part.
  • HE hematoxylin and eosin
  • Ten cross-sections of the injured and non-injured arteries were imaged by CCD, digitized with Image Pro image analysis software (Media Cybernetis, Silver Spring MD), and the lumen area, inner elastic membrane area and outer area were measured. The inner area of the elastic membrane. The percentage of arterial stenosis is counted as the ratio of the area of the intima and the area of the inner elastic membrane.
  • Example 3 Effect of PGE2 receptor modulator on vascular remodeling
  • Endothelial cells were cultured using patch culture (Cell metabolism. 2011; 13: 592-600). C57BL/6 mice were sacrificed 6-8 weeks from the neck and immersed in alcohol for sterilization. The mice were dissected in a biosafety cabinet and the thoracic aorta was taken. Under the stereoscopic microscope, gently separate the fat and connective tissue around the aorta of the dry and thoracic. The blood vessels were longitudinally dissected, the blood vessels were cut into small pieces about 1.5 mm long, and the endothelium side was attached downward to the bottom of the culture dish. Place the culture dish in the incubator for 5-10 minutes.
  • DMEM medium with 20% fetal bovine serum, 1% double antibody (penicillin 10000 U/mL; streptomycin 10000 ⁇ g/mL), 100 ⁇ g/ml heparin, 100 ⁇ g/ml endothelial cell growth factor (ECGS), re-cultured in an incubator. After 3-4 days, after climbing out of the cell , subculture, and used in cell experiments.
  • Cell growth was measured using Cell Counting Kit-8 (CCK-8; Yeasen, Shanghai, China). Briefly, cells were seeded in 96-well flat-bottom plates. After the cells were completely attached to the bottom, the cells were starved for 6-8 hours in a medium containing 3% FBS and no ECGS. The medium was then replaced with a medium-CCK-8 mixture (volume 10:1). After 4 hours, the absorbance was measured at 450 nm as a baseline. Then, the cells were incubated with the drug-containing 3% FBS, medium without ECGS for 48 hours. Finally, the medium was replaced again with the medium-CCK-8 mixture. After 4 hours, the absorbance measured at 450 nm showed cell growth.
  • CCK-8 Cell Counting Kit-8
  • the concentrations of the drugs used were as follows: 10 ng/mL IL-1 ⁇ (Bioengineering (Shanghai) Co., Ltd., China), 1 ⁇ M AE1-329 (Japan ONO PHARMACEUTICAL), 1 ⁇ M GW627368X (Selleck, USA) or 10 ⁇ M misoprostol (China National Institute for the Control of Pharmaceutical and Biological Products, China).
  • the endothelial cells were seeded in black 96-well plates, and cultured for 30 hours in the low serum containing 3% serum endothelial cells before the experiment, and treated with leukocytes 2 hours before the incubation (the drug and concentration were: 1 ⁇ M AE1- 329, 1 ⁇ M GW627368X and 10 ⁇ M misoprostol), ready for use.
  • the leukocyte flow was as follows. The mice were intraperitoneally injected with 1% Brewer modified ⁇ glycolate broth (B&D, USA) 1 mL. After 4-5 hours, the mouse peritoneal cavity was washed with PBS containing 0.1% BSA, and the rinsing liquid was collected. Centrifuge in a centrifuge tube. The supernatant was discarded, and the leukocytes were resuspended in 10% serum in RPMI 1640 medium and incubated with the treated endothelial cells for 30 minutes.
  • B&D Brewer modified ⁇ glycolate broth
  • RPMI1640 medium containing 1 mg/mL rhodamine 6G Biotech (Shanghai) Co., Ltd., China
  • RPMI1640 medium containing 1 mg/mL rhodamine 6G Biotech (Shanghai) Co., Ltd., China
  • microplate reader excitation light: 560 nm
  • emission light 630 nm
  • Example 2 The vascular injury model experiment described in Example 2 was carried out using the endothelium-specific knockout EP4 mice described in Example 1.
  • Sections (5 ⁇ m) from paraffin-embedded tissues were dewaxed, rehydrated, and subjected to antigen retrieval by boiling in EDTA antigen-repairing water (pH 9.0; ZSGB-BIO, Beijing, China) for 2 minutes. Sections were then incubated with goat serum containing 0.3% Triton X-100 for blocking and membrane disruption. After incubation, the antibody was incubated overnight at 4 °C with the primary antibody and the samples were incubated with Alexa Fluor-594 coupled and/or Alexa Fluor-488 conjugated secondary antibody for 3 hours at room temperature. The nuclei were stained with DAPI-containing tablets (ZSGB-BIO, Beijing, China) and mounted.
  • Sections were imaged using a Zeiss inverted fluorescence microscope (AXI0; Zeiss) equipped with Zen software or a laser scanning confocal microscope (SP8; Leica) equipped with a 20x objective. Images were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc. Rockville, MD, USA).
  • the antibodies used included polyclonal anti-vWF (1:800; Sigma), monoclonal antibody anti-F4/80 (1:50; BM8; Abcam).
  • Endothelial-specific knockout of EP4 significantly inhibited the process of re-endothelialization, increased leukocyte infiltration of neointimal membrane, and increased the intimal area and the ratio of intima to medial membrane, which did not affect the medial membrane area.
  • Endothelial-specific knockdown of EP4 inhibits endothelial repair after endothelial injury in mice and increases leukocyte infiltration of angiogenic intima (Fig. 3) and aggravates intimal hyperplasia after endothelial injury in mice (Fig. 4).
  • Example 3 primary cultured endothelial cells as described in Example 3 were seeded in 96-well flat-bottom plates. After the cells were completely attached to the bottom, the cells were starved for 6-8 hours in a medium containing 3% FBS and no ECGS. The medium was then replaced with a medium-CCK-8 mixture (volume 10:1). After 4 hours, the absorbance was measured at 450 nm as a baseline. Then, the cells were incubated with the drug-containing 3% FBS, medium without ECGS for 48 hours. Finally, the medium was replaced again with the medium-CCK-8 mixture. After 4 hours, the absorbance measured at 450 nm showed cell growth. The concentration of the drug used was as follows: 10 ng/mL IL-1 ⁇ , 1 ⁇ M AE1-329 or 1 ⁇ M GW627368X.
  • AE1-329 inhibits vascular remodeling after femoral artery endothelial injury in mice.
  • mPGES-1 (gene: Ptges) (Proc Natl Acad Sci USA. 2003; 100:9044-9049) and IP (gene: Ptgir) (Science. 2002; 296:539-541) defective mice were obtained from Pfizer and University, respectively. The FitzGerald Laboratory in Pennsylvania. Both animals were backcrossed with C57BL/6 background for more than 10 generations and were used to generate IP/mPGES-1 double knockout (DKO) by hybridization of Ptgir -/- Ptges -/- and Ptgir -/- Ptges +/- Mouse and littermate control (IP KO). DKO mice developed normally without obvious abnormalities.
  • tamoxifen (37.5 mg/ml dissolved in sunflower seed oil) was intraperitoneally injected into the test group and littermate at a dose of 150 mg/kg/day for 6 days, of which the third dose After 3 days of interruption.
  • the genetically modified mice used in this study were gender matched. Data from two gender sets were used to show gene-specific effects in each study, and gender-specific subgroup analyses are provided in Tables 1 and 2.
  • the femoral artery was injured using the method described previously (Circulation. 2017; 135: 1253-1264). Briefly, an inguinal incision was made on the side of the anesthetized mouse. The femoral artery and its small branches between the rectus femoris and the femoral muscle are then carefully exposed and separated from the accompanying nerves and blood vessels by blunt dissection. A 6-0 silk wire is then knotted around the proximal femoral artery to stop blood flow during surgery. Another 6-0 silk thread is placed under the branch.
  • a bendable angioplasty guidewire (diameter 0.35 mm; Cook Inc., IN, USA) was inserted from the branch into the femoral artery to a length of not less than 5 mm.
  • the guidewire was left in place for 3 minutes to dilate and denude the artery.
  • the guide wire was taken out, and the branches were connected at the proximal end with a 6-0 silk thread, and the blood flow of the femoral artery was restored by releasing the wire for blood flow control.
  • the skin incision was then closed with a 5-0 silk thread.
  • the arteries were harvested, embedded in paraffin, and stained with hematoxylin and eosin (H&E) to determine the severity of the hyperplasia.
  • H&E hematoxylin and eosin
  • 10-13 transverse sections of the artery were continuously obtained at intervals of 150 ⁇ m, and the sections with the most severe proliferation were used for comparison.
  • vWF endothelial cell marker von Wllebrand Factor
  • the circumference of the vWF positive section and the inner wall of the vessel along the inner wall of the blood vessel was measured using Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA). The ratio of the total length to the circumference of the vWF positive segment was calculated to represent the degree of re-endothelialization.
  • image-Pro Plus 6.0 software Media Cybernetics, MD, USA
  • Hue(H) Saturation(S) Intensity(I)-based color selection strategy Hue(H) Saturation(S) Intensity(I)-based color selection strategy.
  • Immunofluorescence staining was carried out in the same manner as described above (Circulation. 2017; 135: 1253-1264). Briefly, paraffin sections (5 ⁇ m) were dewaxed, rehydrated and antigen-repaired using EDTA antigen-repairing water (pH 9.0; ZSGB-BIO, Beijing, China). After incubation with normal goat serum for 1 hour at room temperature, the samples were incubated with primary antibody overnight at 4 °C, followed by staining with Alexa Fluor-488-conjugated or Alexa Fluor-594-conjugated secondary antibody for 3 hours at room temperature.
  • the sections were then stained and mounted with VectaShield medium containing DAPI and imaged using a Zeiss microscope system (AXI0; Zeiss) or a laser-scattering confocal microscope system (SP8; Leica). To determine re-endothelialization, images were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc. Rockville, MD, USA).
  • mPGES-1KO was hybridized with IP KO to generate double KO (DKO) mice, and littermate IP KO mice were used as controls. Mice were subjected to femoral artery guidewire injury (endothelial ablation) for 28 days. In this IP-deficient background, mPGES-1 deletion increased neointimal area by ⁇ 84%, and increased the ratio of intimal to medial area, with no change in medial thickness (Fig. 7A-D). This reveals a protective effect of mPGES-1 -derived PGE 2 in vascular responses to injury. Urine metabolites of PGE 2 were reduced in DKO mice, while PGI 2 metabolites were increased (Fig. 7E & F).
  • ECs were isolated from IP KO and DKO mice and their proliferative capacity was assessed in vitro (Fig. 8G). DKO in EC proliferation was significantly impaired (FIG. 8H), show that PGE 2 pathway promotes EC proliferation. Moreover, treatment of EC with the PGE analog misoprostol promoted EC proliferation in vitro (Fig. 8I) and also reduced leukocyte adhesion to endothelial monolayers in vitro (Fig. 8J). Primary aortic smooth muscle cells were also isolated from IP KO and DKO. No difference in cell proliferation was detected in these two groups (Fig. 15).
  • Endothelial cells Mouse aortic endothelial cells (MAEC) were isolated as previously described (Circulation. 2017; 135: 1253-1264; Cell metabolism. 2011; 13: 592-600). Briefly, the aorta was collected and cut into 1-2 mm 2 sections. The aortic fragment was attached to the culture dish through its luminal surface, and then cultured in DMEM medium containing 20% fetal bovine serum (FBS) and 100 ug/mL endothelial cell growth supplement (ECGS) for 5-7 days to make endothelial cells Adherent growth. The endothelial cells are then subcultured. 2-6 generations of MAEC were used in this study.
  • FBS fetal bovine serum
  • ECGS endothelial cell growth supplement
  • HMEC human microvascular endothelial cells
  • MASMC Mouse aortic smooth muscle cells
  • Cell proliferation Cell growth was determined using Cell Counting Kit-8 (CCK-8; 40203 ES60; Yeasen, Shanghai, China) according to the manufacturer's instructions, as previously described (Circulation. 2017; 135: 1253-1264). The assay allowed for multiple assays without significant cytotoxicity. Briefly, cells were seeded in 96-well flat-bottom plates. After the cells were attached, the EC was cultured for 6-8 hours in a medium containing 3% FBS. Thereafter, the medium was changed to a 3% FBS medium-CCK-8 mixture (volume 10:1). Thereafter, the cells were cultured in the mixture for no more than 4 hours; the absorption at 450 nm was determined as a baseline.
  • CCK-8 Cell Counting Kit-8
  • 40203 ES60 Yeasen, Shanghai, China
  • the assay allowed for multiple assays without significant cytotoxicity. Briefly, cells were seeded in 96-well flat-bottom plates. After the cells were attached, the EC was cultured
  • the cells were then incubated for an additional 48 hours in fresh 3% FBS medium with indicator reagents.
  • the medium was then replaced with a medium-CCK-8 mixture, and the cultivation was continued for the same time as described above for determining absorption at 450 nm.
  • the change in absorption between measurements was used to define cell growth.
  • MASMC proliferation cells were pre-starved for 24 hours in FBS-free serum, then cultured in medium containing 1% FBS, and cell proliferation status was measured with CCK-8 before and after 1% FBS culture, respectively.
  • the reagents used for the proliferation study and their concentrations are as follows: AE1-329 (0.1-1 ⁇ mol/L; ONO Pharmaceutical Co., Ltd., Osaka, Japan), bupropion (1 ⁇ mol/L; 13740; Cayman Chemical, MI) , USA), thioprostone (1 ⁇ mol/L; 14765; Cayman), GW627368X (0.1-1 ⁇ mol/L; HY-16963; MedChemExpress, NJ, USA), L-798106 (1 ⁇ mol/L; 11129; Cayman), Prostaglandin (1 ⁇ mol/L; 18215; Cayman), Cay 10441 (10 ⁇ mol/L; 10005186; Cayman), PF-04418948 (1 ⁇ mol/L; S7211; Selleck), ONO-8130 (1 ⁇ mol/L; 19118; Cayman), SQ22536 (200 ⁇ mol/L; S8283; Selleck), H89 2HCl (10 ⁇ mol/L; S1582; Sell
  • Endothelial-leukocyte adhesion assay For endothelial-leukocyte adhesion assays, MAECs were plated in 96-well plates, pre-starved for 6-8 hours in DMEM containing 3% FBS, and incubated with test reagent for 2 hours. White blood cells were collected from the mouse peritoneum. Briefly, 4% Brewer Modified Thioglycollate Medium (211716; BD Biosciences, NJ, USA) was injected into the mouse peritoneum (1 mL/mouse). After 4-5 hours, the peritoneal leukocytes were washed with 0.1% bovine serum albumin, centrifuged, and resuspended in 1640 medium containing 10% FBS.
  • the medium with the indicated drug was replaced with 1640 medium (3 ⁇ 10 4 /well) containing leukocytes. Endothelial cells and white blood cells were then co-cultured for 30 minutes. Thereafter, the cells were washed once with 1640 medium containing rhodamine 6G (200 ⁇ g/mL; 252433; Sigma, Darmstadt, Germany), followed by washing 3 times with fresh 1640 medium. Finally, the fluorescent signal was detected by a microplate reader (excitation wavelength: 560 nm, emission wavelength: 630 nm; Infinite M200, Tecan, Hombrechtikon, Switzerland).
  • misoprostol (10 ⁇ mol/L; 410004; National Institutes for Food and Drug Control, Beijing, China)
  • AE1-329 (1 ⁇ mol/L; Gifted by ONO Pharmaceutical Co., Ltd., Osaka, Japan)
  • GW627368X (1 ⁇ mol/L; HY-16963; MedChemExpress, NJ, USA)
  • db-cAMP (30 ⁇ mol/L; D0260; Sigma, Darmstadt, Germany).
  • EP4 activation promotes endothelial cell proliferation and reduces endothelial-leukocyte adhesion
  • SQ22536 [adenylate cyclase (AC) inhibitor], H 89 2HCl (PKA inhibitor) and ESI-09 (EPAC inhibitor) were used.
  • Treatment with SQ22536 or H89 2HCl abolished the antiproliferative effect of AE1-329 proliferative (Fig. 9G) and GW627368X (Fig. 9H), but with ESI-09 no, it indicated that the cAMP-PKA axis constitutes an enhancement driven by EP4 activation.
  • EP4 AE1-329 by activation, but not other PGE 2 receptor, to promote proliferation of primary human endothelial cells (FIG. 13A).
  • EP4 blockade of GW627368X Fig. 13B.
  • mice C57BL/6 mice were randomly divided into 3 groups: (1) control group (Ctl), in which mice Two times a day, intraperitoneal injection of control solvent (dimethyl sulfoxide (DMSO) in normal saline (volume 1:100)), (2) one dose of FSK group (FSK1), in which mice were injected once daily intraperitoneally with solvent One dose of FSK (2 mg/Kg) was added, and (3) two doses of FSK group (FSK2), in which mice were intraperitoneally injected twice daily with FSK (2 mg/Kg).
  • control solvent dimethyl sulfoxide (DMSO) in normal saline (volume 1:100)
  • FSK1 dimethyl sulfoxide
  • FSK2 two doses of FSK group
  • arteries were harvested 28 days after injury.
  • arteries were collected at 7 days to study early vascular changes caused by injury and FSK.
  • the collected blood vessels were all embedded in paraffin, and were sequentially sliced to 12 layers, and each two adjacent layers were separated by 200 ⁇ m.
  • sections of all layers were stained with hematoxylin and Yinhong (H&E). Sections with the most severe neointimal hyperplasia in each layer were selected as representative sections for further study.
  • mice 10 week old male genetically modified mice (referred to as Konck in mice (Kin)) were used.
  • Konck in mice By encoding the sequence between exons 2 and 4 of the ApoE gene with a scavenger receptor class 1 (SR-B1) knockdown element containing (1) an interfering RNA encoding for SR-BI, and (2) encoding PDZK1 (which can be down-regulated)
  • SR-B1 knockdown element containing (1) an interfering RNA encoding for SR-BI, and (2) encoding PDZK1 (which can be down-regulated)
  • This mouse was generated by sequence replacement of the SR-BI expression) truncated form of the expression cassette and (3) AngII expression cassette followed by Gaussia luciferase and the Tet-on operon in the context of C57BL/6 mice.
  • the apolipoprotein E (ApoE) gene was knocked out, SR-BI expression was down-regulated in cells in which the ApoE promoter was active, and AngII expression was achieved by administration of doxycycline (Dox) conditions.
  • Dox doxycycline
  • mice were randomly divided into 2 groups, control group (Ctl) and two doses of FSK group (FSK2).
  • the solvent or FSK was administered immediately after the mice started receiving a high fat diet (HFD) containing 21% fat and 0.2% cholesterol and Dox (1 mg/ml).
  • HFD high fat diet
  • Dox 1 mg/ml
  • mice were sacrificed by administering an excess of sodium pentobarbital (100 mg/mL) and perfused with left ventricle by continuous injection with 4% paraformaldehyde (PFA). The heart and aorta were then collected and fixed in 4% PFA.
  • Both the thoracic aorta and the aortic root were used to determine the effect of FSK on atherosclerotic plaque regions in Kin mice. Briefly, after carefully removing the perivascular tissue, the thoracic aorta was incised and stained with oil red O for face. The heart is transected through the middle layer of the ventricle. The upper portion of the heart was then embedded in OCT and frozen at -80 °C after overnight in a solution containing 20% sucrose. Slice from the ventricle to the direction of the aorta. When the aortic root was reached, 4 layers of frozen sections separated by 40 ⁇ M were collected. Sections of the different layers were then stained with Oil Red O. For aortic roots, the layer with the largest plaque area is used for comparison.
  • the aorta and frozen sections were stained with Oil Red O according to the following method. Briefly, the aorta was stained with Oil Red O and sectioned for 30 minutes, and washed briefly with 80% methanol twice before and immediately after Oil Red O staining. Photos were taken using a Zeiss optical microscope (AXI0; Zeiss, Oberkochen, Germany) and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, MD). These processes are operated blindly. During the organization collection and data collection, the operator does not know the mouse group information.
  • vascular remodeling is one of the important pathophysiological processes of atherosclerosis progression.
  • FSK forskolin

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Abstract

本发明提供了一种调节对象中血管重构的方法，包括给予对象有效量的能够上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质；本发明还提供了能够上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质在制备用于调节对象中血管重构的药物中的应用；本发明还提供了用于调节对象中血管重构的制品，其包含能够上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质。

Description

靶向前列腺素E2及其受体的药物与应用 技术领域
本发明涉及用于调节血管重构的药物和方法。具体而言，本发明涉及通过PGE2信号通路调节血管重构的药物、方法和制品及其应用。
背景技术
前列腺素类通过环氧酶级联合成并被非类固醇类抗炎药抑制。前列腺素E2(PGE2)是一种重要的细胞生长和调节因子，是花生四烯酸环氧合酶代谢产物，为二十碳不饱和脂肪酸，是前列腺素(PG)的一种。目前己知至少有四种7次跨膜G蛋白偶联受体，即EP1、EP2、EP3和EP4，介导了PGE2的生物学功能。类似的PGI2通过受体IP介导PGI2的生物学功能。
发明概述
本发明提供了一种调节对象中血管重构的方法，包括给予对象有效量的上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质。
本发明还提供了上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质在制备用于调节对象中血管重构的药物中的应用。
本发明还提供了上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质，其用于调节对象中的血管重构。
本发明还提供了用于调节对象中血管重构的制品，其包含上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质。
本发明提供了一种调节对象中血管重构的方法，包括给予对象有效量的PGE2受体的激动剂或者增加内皮细胞PGE2受体表达的药物。
本发明还提供了PGE2受体的激动剂在制备用于调节对象中血管重构的药物中的应用。
本发明还提供了PGE2受体的激动剂，其用于调节对象中的血管重构。
本发明还提供了用于调节对象中血管重构的制品，其包含PGE2受体的激动剂。
附图简述
图1：与PGI受体敲除小鼠(IPKO)相比，同时敲除PGI受体与mPGES (DKO)加重小鼠内皮损伤后血管内膜增生。Bar＝100μm，n＝14IPKO,n＝16DKO。
图2：米索前列醇(Misoprostol，10μM)促进内皮细胞增殖(A)并抑制白细胞与内皮细胞粘附(B)。EP4阻断剂GW627368X(1μM)可反转米索前列醇的作用，即抑制内皮细胞增殖并增加白细胞与内皮细胞粘附。*，p<0.05，**，p<0.01。
图3：小鼠内皮损伤后7天，对照小鼠(Ctl)与内皮EP4特异性敲除小鼠(cKO)的内皮修复程度(A&B)及内膜白细胞浸润(C&D)。cKO减缓内皮修复并增加新生内膜白细胞(多为巨噬细胞，F4/80+)浸润。n＝6(Ctl)，n＝7(cKO)(A&B)。n＝8(C&D)。*,p<0.05。
图4：内皮特异性敲除EP4加重小鼠内皮损伤7天(A,B,C,D)与28天(A,E,F,G)后血管内膜面积及内膜与中膜比例，并不改变中膜厚度。n＝8(A,B,C,D)，n＝14(Ctl),n＝10(cKO)(A,E,F,G)。*,p<0.05。
图5：在有/无炎症因子(IL-1β)刺激的情况下，EP4激动剂AE1-329浓度依赖性地促进野生小鼠内皮细胞增殖(A)，而在存在IL-1β刺激时，EP4阻断剂GW627368X浓度依赖性地抑制野生小鼠内皮细胞增殖(B)。在mPGES敲除的内皮细胞中，AE1-329促进内皮细胞增殖，而GW627368X不能抑制内皮细胞增殖(C)。AE1-329(1μM)处理野生内皮细胞2小时，抑制白细胞与内皮细胞粘附，而GW627368X(1μM)作用2小时，促进白细胞与内皮细胞粘附。
图6：野生小鼠接受股动脉损伤后，每天给予AE1-329(0.3mg/Kg)显著降低血管损伤28天后的新生内膜面积(A&B)、内膜与中膜比例(C)，且不影响中膜厚度(D)。n＝14(载体),n＝10(AE1-329)。*，p<0.05。
图7：IP缺失揭示mPGES-1衍生的PGE2针对导丝损伤诱导的新生内膜形成的保护性作用。DKO(Ptgir -/-Ptges -/-)和同窝IPKO(Ptgir -/-)小鼠在股动脉经受导丝损伤，并在损伤后28天收集血管及定量新生内膜形成。示出苏木精和尹红(H&E)染色的代表性图像(A；Bar＝100μm)。确定了新生内膜面积(B),内膜与中膜比例(C)和中膜厚度(D)(n＝14IPKO,16DKO)。通过HPLC-MS/MS确定了尿中PGE2(E)和PGI2(F)代谢物，如方法中所详述(n＝8).*P<0.05；Student’s unpaired t-test。
图8：IP缺陷小鼠中缺失mPGES-1抑制内皮剥蚀(denudation)损伤后的 再内皮化。术后7天收集DKO和IPKO的损伤的股动脉。H&E染色切片上评估新生内膜形成和白细胞浸润，示出代表性H&E图像(A；Bar＝100μm)。定量新生内膜面积，内膜与中膜比例和中膜厚度(B；n＝14IPKO,16DKO)。通过用IPP软件分析H&E染色组织切片定量新生内膜白细胞数量(C；n＝8IPKO,10DKO)。损伤血管针对F4/80(巨噬细胞标记，红色)和vWF(EC标记，绿色)(D；Bar＝100μm)以及针对α-SMA(SMC标记)(E；Bar＝100μm)免疫染色。DAPI将核染成蓝色。定量vWF-阳性细胞数量以确定再内皮化(F；n＝7)。自IPKO和DKO小鼠的降主动脉分离EC，体外分析其细胞增殖(G)并比较(H)。基于用3％FBS处理48小时前后细胞数差异计算EC增殖率。10μmol/L的PGE类似物米索前列醇促进内皮增殖(I,n＝9来自两次独立实验)并抑制内皮-白细胞粘附(J,n＝9来自三次独立实验)。*P<0.05,**P<0.01；Student’s unpaired t-test.
图9：PGE2通过EP4或EP2/cAMP/PKA信号传导途径在体外促进内皮细胞增殖。小鼠动脉内皮细胞(MAEC)用于研究体外细胞增殖。来自DKO(A)和野生型(WT)小鼠(B)的细胞用PGE2受体激动剂[AE1-329(AE1),EP4激动剂；硫前列酮(Sul),EP1/3激动剂；布他前列腺素(Buta),EP2激动剂]刺激，并显示相对增殖。WT EC用不同浓度的AE1处理，有或无IL-1β(10ng/mL)，并确定增殖(C)。GW627368X(GW,EP4拮抗剂)抑制IL-1β-刺激的MAEC增殖(D)。显示AE1或GW对于用或不用IL-1β(10ng/mL)刺激的Ptges-/-EC的增殖的作用(E)。在IL-1β(10ng/mL)刺激下，1μmol/L浓度的GW(EP4拮抗剂)抑制内皮细胞增殖，但是L-798106(EP3拮抗剂),PF-04418948(EP2拮抗剂)或ONO-8130(EP1拮抗剂)则否(F)。AE1(G)的促增殖作用和GW(H)的抗增殖作用被SQ(SQ22536,腺苷酸环化酶抑制剂；200μmol/L)或H 89(H 89 2HCl,PKA抑制剂；10μmol/L)阻止，但是不被ESI-09(EPAC抑制剂；10μmol/L)阻止。双丁酰环磷酸腺苷(dibutyryl cyclic AMP，Db-cAMP)(I；细胞可透过cAMP类似物,30μmol/L)促进内皮增殖，并且这也被H89 2HCl(PKA抑制剂)钝化。内皮增殖被伊洛前列素(Iloprost，IP激动剂,1μmol/L)刺激且被Cay10441(IP拮抗剂,10μmol/L)抑制(J)。白细胞与EC的粘附被EP4激动剂AE1-329抑制，并被EP4拮抗剂GW促进(K)。Db-cAMP处理减少白细胞粘附EC(L)。所有结果来自至少三组独立数据。*P<0.05.**P<0.01；One-way ANOVA用于使用Bonferroni's(A,G-K), Dunnett's(B)或Turkey’s(C-F)post-test的数据比较。L中使用Student’s unpaired t-test。
图10：诱导缺失内皮EP4促进新生内膜形成。通过他莫西芬处理在小鼠中诱导内皮EP4基因的后天缺失(A)。用Western印迹检测分离自cKO和Ctl小鼠的原代EC中EP4的表达(B)，并通过免疫荧光染色确定其在股动脉中的表达(C；Bar＝100μm)。显示代表性图像(EP4,绿色；vWF,红色)。术后28天收集损伤的血管用H&E染色，示出代表性图像(D；Bar＝100μm)。定量新生内膜面积(E),内膜与中膜的比例(F)和中膜厚度(G)。n＝14Ctl,10cKO.*P<0.05.**P<0.01；Student’s unpaired t-test.
图11：诱导缺失内皮EP4损害再内皮化。导丝损伤后7天收集对照额cKO小鼠的损伤的股动脉内皮细胞针对vWF免疫染色(绿色)并定量血管覆盖(再内皮化)(分别示于A&B；n＝7Ctl,6cKO)。新生内膜形成和白细胞浸润用H&E染色评估。定量新生内膜白细胞的数量(C；n＝8Ctl,9cKO)。示出代表性H&E图像(D；Bar＝100μm)。统计学定量新生内膜面积、内膜与中膜比例和中膜厚度(E；n＝8)。DAPI将核染成蓝色。*P<0.05；Student’s unpaired t-test.
图12：药理学激活EP4促进内皮修复并针对新生内膜形成具有保护作用。血管损伤后，C57BL/6小鼠i.p.注射载体(Veh)或AE1-329(AE1,EP4选择性激动剂)，剂量0.3mg/kg/day，28天(n＝12)。损伤的血管用H&E染色，示出代表性图像(A；A1,A2)。定量新生内膜面积、内膜与中膜比例和中膜厚度(B)。血管损伤后，另一批次小鼠通过i.p.注射载体(Veh)或米索前列醇(Miso,PGE类似物)(剂量100μg/Kg,一天三次，共7天(n＝7Veh,5Miso))处理。损伤的血管用H&E染色，示出代表性图像(A；A3,A4)。定量新生内膜面积、内膜与中膜比例和中膜厚度(C)。通过vWF的免疫染色检查再内皮化并定量，如前详述(D&E)。*P<0.05,**P<0.01；Student’s unpaired t-test.
图13：EP4信号传导参与人内皮细胞的增殖。人微血管内皮细胞用1μmol/LPGE2受体的激动剂(A)或拮抗剂(B)处理，并确定相对增殖。激动剂:AE1-329(EP4),硫前列酮(EP1/3)和布他前列腺素(EP2)。拮抗剂:GW627368X(EP4),L-798106(EP3),PF-04418948(EP2)和ONO-8130(EP1)。两种结果来自三组独立数据。*P<0.05,**P<0.01；One-way ANOVA with Dunnett's post-test用于数据比较。
图14：损伤后7天定量IP KO和DKO小鼠的血管中F4/80阳性区域。n＝4IPKO,5DKO；*P<0.05；Student’s unpaired t-test.
图15：确定分离自IPKO和DKO小鼠的动脉平滑肌细胞的增殖能力。平滑肌细胞(SMC)分离自IPKO和DKO小鼠的降主动脉，并体外分析其细胞增殖(A)和进行比较(B)。n＝8孔，来自2个独立实验。
图16：AE1-329(A),伊洛前列素(B),布他前列腺素(C)和硫前列酮(D)对内皮增殖的浓度应答。n＝9孔，来自3个独立实验；*p<0.05,**p<0.01,One-way ANOVA with Turkey’s post tests.
图17：通过HPLC-MS/MS方法确定培养的小鼠动脉内皮细胞(MAEC)的上清PGE2水平。MAEC用载体(对照)或IL-1β(10ng/mL)处理12小时，示出PGE2水平(A)。与野生型(WT)细胞相比，mPGES-1缺陷(mPGES-1KO)MAEC中PGE2的上清水平显著降低(B)。n＝4,**P<0.01；Student’s unpaired t-test.
图18：佛司可林(Forskolin)(腺苷酸环化酶激活剂,3μmol/L)促进内皮增殖，被H89 2HCl(PKA抑制剂)钝化。n＝7；**P<0.01；One-way ANOVA with Bonferroni's post-test.
图19：在PKA抑制情况下，AE1-329,GW627368X和db-cAMP不影响EC增殖。当MAEC用PKI(PKA抑制剂,10μmol/L)处理时，EP4激动剂(AE1-329,简写为AE1,1μmol/L),EP4拮抗剂(GW627368X,简写为GW,1μmol/L),或db-cAMP(细胞可透过cAMP类似物,30μmol/L)不影响细胞增殖。每个结果来自三组独立数据。
图20：如血管损伤后7天所检查，施用米索前列醇(100μg/Kg,i.p.,一日三次)降低了浸润内膜的白细胞数。**P<0.01；Student’s unpaired t-test.
图21：血管损伤后，佛司可林抑制血管重构。图21A示出损伤后7天，对照(0mg/Kg/day FSK)、FSK1(2mg/Kg/day FSK)和FSK2(4mg/Kg/day FSK)处理组的新生内膜面积、内膜与中膜比例和中膜面积。图21B示出损伤后28天，对照(载体(vehicle))和佛司可林(2mg/Kg/day)组的新生内膜面积、内膜与中膜比例、中膜面积和体重。*p<0.05,**p<0.01
图22：血管的损伤应答中mPGES-1衍生的PGE2的机制的图示说明。敲除mPGES-1降低PGE2并增加PGI2。IP缺陷小鼠中，发现mPGES-1衍生的PGE2的保护性作用。PGE2通过内皮细胞上的EP4受体刺激内皮增殖， 抑制白细胞粘附内皮细胞，并保护受伤的血管不发生新生内膜增生。箭头指示刺激作用，而平端线指示抑制作用。
图23：FSK减少了动脉粥样硬化斑块形成。图23A示出胸主动脉的油红O染色，图23B示出心脏的油红O染色，其中NS＝对照(溶剂)，FSK＝2剂FSK(共4mg/Kg/day)，lesion area＝动脉粥样硬化斑块区域，lesion area ratio＝动脉粥样硬化斑块区域比例。*p<0.05
N.S.，无显著差异
发明详述
本发明人首次发现了前列腺素E2(PGE2)信号通路对于血管重构具有调节作用。本发明人发现上调PGE2信号通路，例如使用PGE2受体激动剂(例如PGE2类似物米索前列醇和PGE2受体激动剂AE1-329)，能够促进内皮细胞增殖，改善损伤血管的新生内膜形成，抑制白细胞与内皮细胞粘附，抑制血管重构。
除非另有说明，所有技术和科学术语都具有本领域技术人员常见的含义。所有专利，专利申请，公开出版物，GenBank序列，网站以及其他公开材料除非另有说明包含在此作为参考。如果本发明的术语有多种定义，以本文为准。
在一个方面，本发明提供了一种调节例如抑制对象中血管重构的方法，包括给予对象有效量的上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质、和/或增加PGE2或其功能性片段和/或血管内皮细胞PGE2受体EP4和/或EP2或其功能性片段的物质。
本文中，上调PGE2受体EP4或EP2信号通路信号传导活性的物质是指与不用所述物质处理相比，在相同条件下，用所述物质处理的细胞中PGE2受体EP4或EP2信号通路的活性升高，导致例如增加的血管内皮细胞增殖、和/或降低的白细胞与血管内皮细胞粘附等。
在一个实施方案中，所述上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质包括但不限于PGE2及其功能性片段、PEG2受体EP4或EP2及其功能性片段、EP4或EP2受体激动剂、降解或下调PGE2和/或EP4或EP2受体的分子例如降解酶的抑制剂，例如PGE2降解酶(例如15-PDGH)的抑制剂(例如SW033291)；PGE2的功能衍生物和功能类似物，例如米 索前列醇；受体EP4或EP2激动剂，例如布他前列腺素、AE1-329或KMN-80；以及上调cAMP-PKA信号通路信号传导活性的物质，例如cAMP类似物如双丁酰环磷酸腺苷、cAMP激动剂如腺苷酸环化酶(AC)激活物，例如佛司可林，PGI2受体激动剂，如伊洛前列腺素。
本文中，所述上调cAMP-PKA信号通路信号传导活性的物质是指与不用所述物质处理相比，在相同条件下，用所述物质处理的细胞中cAMP-PKA信号通路的活性升高。所述上调cAMP-PKA信号通路信号传导活性的物质包括但不限于cAMP、cAMP类似物、cAMP合成酶(例如腺苷酸环化酶(AC))的激动剂、cAMP降解酶的抑制剂、PKA的激动剂、PGI2受体激动剂等。
本文中，降解或下调PGE2和/或EP4或EP2受体的分子是指，与不存在所述分子相比，所述分子使得PGE2或其EP4或EP2受体降解从而丧失部分或全部活性、或者使得PGE2或其EP4或EP2受体的活性和/或水平降低，使得PGE2与其受体EP4或EP2的结合降低。降解或下调PGE2和/或EP4或EP2受体的分子的抑制剂阻止所述分子降解或下调PGE2和/或EP4或EP2受体，在相同条件下，使用抑制剂与不使用抑制剂相比，PGE2与其受体EP4或EP2的结合升高。
在一个实施方案中，所述能够增加PGE2或其功能性片段和/或血管内皮细胞PGE2受体EP4或EP2或其功能性片段表达的物质包括能够表达PGE2或其功能性片段和/或PGE2受体EP4或EP2或其功能性片段的表达或基因治疗载体。在一个实施方案中，所述表达或基因治疗载体可包含与启动子可操纵地连接的编码PGE2或其功能性片段和/或PGE2受体EP4或EP2或其功能性片段的核苷酸序列。
在一个方面，本发明提供了一种调节对象中血管重构的方法，包括给予对象有效量的PGE2受体的激动剂或者增加血管内皮细胞PGE2受体表达的物质。在一个实施方案中，所述方法抑制对象中的血管重构。
如本文所用，“对象”或“个体”或“患者”在本文可互换使用，是指哺乳动物，优选灵长类动物，更优选人。
在一个实施方案中，所述“对象”指非人哺乳动物，例如非人灵长类动物、啮齿类动物等，例如牛、马、山羊、绵羊、大鼠、小鼠、兔、狗。
在本发明中，术语“血管重构”是指血管为适应内外环境变化而发生 的结构和功能改变，包括血管壁细胞的增生、肥大、凋亡、细胞迁移、细胞外基质的产生及降解等细胞生物学变化。血管重构既是动脉粥样硬化和高血压等血管相关疾病恶化的重要病理基础或也是此类疾病发生发展的病因。
在一个实施方案中，本发明所述血管重构是与血管相关疾病或状况相关的血管重构。在本发明中，术语“与血管相关疾病或状况相关的血管重构”是指由于血管重构导致血管相关疾病或状况，或者由于血管相关疾病或状况导致血管重构。
在一个实施方案中，本发明所述血管重构是与血管内皮损伤性疾病或状况相关的血管重构。
在一个实施方案中，本发明所述所述血管相关疾病或状况选自血管壁损伤(如介入支架导致的物理性损伤、动脉粥样硬化导致的血管损伤、高血脂导致的血管损伤、高血压导致的血管损伤、糖尿病造成的血管损伤、自身免疫病造成的血管损伤)、损伤后血管狭窄、损伤后血流功能障碍、血栓、PCI和Bypass术后血管再狭窄、冠心病、心肌缺血、心肌梗死、心梗后心力衰竭、心梗后心律失常、动脉粥样硬化、脑梗及其任意组合。
如本文所用，“有效量”或“治疗有效量”指施用于对象之后至少足以产生疗效的物质、化合物、材料、药物或包含化合物的组合物的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所需的量。对患者施用本发明组合物的实际剂量可根据以下身体和生理因素来确定：体重、性别、症状严重程度、所治疗疾病的类型、先前或当前的治疗干预、患者的未知病因疾病、施用时间、具体化合物的排泄率以及施用途径。在任何情况下，将由负责施用的医务人员确定组合物中活性成分的浓度以及用于个体对象的合适剂量。
在本发明中，术语“PGE2受体”是指细胞表面上存在的、PGE2与其相互作用并产生相应生物学作用的分子。已知PGE2受体包括EP1、EP2、EP3和EP4。在一个实施方案中，本发明所述PGE2受体是EP2和/或EP4。
“激动剂”在本文是指模拟生物学活性、优选一种多肽、受体或其配体的生物学活性的任何分子。本文所述PGE2受体激动剂包括能够模拟和/或增加PGE2与其受体相互作用产生的相应生物学效果的分子。例如，PGE2受体激动剂包括但不限于PGE2受体的天然配体及其功能性片段、肽模拟 物、激动性抗体或抗体片段、增加PGE2表达或活性的分子、增加PGE2受体表达或其激活的分子。在本文中，所述PGE2受体激动剂包括PGE2及其衍生物以及PGE2受体及其衍生物。
本文所述PGE2受体激动剂也包括能够增加或增强细胞中PGE2与其受体例如EP4相互作用后产生的相应生物学作用或生物学效果的物质，例如增加或增强PGE2-EP4下游信号通路活性的物质。
在PGE2及其受体的上下文中，PGE2与其受体相互作用产生的“生物学作用”和“生物学效果”是指PGE2或其功能类似物与其受体例如EP4或其功能片段相互作用例如结合后对于细胞生长状况例如内皮细胞增殖、白细胞与内皮细胞粘附等产生的作用或效果，包括例如促进或抑制生长或增殖速度、增加或减少细胞数量、升高或降低细胞存活等。本文中，PGE2受体激动剂例如EP4受体激动剂涵盖了能够促进内皮细胞增殖、抑制白细胞与内皮细胞粘附的任何物质和分子。
在一个实施方案中，PGE2受体的激动剂包括PGE2或其功能性片段；PGE2的功能衍生物和功能类似物，例如米索前列醇；PGE2降解酶(例如15-羟基前列腺素脱氢酶(15-PDGH))的抑制剂，例如SW033291；受体EP1的激动剂，例如ONO-DI-004；受体EP2的激动剂，例如布他前列腺素(Butaprost)；受体EP3的激动剂，例如硫前列酮(Sulprostone)；以及受体EP4的激动剂，例如AE1-329或KMN-80。
在本文中，术语“功能性片段”是指分子的片段，其与所述分子具有或产生相同或相似的生物学功能。例如，PGE2的功能性片段是PGE2的片段，其能够结合PGE2受体并导致与PGE2结合PGE2受体产生的生物学功能相同或相似的生物学功能。
本文中所用的术语“类似物”指的是结构上与一种化合物相似但组成上稍有不同的另一种化合物(比如一个原子由另一元素的原子替代，或存在特定官能团，或一个官能团由另一个官能团替代)。因此，类似物是与对照化合物在功能和外观上相似或相当，但结构或来源不同的化合物。
在本文中，术语“肽模拟物”是指特异性结合PGE2受体的并可用于本发明的任何肽样分子。所述肽模拟物可以使用本领域已知的方法鉴别，例如筛选肽模拟物、肽的文库、DNA或cDNA表达文库、组合化学及特别有用的噬菌体展示文库。这些文库可以筛选本发明所述的PGE2受体的激 动剂，通过将该文库与基本纯化的PGE2多肽、PGE2多肽受体、其片段或结构类似物相接触而进行。
在一个实施方案中，PGE2受体的激动剂包括PGE2或其功能性片段、PGE2的功能衍生物和功能类似物。
本文中，PGE2的功能衍生物是从PGE2多肽产生的、可以结合PGE2受体并产生与PGE2结合相应受体产生的生物学作用相同或相似的生物学作用的分子。PGE2的功能类似物是与PGE2分子结构类似、并且可以结合PGE2受体并产生与PGE2结合相应受体产生的生物学作用相同或相似的生物学作用的分子。PGE2的功能衍生物和功能类似物包括但不限于例如截短的PGE2、修饰的PGE2或其变体等。所述修饰可以是本领域常规用于修饰蛋白以例如增加其稳定性、半衰期、可溶性等的修饰形式，包括但不限于糖基化、磷酸化、白蛋白化、羧基化等。
在一个实施方案中，PGE2受体的激动剂还包括PGE2降解酶的抑制剂。在本文中，PGE2降解酶是指在PGE2代谢途径中降解PGE2的酶。本领域已知15-羟基前列腺素脱氢酶(15-PGDH)是前列腺素生物降解的关键酶。因此，在进一步的实施方案中，本发明PGE2受体的激动剂包括15-PGDH的抑制剂。
本文所用术语“PGE2降解酶的抑制剂”是指能够降低或消除酶降解PGE2的活性的任何物质，包括但不限于拮抗性抗体或抗体片段、干扰RNA分子、双链RNA(dsRNA)、小分子例如SW033291。
在本发明的一个实施方案中，所述抑制剂是选择性或特异性抑制剂。在本发明中，用于抑制剂时，术语“选择性”和“特异性”可互换使用，意指所述抑制剂仅对于所述靶具有抑制作用，或者对于所述靶的抑制作用相对于对其它化合物或分子具有更高的抑制作用，例如高至少大约1、2、3、4、5、6、7、8、9、10、20、30、40、50、100、500、1000、10000倍等。
本领域已知的PGE2受体的激动剂、特别是选择性激动剂均可以用于本发明中。受体EP1的激动剂包括例如ONO-DI-004；受体EP2的激动剂包括例如布他前列腺素；受体EP3的激动剂包括例如硫前列酮；以及受体EP4的激动剂包括例如AE1-329及KMN-80。
在一个实施方案中，本发明所述PGE2受体的激动剂是EP2和/或EP4受体的激动剂、特别是选择性激动剂。
在一个实施方案中，本发明所述PGE2受体的激动剂选自PGE2、PGE2类似物例如米索前列醇、布他前列腺素、KMN-80和AE1-329。
在一个实施方案中，PGE2受体的激动剂也涵盖增加PGE2-EP4受体信号通路下游分子(cAMP、PKA)信号传导活性、从而增强相应生物学作用例如细胞生长或增殖的物质。在一个实施方案中，PGE2受体的激动剂包括cAMP-PKA信号通路激动剂，包括例如cAMP类似物如Db-cAMP、cAMP激动剂如腺苷酸环化酶(AC)激活物，例如佛司可林、PGI2受体激动剂如伊洛前列腺素。
在一个实施方案中，AE1-329(PubChem CID：9846782)是式I所示的化合物。
Figure PCTCN2018085035-appb-000001
在一个实施方案中，佛司可林(CAS登录号：66575-29-9)是式II所示的化合物。
Figure PCTCN2018085035-appb-000002
本文中，所述“增加PGE2或其功能性片段和/或血管内皮细胞PGE2 受体EP4和/或EP2或其功能性片段表达的物质”是指给予对象后，对象中PGE2或其功能性片段、或者血管内皮细胞上的PGE2受体或其功能性片段的表达与没有给予该物质相比是增加的。所述物质包括例如但不限于如下文所述的表达或基因治疗载体，其包含与启动子可操纵地连接的编码PGE2或其功能性片段和/或PGE2受体EP4和/或EP2或其功能性片段的核酸序列。
所述“增加血管内皮细胞PGE2受体表达的物质”是指给予对象后，对象中血管内皮细胞上的PGE2受体的表达与没有给予该物质相比是增加的。所述物质包括例如但不限于如下文所述的表达或基因治疗载体，其包含与启动子可操纵地连接的编码PGE2受体例如EP2、EP4或其功能性片段的核酸序列。
在一个实施方案中，本发明所述方法还可以包括给予对象其他药物，例如治疗或预防血管相关疾病或状况的药物。在一个实施方案中，本发明所述方法还可以包括给予对象例如但不限于抗细菌剂、抗真菌剂、抗生素、抗病毒剂、抗凝血剂、抗血栓形成剂、抗高血脂剂、强心药、抗高血压剂、胆碱能药、抗胆碱能药、解痉药、局部麻醉剂、镇痛药、麻醉性拮抗药、抗氧化剂和血管活性剂。
在一个实施方案中，本发明所述方法还可以包括给予对象炎症因子例如IL-1β。
在本发明的调节对象中血管重构的方法中，通过口服、含服、吸入、静脉注射、动脉注射、肌肉注射、皮下注射、腹腔注射或局部施用的方式给对象施用所述PGE2受体的激动剂或者增加血管内皮细胞PGE2受体表达的物质。
在一个实施方案中，本发明所述方法可以局部(例如血管损伤部位)给予所述PGE2受体的激动剂或者增加血管内皮细胞PGE2受体表达的物质。
在一个实施方案中，本发明的方法通过血管内给药或将所述PGE2受体的激动剂或者增加血管内皮细胞PGE2受体表达的物质包含或涂覆在血管支架上或带有球囊的导管的球囊上而实现所述局部施用。
在一个方面，本发明提供了上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质和/或能够增加PGE2或其功能性片段和/或血管内皮细胞 PGE2受体EP4和/或EP2或其功能性片段表达的物质在制备用于抑制对象中血管重构的药物中的应用。
在一个方面，本发明提供了上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质和/或能够增加PGE2或其功能性片段和/或血管内皮细胞PGE2受体EP4和/或EP2或其功能性片段表达的物质，其用于抑制对象中血管重构或作为抑制对象中血管重构的药物。
在一个方面，本发明还提供了本发明所述PGE2受体的激动剂在制备用于调节对象中血管重构的药物中的应用。在一个实施方案中，所述药物抑制对象中的血管重构。
在一个实施方案中，所述PGE2受体的激动剂包括PGE2或其功能性片段；PGE2的功能衍生物和功能类似物，例如米索前列醇；PGE2降解酶(例如15-PDGH)的抑制剂，例如SW033291；受体EP1的激动剂，例如ONO-DI-004；受体EP2的激动剂，例如布他前列腺素；受体EP3的激动剂，例如硫前列酮；以及受体EP4的激动剂，例如AE1-329或KMN-80。
在一个实施方案中，PGE2受体的激动剂也涵盖增加PGE2-EP4受体信号通路下游分子(cAMP、PKA)信号传导活性、从而增强相应生物学作用例如细胞生长或增殖的物质。在一个实施方案中，PGE2受体的激动剂包括cAMP-PKA信号通路激动剂，包括例如cAMP类似物如Db-cAMP、cAMP激动剂如腺苷酸环化酶(AC)激活物，例如佛司可林，PGI2受体激动剂，如伊洛前列腺素。
在一个实施方案中，所述药物还可以包含其他活性物质，例如治疗或预防血管相关疾病或状况的物质。
在一个实施方案中，所述药物例如包括但不限于抗细菌剂、抗真菌剂、抗生素、抗病毒剂、抗凝血剂、抗血栓形成剂、抗高血脂剂、强心药、抗高血压剂、胆碱能药、抗胆碱能药、解痉药、局部麻醉剂、镇痛药、麻醉性拮抗药、抗氧化剂和血管活性剂。
在一个实施方案中，所述药物还可以包含炎症因子例如IL-1β。
所述药物优选除了活性成分例如PGE2受体的激动剂之外至少包含一种药物可接受的载体，还可以任选包括药物可接受的佐剂、缓冲剂、分散剂 等。药物载体可以是适于将多肽、抗体或基因治疗载体等输送给患者的任何相容的非毒性物质。无菌水、醇、脂肪、蜡及惰性固体都可用作载体。
所述药物可以制备为或者在重建后是用于通过口服、含服、吸入、静脉注射、动脉注射、肌肉注射、皮下注射、腹腔注射或局部施用的方式给对象施用的形式。
本发明所述药物可以用于治疗或预防血管壁损伤(如介入支架导致的物理性损伤、动脉粥样硬化导致的血管损伤、高血脂导致的血管损伤、高血压导致的血管损伤、糖尿病造成的血管损伤、自身免疫病造成的血管损伤)、损伤后血管狭窄、损伤后血流功能障碍、血栓、PCI和Bypass术后血管再狭窄、冠心病、心肌缺血、心肌梗死、心梗后心力衰竭、心梗后心律失常、动脉粥样硬化、脑梗及其任意组合。
在一个方面，本发明还提供了用于调节对象中血管重构的制品，其包含或涂覆有本发明所述的PGE2受体的激动剂或能够表达PGE2受体的激动剂、和/或PGE2受体或其功能性片段的核酸的表达或基因载体。在一个实施方案中，所述制品抑制对象中的血管重构。
在一个实施方案中，所述PGE2受体的激动剂包括PGE2或其功能性片段；PGE2的功能衍生物和功能类似物，例如米索前列醇；PGE2降解酶的抑制剂，例如15-PDGH的抑制剂，例如SW033291；受体EP1的激动剂，例如ONO-DI-004；受体EP2的激动剂，例如布他前列腺素；受体EP3的激动剂，例如硫前列酮；以及受体EP4的激动剂，例如AE1-329或KMN-80。
在一个实施方案中，PGE2受体的激动剂也涵盖增加PGE2-EP4受体信号通路下游分子(cAMP、PKA)信号传导活性、从而增强相应生物学作用例如细胞生长或增殖的物质。在一个实施方案中，PGE2受体的激动剂包括cAMP-PKA信号通路激动剂，包括例如cAMP类似物如Db-cAMP、cAMP激动剂如腺苷酸环化酶(AC)激活物，例如佛司可林，PGI2受体激动剂，如伊洛前列腺素。
在一个实施方案中，所述调节对象中血管重构的制品包括血管支架或带有球囊的导管，其包含或涂覆有本发明所述的PGE2受体的激动剂或包含能够表达PGE2受体的激动剂、和/或PGE2受体或其功能性片段的核酸的表达或基因载体。
在一个实施方案中，本发明所述PGE2受体是EP2和/或EP4。
在一个实施方案中，所述PGE2受体的激动剂是EP2和/或EP4受体的激动剂，例如选自PGE2、PGE2类似物例如米索前列醇、布他前列腺素、KMN-80和AE1-329。
在一个实施方案中，本发明所述制品包含可以缓释形式释放的PGE2受体的激动剂，例如PGE2多肽或其功能性片段、PGE2类似物如米索前列醇、布他前列腺素、KMN-80和/或AE1-329。
本发明的血管支架或带有球囊的导管用于治疗或预防对象中选自如下的血管相关疾病或状况：血管壁损伤(如介入支架导致的物理性损伤、动脉粥样硬化导致的血管损伤、高血脂导致的血管损伤、高血压导致的血管损伤、糖尿病造成的血管损伤、自身免疫病造成的血管损伤)、损伤后血管狭窄、损伤后血流功能障碍、血栓、PCI和Bypass术后血管再狭窄、冠心病、心肌缺血、心肌梗死、心梗后心力衰竭、心梗后心律失常、动脉粥样硬化、脑梗及其任意组合。
在一个方面，本发明还提供了如下的表达或基因治疗载体(例如病毒载体，如腺病毒载体、腺伴随病毒载体等)，其中核苷酸序列在能驱动核苷酸序列在内皮细胞优选在血管内皮细胞中表达的启动子的控制下：
(a)一种表达或基因治疗载体，其中编码PGE2多肽或其功能性片段的核苷酸序列可操纵地与启动子连接；
(b)一种表达或基因治疗载体，其中编码PGE2受体例如EP1、EP2、EP3、EP4、特别是EP2和/或EP4、或其功能性片段的核苷酸序列与启动子可操纵地连接；
(c)一种表达或基因治疗载体，其中编码PGE2受体例如EP1、EP2、EP3、EP4、特别是EP2和/或EP4的激动剂的核苷酸序列与启动子可操纵地连接。
基因治疗载体可任选地包含编码另外蛋白质的一或多个另外的核苷酸序列。所述另外蛋白质可以是另一治疗性分子、或者(可选择的)标记蛋白，其使得可以鉴别、选择和/或筛选含有表达构建体的细胞，这些标记可参见Sambrook和Russel(2001)“Molecular Cloning：A Laboratory Manual(3rd edition)”，Cold Spring Harbor Laboratory，Cold Spring Harbor Laboratory Press，New York提供。
在一个实施方案中，所述制品例如包括但不限于抗细菌剂、抗真菌剂、 抗生素、抗病毒剂、抗凝血剂、抗血栓形成剂、抗高血脂剂、强心药、抗高血压剂、胆碱能药、抗胆碱能药、解痉药、局部麻醉剂、镇痛药、麻醉性拮抗药、抗氧化剂和血管活性剂。
在一个实施方案中，所述制品可以包括炎症因子例如IL-1β。
如本文所用，范围和量可以表示为“大约”特定数值或范围。大约也包括精确量。因此“大约5％”意味着“大约5％”以及“5％”。
如本文所用，“任选存在的”或“任选地”意味着随后描述的事件或情况发生或不发生，以及所述描述包括所述事件或情况发生的情况和其不发生的情况。例如，任选存在的药物可接受的载体意味着包括或不包括所述药物可接受的载体。
实施例
以下实施例意图说明本发明的多种实施方案。因此，讨论的具体实施方案不应当理解为限制本发明的范围。本领域技术人员会清楚可以进行各种等价、改变和修改而不背离本发明的范围，并且应当理解这类等价实施方案包括在本文中。此外，本公开中引用的所有参考文献均以其全文加入本文参考，就如在本文中完整示出一样。
实施例1：小鼠模型
1.敲除前列环素(PGI)受体(IPKO)
PGI受体(IP)敲除小鼠的制备见文献(Nature.1997 Aug 14；388(6643):678-82)。使用C57BL/6小鼠，获得破坏IP编码基因的胚胎干细胞，注入孕鼠囊胚腔，获得嵌合体小鼠。用获得的小鼠与C57BL/6小鼠回交，获得IP +/-小鼠，通过IP +/-小鼠自交获得IP敲除(IP -/-)鼠。
2.同时敲除PGI受体与膜结合型前列腺素E合成酶(mPGES)(DKO)
DKO小鼠(IP -/-mPGES -/-)由IP敲除C57BL/6小鼠(IP -/-mPGES +/-)与mPGES敲除C57BL/6小鼠(mPGES -/-)杂交得到。实验用mPGES -/-C57BL/6小鼠及其对照鼠源于mPGES +/-C57BL/6小鼠自交(Circulation.2011 Feb 15；123(6):631-9)。
3.内皮特异性敲除受体EP4
使用他莫西芬-CreERT2策略构建内皮EP4缺失的小鼠。简言之，如前所述(Circulation.2017 Mar 28；135(13):1253-1264)，用EP4基因两侧含有 loxP-位点的C57BL/6小鼠(EP4f/f)与由Ralf Adams提供的Cdh5-启动子驱动的CreERT2(Cdh5(PAC)-CreERT2+)C57BL/6小鼠(Nature.2010；465:483-486)杂交。产生的后代EP4f/f Cdh5-CreERT2+雄鼠与EP4f/f Cdh5-CreERT2-雌鼠互交产生可实施内皮EP4条件性敲除的动物(EP4f/f Cdh5-CreERT2+，简称cKO)和同窝对照(EP4f/fCdh5-CreERT2-，简称Ctl)。为了诱导Cre酶介导的EP4缺失，给实验小鼠和同窝对照腹腔内注射他莫西芬(Alfa Aesar,Heysham,England)(37.5mg/ml溶于向日葵种子油中)，剂量为150mg/kg体重每天，连续三天。然后，让小鼠休息三天，之后再进行另外三天注射。
C57BL/6小鼠购自中国食品药品检定研究院并用于评估在EP4激动剂AE1-329对血管重构的影响。
所有动物方案均获中国国家心血管疾病中心，阜外医院，实验动物中心，机构动物护理和使用委员会的批准。
实施例2：血管损伤模型
使用实施例1制备的IPKO和DKO小鼠进行血管损伤模型实验。
小鼠股动脉内膜损伤模型参照文献(Circulation.2011 Feb 15；123(6):631-9)描述的方法进行。首先用戊巴比妥麻醉小鼠，剂量90mg/Kg。然后在严格无菌的条件下，用钝性分离的方法暴露其左侧股动脉。将股直肌和股内肌之间的一个小的动脉分支单独分离出来，在此分支上进行横向动脉切开术，把一根可弯曲的血管成形导丝(直径0.35mm)顺着动脉的方向插入股动脉，插入深度超过5mm。留置导丝3分钟来损伤和扩张股动脉。通过去除股动脉近端和远端的逢合线恢复股动脉的血流，然后用5号的缝合丝线关闭皮肤切口。
损伤28天后，再次麻醉动物，然后在恒定100mmHg压力下依次用0.9％的氯化钠溶液和4％的缓冲福尔马林(pH7.0)进行灌注。收集左侧和右侧股动脉。每根股动脉包埋入石蜡中，切片取自股动脉分支处，厚度为4.5μm，间隔30μm，向近心端切400μm。切取部分进行常规的苏木精和伊红(HE)染色。与其临近的部分进行生物标志物的免疫荧光检测。取自损伤和非损伤的动脉的10个横切部分，经CCD成像后，用Image Pro图像分析软件(Media Cybernetis,Silver Spring MD)进行数字化，并测定管腔面积、内弹力膜内侧面积和外弹力膜内侧面积。动脉狭窄百分比计作内膜面积和内弹力 膜内侧面积的比率。
结果如图1所示。与PGI受体敲除小鼠(IPKO)相比，同时敲除PGI受体与mPGES(DKO)加重小鼠内皮损伤后血管内膜增生。在IPKO小鼠中缺失mPGES明显抑制再内皮化过程，增加白细胞浸润新生内膜，并加重血管内膜面积及内膜与中膜比例，并不影响中膜面积。
实施例3：PGE2受体调节剂对血管重构的作用
小鼠主动脉内皮细胞原代培养
采用贴块儿培养，培养内皮细胞(Cell metabolism.2011；13:592-600)。脱颈处死6-8周C57BL/6小鼠，浸酒精做杀菌处理。在生物安全柜中解剖小鼠，取胸主动脉。体视镜下，轻轻分离干静胸主动脉周围脂肪及结缔组织。纵向剖开血管，将血管剪成约1.5mm长的小段儿，内皮侧向下贴附于培养皿底部。将培养皿置于培养箱中5-10分钟，待血管块儿周转残留液体蒸发后，取出培养皿，并向皿中加入内皮细胞培养基(DMEM培养基，具有20％胎牛血清、1％双抗(青霉素10000U/mL；链霉素10000μg/mL)、100μg/ml肝素、100μg/ml内皮细胞生长因子(ECGS)。重新置于培养箱中培养。3-4天后，待爬出细胞后，进行传代培养，并用于细胞实验研究。
细胞增殖检测
使用细胞计数试剂盒-8(CCK-8；上海翊圣生物科技有限公司(Yeasen,Shanghai,China))测量细胞生长。简言之，将细胞种植在96孔平底平板中。细胞完全附着在底部后，将细胞在含有3％FBS、没有ECGS的培养基中饥饿6-8小时。然后，将培养基更换为培养基-CCK-8混合物(体积10:1)。4小时后，在450nm测量吸收作为基线。然后，将细胞与含有药物的3％FBS、没有ECGS的培养基孵育48小时。最后，将培养基再次更换为培养基-CCK-8混合物。4小时后，在450nm测量吸收显示细胞生长。所用药物浓度如下：10ng/mL IL-1β(生工生物工程(上海)股份有限公司，中国)、1μM AE1-329(日本ONO PHARMACEUTICAL公司)、1μM GW627368X(Selleck,美国)或10μM米索前列醇(中国药品生物制品检定所，中国)。
内皮细胞与白细胞粘附性检测
将内皮细胞种于黑色96孔板中，于实验前换含3％血清内皮细胞培养基低血清培养30小时，于加入白细胞进行孵育前2小时进行药物处理(所 用药物和浓度为：1μM AE1-329、1μM GW627368X和10μM米索前列醇)，备用。
取白细胞流程如下，向小鼠腹腔注射4％Brewer改良巯乙醇酸盐肉汤(B&D，美国)1mL，4-5小时后，用含0.1％BSA的PBS冲洗小鼠腹腔，将冲洗液收集至离心管中，离心。弃上清，使用10％血清的RPMI 1640培养基重悬白细胞，并与处理好的内皮细胞共孵育30分钟。30分钟后，加入含1mg/mL罗丹明6G(生工生物工程(上海)股份有限公司，中国)的RPMI1640培养基1分钟，随后洗板3次，最后进行酶标仪检测(激发光：560nm,发射光：630nm)，定量内皮细胞上粘附白细胞的数目。
结果如图2和图5所示。在体外，用米索前列醇(PGE2类似物)或AE1-329(EP4激动剂)处理内皮细胞均显著促进细胞增殖。而GW627368X(强力EP4拮抗剂)处理显著抑制野生型内皮细胞的增殖，但不抑制mPGES敲除的内皮细胞增殖。米索前列醇或AE1-329处理明显减少了白细胞对内皮细胞的粘附，但是GW627368X处理则增加了白细胞对内皮细胞的粘附。最后，在野生型小鼠中，AE1-329处理显著改善了用导丝损伤28天的股动脉的新生内膜形成。
实施例4：EP4受体对于血管重构的作用
使用实施例1所述的内皮特异性敲除EP4的小鼠进行实施例2所述的血管损伤模型实验。
免疫荧光
来自石蜡包埋的组织的切片(5μm)经脱蜡、再水化，并通过在EDTA抗原修复水(PH 9.0；ZSGB-BIO，北京，中国)中煮沸2分钟进行抗原修复。然后将切片与含0.3％Triton X-100的山羊血清孵育，进行封闭和膜破裂。孵育后，与一抗在4℃孵育过夜，样品与Alexa Fluor-594偶联和/或Alexa Fluor-488偶联的二抗在室温孵育3小时。用含有DAPI封片剂(ZSGB-BIO，北京，中国)染核并封片。使用配有Zen软件的蔡司倒置荧光显微镜(AXI0；Zeiss)或配有20×物镜的激光扫描共聚焦显微镜(SP8；Leica)对切片进行成像。使用Image-Pro Plus 6.0软件(Media Cybernetics,Inc.Rockville,MD,USA)对图像进行分析。所用抗体包括多抗抗-vWF(1:800；Sigma)，单抗抗-F4/80(1:50；BM8；Abcam)。
结果如图3和图4所示。内皮特异性敲除EP4明显抑制再内皮化过程，增加白细胞浸润新生内膜，并加重血管内膜面积及内膜与中膜比例，并不影响中膜面积。内皮特异性敲除EP4抑制小鼠内皮损伤后内皮修复并增加血管新生内膜的白细胞浸润(图3)以及加重小鼠内皮损伤后血管内膜增生(图4)。
实施例5.体外激动或抑制EP4受体
简言之，将如实施例3所述的内皮细胞原代培养细胞种植在96孔平底平板中。细胞完全附着在底部后，将细胞在含有3％FBS、没有ECGS的培养基中饥饿6-8小时。然后，将培养基更换为培养基-CCK-8混合物(体积10:1)。4小时后，在450nm测量吸收作为基线。然后，将细胞与含有药物的3％FBS、没有ECGS的培养基孵育48小时。最后，将培养基再次更换为培养基-CCK-8混合物。4小时后，在450nm测量吸收显示细胞生长。所用药物浓度如下：10ng/mL IL-1β、1μM AE1-329或1μM GW627368X。
结果如图5所示。体外激动或抑制受体EP4影响内皮细胞增殖及内皮细胞与白细胞粘附。(图5)
实施例6.体内激动受体EP4
取8周龄C57小鼠，行股动脉损伤术(方法见前述实施例2)。于术后第二天始，腹腔注射AE1-329(0.3mg/Kg/次，1次/日)，28天后取股动脉，行石蜡切片定量分析(见前述实施例2)。
结果如图6所示。AE1-329抑制小鼠股动脉血管内皮损伤后的血管重构。
实施例7.动物研究
mPGES-1(基因:Ptges)(Proc Natl Acad Sci USA.2003；100：9044-9049)和IP(基因:Ptgir)(Science.2002；296：539-541)缺陷小鼠分别得自Pfizer和University of Pennsylvania的FitzGerald实验室。这两种动物与C57BL/6背景回交10代以上，并通过将Ptgir -/-Ptges -/-和Ptgir -/-Ptges +/-杂交用于产生IP/mPGES-1双敲除(DKO)小鼠和同窝对照(IP KO)。DKO小鼠正常发育，没有明显异常。全面缺失EP4由于动脉导管开放(Nature.1997；390：78-81) 在围产期是致命的。为克服这一缺陷，使用他莫西芬-CreERT2策略(Circulation.2017；135：1253-1264)产生内皮特异性EP4(基因:Ptger4)敲除小鼠。简言之，C57BL/6 Ptger4 flox/flox小鼠(Genesis.2004；40：7-14)与Cdh5-启动子驱动的CreERT2(Cdh5(PAC)-CreERT2 +)小鼠(Nature.2010；465：483-486)(Ralf Adams惠赠)杂交。所得Ptger4 f/f Cdh5-CreERT2 +和Ptger4 f/f Cdh5-CreERT2 -小鼠然后杂交，产生本研究中使用的动物—内皮EP4条件敲除(Ptger4 f/f Cdh5-CreERT2 +:简写为cKO)和同窝对照(Ptger4 f/f Cdh5-CreERT2 -:简写为Ctl)。
为诱导内皮EP4缺失，他莫西芬(37.5mg/ml溶于向日葵种子油中)以150mg/kg/天的剂量腹膜内注射实验小鼠和同窝对照，共6天，其中第三次剂量后中断3天。用于本研究的基因修饰小鼠是性别匹配的。从两种性别集合的数据用于显示每个研究中的基因特异性作用，性别特异性亚组分析在表1和2中提供。来自National Institutes for Food and Drug Control(Beijing,China)的6-8周龄雄性C57BL/6小鼠用于确定AE1-329(ONO Pharmaceutical Co.,Ltd.,Osaka,Japan惠赠)或迷索前列醇(410004；纯度,98.9％；National Institutes for Food and Drug Control,Beijing,China)在血管重构中的作用。所用动物实验方案均根据中国国家心血管疾病中心阜外医院实验动物中心机构动物护理和使用委员会的指南进行。
股动脉损伤模型
使用之前描述的方法(Circulation.2017；135：1253-1264)损伤股动脉。简言之，在麻醉小鼠的一侧制造腹股沟切口。然后小心暴露股直肌和股内肌之间的股动脉和其小分支并通过钝性剥离与伴随神经和血管分离。然后6-0丝线绕近端股动脉打结以在手术期间停止血流。另一6-0丝线置于分支下。然后在分支进行横向动脉切开术，将可弯曲的血管成形导丝(直径0.35mm；Cook Inc.,IN,USA)从分支插入股动脉，向髂动脉的长度不少于5mm。留置导丝3分钟以扩张和剥蚀(denude)动脉。然后，取出导丝，将分支在近端用6-0丝线连接，通过释放用于血流控制的丝线恢复股动脉血流。然后用5-0丝线闭合皮肤切口。
损伤7或28天后，收集动脉，在石蜡中包埋，并用苏木精和伊红(H&E)染色，以确定增生严重性。详细地，以150μm间隔连续获得10-13个动脉横切片，并将具有最严重增生的切片用于比较。为评估再内皮化，损伤后7 天的动脉切片用内皮细胞标记von Wllebrand Factor(vWF)免疫染色，并用与显微镜系统相连的CCD相机照相(AXI0；Zeiss,Oberkochen,Germany)。沿血管内壁的vWF阳性的区段和血管内壁的周长使用Image-Pro Plus 6.0软件(Media Cybernetics,MD,USA)测量。计算vWF阳性区段的总长度与周长的比率以代表再内皮化程度。为评估白细胞浸润/迁移，损伤7天的动脉代表性切片用H&E染色分析。简言之，捕获图像后，内膜白细胞使用Hue(H)Saturation(S)Intensity(I)-基于颜色选择策略用Image-Pro Plus 6.0软件(Media Cybernetics,MD,USA)客观确定。
免疫荧光染色
免疫荧光染色与如前所述(Circulation.2017；135：1253-1264)相同的方案进行。简言之，石蜡切片(5μm)脱蜡、再水化并使用EDTA抗原修复水(PH9.0；ZSGB-BIO,Beijing,China)进行抗原修复。与正常山羊血清在室温保温1小时后，样品与一抗在4℃保温过夜，随后用Alexa Fluor-488-偶联的或Alexa Fluor-594-偶联的二抗在室温染色3小时。然后将切片用含有DAPI的VectaShield培养基对核染色并封片，使用Zeiss显微镜系统(AXI0；Zeiss)或激光散射共聚焦显微镜系统(SP8；Leica)成像。为确定再内皮化，使用Image-Pro Plus 6.0软件(Media Cybernetics,Inc.Rockville,MD,USA)分析图像。用于本研究的一抗和稀释倍数是多克隆抗-EP4抗体(2.5μg/mL；101775；Cayman Chemical,MI,USA)、兔多克隆抗-vWF抗体(1μg/mL；F3520；Sigma,Darmstadt,Germany)、绵羊多克隆抗-vWF抗体(10μg/mL；Ab11713；Abcam,Cambridge,UK)、单克隆抗-α-SMA抗体(5μg/mL；A5228；Sigma,Darmstadt,Germany)和多克隆抗-F4/80抗体(2μg/mL；Ab16911；Abcam,Cambridge,UK)。
Western印迹分析
对于Western印迹分析，细胞在含有蛋白酶抑制剂(4693116001,Roche,Basel,Switzerland)的RIPA缓冲液中裂解。离心(15800g,10min)后，细胞裂解物与上样缓冲液混合，用10％SDS-PAGE分级分离，转移至聚偏二氟乙烯膜。然后膜用初级多克隆EP4抗体(2.5μg/mL；101775；Cayman,Michigan,USA)在4℃过夜探查，随后用山羊抗兔二抗在室温保温1小时。最终，膜与ECL发光液体(P1020；PPLYGEN,Beijing,China)保温，免疫反应条带的信号使用FluorChem System(ProteinSimple,CA,USA)显像。
类前列腺素确定
如前所述(Methods in Enzymology.2007；433：51-72)，PGE 2和PGI 2水平通过液相(L)层析(C)-串联质(M)谱(S)测量其主要的尿代谢产物tetranor-PGEM和2,3-dinor-6-keto-PGF1α确定。
统计分析
使用GraphPad Prism 5软件(GraphPad Software Inc.,San Diego,California,USA)进行统计分析。当仅涉及两组时，使用Student’s two-tailed unpaired t-检验进行比较。使用one-way ANOVA分析比较多个组。对于具有相等方差的数据使用Tukey's或Bonferroni's post hoc检验，而对于具有不等方差的数据使用Dunnett's post hoc检验。使用Bartlett's检验分析数据方差。结果表示为平均值±SEM。p<0.05时差异被认为是统计学显著的。
结果：
小鼠中mPGES-1的缺失增强了PGI 2产生，导致弱化的血管重构(Circulation.2011；123:631-639)。为阐明mPGES-1衍生的PGE 2在血管重构中的作用，将mPGES-1KO与IP KO杂交产生双KO(DKO)小鼠，并以同窝IP KO小鼠作为对照。小鼠经受股动脉导丝损伤(内皮剥蚀)28天。在此IP缺陷背景，mPGES-1缺失增加了新生内膜面积～84％，还增加了内膜与中膜面积的比例，中膜厚度未变(图7A-D)。这揭示mPGES-1衍生的PGE 2在对于损伤的血管应答中的保护性作用。PGE 2的尿代谢产物在DKO小鼠中降低，而PGI 2代谢产物增加(图7E&F)。
手术后7天收集血管用于组织学检查。新生内膜面积以及内膜与中膜的比例在DKO中显著增加，而中膜面积不变(图8A&B)。内膜白细胞，主要是巨噬细胞(F4/80染色阳性)，在DKO中增加(图8C,D和图14)。α-SMA的表达主要在中膜的平滑肌细胞(SMC)中检测到，但极少在新生内膜区域中检测到，与基因型无关(图8E)。当vWF(内皮标记)染色时，在DKO中观测到内皮细胞(EC)数的显著减少(图8D&F)，表明导丝剥蚀损伤后受抑制的再内皮化。
自IP KO和DKO小鼠分离EC，在体外评估它们的增殖能力(图8G)。DKO中EC增殖显著受损(图8H)，表明PGE 2信号通路促进EC增殖。而且，用PGE类似物米索前列醇处理EC在体外促进了EC增殖(图8I)，以及在体外也降低了白细胞对内皮单层的粘附(图8J)。也自IP KO和DKO分离原代 主动脉平滑肌细胞。在这两组中检测到细胞增殖无差异(图15)。
表1 IPKO和DKO中性别信息的数据分析
Figure PCTCN2018085035-appb-000003
注:面积(×10 3μm 2).
表2 EP4对照和EP4cKO中性别信息的数据分析
Figure PCTCN2018085035-appb-000004
注:面积(×10 3μm 2).
实施例8.细胞研究
内皮细胞：小鼠主动脉内皮细胞(MAEC)如前所述分离(Circulation.2017；135：1253-1264；Cell metabolism.2011；13：592-600)。简言之，收集主动脉并切成1-2mm 2切片。主动脉片段通过其管腔面附着于培养皿，然后在含有20％胎牛血清(FBS)和100ug/mL内皮细胞生长补充剂(ECGS)的DMEM培养基中培养5-7天，使得内皮细胞贴壁生长。然后，将内皮细胞传代培养。本研究中使用2-6代的MAEC。在第2代，比较分离自IP KO或DKO的MAEC的内皮细胞增殖。原代人微血管内皮细胞(HMEC)购自 ScienCell(6000；Carlsbad,CA,USA)，并在与用于MAEC培养相同的培养基中培养。
平滑肌细胞：小鼠主动脉平滑肌细胞(MASMC)分离自IP KO和DKO。简言之，分离主动脉，在内表面刮三次，并切成1-2mm 2切片。主动脉片段然后通过其管腔面附着于培养皿，盖上盖玻片，在含有10％FBS的DMEM培养基中培养5-7天。然后传代培养MASMC。比较第2代的MASMC的增殖。
细胞增殖：使用细胞计数试剂盒-8(CCK-8；40203ES60；Yeasen,Shanghai,China)根据厂商说明确定细胞生长，如前所述(Circulation.2017；135：1253-1264)。测定允许多重检测，而没有明显细胞毒性。简言之，细胞种植在96孔平底平板中。细胞附着后，EC在含有3％FBS的培养基中培养6-8小时。之后，培养基更换为3％FBS培养基-CCK-8混合物(体积10:1)。之后，细胞在混合物中培养不超过4小时；确定450nm处的吸收作为基线。然后细胞在具有指示试剂的新鲜3％FBS培养基中再培养48小时。培养基然后替换为培养基-CCK-8混合物，培养继续进行如上述用于确定450nm处吸收的相同时间。两次测量之间吸收的变化用于限定细胞生长。为确定MASMC增殖，在无FBS血清中预先饥饿细胞24小时，然后在含有1％FBS的培养基中培养，分别于1％FBS培养前后用CCK-8检测细胞增殖状态。
用于增殖研究的试剂及其浓度如下：AE1-329(0.1-1μmol/L；ONO Pharmaceutical Co.,Ltd.,Osaka,Japan惠赠),布他前列素(1μmol/L；13740；Cayman Chemical,MI,USA),硫前列酮(1μmol/L；14765；Cayman),GW627368X(0.1-1μmol/L；HY-16963；MedChemExpress,NJ,USA),L-798106(1μmol/L；11129；Cayman),伊洛前列素(1μmol/L；18215；Cayman),Cay10441(10μmol/L；10005186；Cayman),PF-04418948(1μmol/L；S7211；Selleck),ONO-8130(1μmol/L；19118；Cayman),SQ22536(200μmol/L；S8283；Selleck),H89 2HCl(10μmol/L；S1582；Selleck),ESI-09(10μmol/L；19130；Cayman),db-cAMP(3-100μmol/L；D0260；Sigma,Darmstadt,Germany)和米索前列醇(10μmol/L；410004；National Institutes for Food and Drug Control,Beijing,China).
内皮-白细胞粘附研究：对于内皮-白细胞粘附测定，MAEC种植在96-孔平板，在含有3％FBS的DMEM中预先饥饿6-8小时，与测试试剂保温 2小时。自小鼠腹膜收集白细胞。简言之，4％Brewer Modified Thioglycollate Medium(211716；BD Biosciences,NJ,USA)注射小鼠腹膜(1mL/只小鼠)。4-5小时后，腹膜白细胞用0.1％牛血清白蛋白冲洗，离心，重悬于含有10％FBS的1640培养基中。当完成内皮细胞制备，具有指示的药物的培养基替换为含有白细胞的1640培养基(3×10 4/孔)。然后内皮细胞和白细胞共培养30分钟。之后，细胞用含有罗丹明6G(200μg/mL；252433；Sigma,Darmstadt,Germany)的1640培养基冲洗一次，之后用新鲜1640培养基洗涤3次。最终用酶标仪检测荧光信号(激发波长：560nm,发射波长：630nm；Infinite M200,Tecan,Hombrechtikon,Switzerland)。
用于本研究的试剂及其浓度如下：米索前列醇(10μmol/L；410004；National Institutes for Food and Drug Control,Beijing,China),AE1-329(1μmol/L；Gifted by ONO Pharmaceutical Co.,Ltd.,Osaka,Japan),GW627368X(1μmol/L；HY-16963；MedChemExpress,NJ,USA)和db-cAMP(30μmol/L；D0260；Sigma,Darmstadt,Germany)。
结果
1. EP4激活促进内皮细胞增殖和减少内皮-白细胞粘附
然后在体外研究了可能介导PGE 2对于EC增殖的作用的受体。来自DKO小鼠的EC用激动剂(Drug Discov Today.2017；22：57-71；Pharmacol Rev.2011；63：471-538)处理，对于EP1/3(硫前列酮,对于EP3的EC50＝0.42nmol/L,也是EP1的弱激动剂)、EP2(布他前列素,EC50＝32nmol/L)或EP4(AE1-329,EC50＝3.1nmol/L)受体，所有浓度均为1μmol/L。AE1-329促进了DKO EC的增殖，但是硫前列酮或布他前列素则否(图9A)。用野生型EC观测到相似作用(图9B)。进一步测试每种药物在多种浓度对于细胞增殖活性(图16)。AE1-329剂量依赖性促进EC增殖，而布他前列素在较高浓度即10μmol/L显示促增殖作用。AE1-329在存在或不存在PGE 2产生刺激物IL-1β时均增强EC增殖(图9C&图17A)。相反，GW627368X(选择性EP4拮抗剂)在IL-1β刺激下显著抑制EC增殖(图9D)。当使用mPGES-1KO EC时，在PGE 2产生降低时(图17B)，EP4激动的作用保持显著，但是EP4拮抗的作用则否(图9E)。因此，衍生自EC mPGES-1的内源性PGE 2在促进IL-1β刺激的EC的增殖中起活性作用，其可被EP4的拮抗阻断，但不被其它PGE 2受体的拮抗阻断(图9F)。这进一步证实在EC中EP4激活的增殖作 用(图9A&B)。
为阐述EP4下游信号传导，使用SQ22536[腺苷酸环化酶(AC)抑制剂],H 89 2HCl(PKA抑制剂)和ESI-09(EPAC抑制剂)。用SQ22536或H 89 2HCl处理消除了AE1-329促增殖作用(图9G)和GW627368X的抗增殖作用(图9H)，但用ESI-09则否，表明cAMP-PKA轴构成通过EP4激活驱动的增强的EC增殖的作用基础。与这个发现一致，Db-cAMP(细胞可透过cAMP类似物)和福司柯林(强力AC激活剂)均促进EC增殖，这种增殖作用被用H892HCl或PKI的PKA抑制所钝化(图9I和图18&19)。已知PGI 2通过IP受体(Smyth EM,et al.Prostanoids in health and disease.J Lipid Res.2009；50Suppl:S423-428)升高cAMP信号传导。实际上，IP激动剂伊洛前列素(1μmol/L)促进内皮增殖，而IP拮抗剂Cay10441(10μmol/L)抑制这种反应(图9J)，表明PGI 2对于EC的增殖作用。也体外研究了EP4对于内皮-白细胞粘附的作用。EC单层与EP4选择性激动剂AE1-329保温显著抑制了内皮-白细胞粘附，而GW627368X抑制内皮EP4增强了白细胞对于EC的粘附(图9K)。这与cAMP介导的作用一致，因为Db-cAMP(细胞可透过cAMP类似物，30μmol/L)处理类似地减少白细胞粘附(图9L)。
2.内皮限制缺失EP4损害了再内皮化并加重了新生内膜形成
内皮EP4在血管重构中的作用然后在该受体仅在EC中缺陷的小鼠中检查(图10A)，如EC中EP4蛋白表达显著抑制证实(图10B&C)。小鼠经历导丝损伤并在28天后研究。新生内膜面积和内膜与中膜面积的比例在缺少内皮EP4的小鼠中均增加，而中膜面积不变(图10D-G)。血管损伤7天后，在仅在EC中缺少EP4的小鼠中再内皮化被显著抑制(图11A&B)。这与cKO中内膜白细胞数增加和增加的新生内膜形成一致(图11C-E)。
3.EP4激活保护免于导丝损伤诱导的新生内膜形成
全身给予EP4激动剂AE1-329也改善了新生内膜形成，而不影响中膜厚度(图12A&B)。体外促进内皮增殖的米索前列醇(图8I)在血管损伤后7天在体内减弱了新生内膜形成(图12A&C)并促进了再内皮化(图12D&E)。米索前列醇也降低了白细胞浸润(图20)。
5.EP4参与人内皮细胞增殖
通过AE1-329的EP4激活、而不是其它PGE 2受体，促进了人原代内皮细胞的增殖(图13A)。相反，在4种EP的拮抗剂中，仅通过GW627368X 的EP4阻断抑制了EC增殖(图13B)。
实施例9.腺苷酸环化酶激动剂佛司可林(FSK)的作用
为确定FSK对于内皮重构的作用，用金属导丝损伤C57BL/6小鼠的股动脉，并以2mg/Kg的剂量一日一次或两次给予FSK。损伤后，收集血管做切片及H&E染色并比较。
导丝损伤研究
根据如前所述方法，使用金属导丝(0.35mm直径；Cook Inc.,IN,USA)损伤小鼠的股动脉。为研究佛司可林(FSK，S2449,Selleck company,TX,USA)对血管重构的潜在作用，将C57BL/6小鼠随机分为3组：(1)对照组(Ctl)，其中小鼠一日两次腹腔内注射对照溶剂(二甲亚砜(DMSO)于生理盐水中(体积1:100))，(2)一剂FSK组(FSK1)，其中小鼠腹腔内每日注射一次溶剂加上一次FSK(2mg/Kg)，(3)两剂FSK组(FSK2)，其中小鼠腹腔内一日注射两次FSK(2mg/Kg)。
在动脉损伤后，开始给予对照溶剂或FSK，持续7或28天，根据如上所述处理策略进行。为确定FSK对于稳定新生内膜增生的作用，损伤后28天收集动脉。在另一组实验中，在7天收集动脉以研究损伤和FSK导致的早期血管改变。收集的血管均包埋在石蜡中，并依次切片～12层，每两个邻近层间隔200μm。对于每个动脉，用苏木精和尹红(H&E)染色所有层的切片。选择各层中具有最严重新生内膜增生的切片作为代表性切片，用于进一步研究。
定量动脉粥样硬化斑块
对于动脉粥样硬化研究，使用10周龄的雄性基因修饰小鼠(称为Konck in小鼠(Kin))。通过将ApoE基因的外显子2和4之间的序列用含有(1)编码用于SR-BI的干扰RNA的scavenger receptor class1(SR-B1)knockdown元件、(2)编码PDZK1(其可下调SR-BI表达)截短形式的表达盒和(3)AngII表达盒、之后是Gaussia荧光素酶和在C57BL/6小鼠背景中的Tet-on操纵子的序列置换产生这种小鼠。在所得小鼠中，载脂蛋白E(ApoE)基因被敲除，在ApoE启动子具有活性的细胞中SR-BI表达被下调，并且AngII的表达可通过给予多西环素(Dox)条件性增强。
为研究FSK对于动脉粥样硬化斑块形成的作用，将Kin小鼠随机分为 2组，对照组(Ctl)和两剂FSK组(FSK2)。在小鼠开始接受含有21％脂肪和0.2％胆固醇和Dox(1mg/ml)的高脂饮食(HFD)之后立即给予溶剂或FSK。给予溶剂或两剂FSK的形式与之前所述的导丝损伤研究中相同。28天后，通过给予过量戊巴比妥钠(100mg/mL)处死小鼠并用4％多聚甲醛(PFA)通过连续注射入左心室灌注。然后收集心脏和主动脉并在4％PFA中固定。
胸主动脉和主动脉根均用于确定FSK对于Kin小鼠中动脉粥样硬化斑块区域的作用。简言之，小心除去血管周围组织后，切开胸主动脉并用油红O做en face染色。通过心室中间层横切心脏。然后在含有20％蔗糖的溶液中过夜之后，将心脏的上部分包埋在OCT中并在-80℃冷冻。从心室到主动脉的方向切片。当达到主动脉根时，收集间隔40μM的4层冷冻切片。然后将不同层的切片用油红O染色。对于主动脉根，具有最大斑块区域的层用于比较。
根据如下方法用油红O染色主动脉和冷冻切片。简言之，用油红O染色主动脉和切片30分钟，在油红O染色之前和之后立即用80％甲醇两次简单洗涤。使用Zeiss光学显微镜(AXI0；Zeiss,Oberkochen,Germany)拍照，并使用Image-Pro Plus 6.0软件(Media Cybernetics,MD)分析。这些过程均盲法操作。在组织收集和数据采集全程，操作者不了解小鼠组别信息。
结果
如图21所示，给予FSK 7天通过减少新生内膜面积和内膜与中膜比例而显著抑制血管重构。FSK处理未显著影响中膜面积。在另一组实验中，在损伤后第28天收集血管。新生内膜面积趋于减小。内膜与中膜比例显著降低。而中膜面积未改变。
如图23所示，给予佛司可林(FSK)在脂质代谢失调的小鼠中减少了动脉粥样硬化斑块的面积。血管重构是动脉粥样硬化进展的重要病理生理学过程之一。如我们在方法中详述的，使用倾向于动脉粥样硬化和高血压的基因修饰小鼠评估FSK对于动脉粥样硬化的作用。给予FSK，一日两剂，共28天，显著减少了胸主动脉和主动脉根中动脉粥样硬化斑块的区域。

Claims (18)

  1. 一种抑制对象中血管重构的方法，包括给予对象有效量的上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质。
  2. 权利要求1的方法，其中所述上调PGE2受体EP4和/或EP2信号通路的物质选自：PGE2或其功能性片段；PGE2的功能衍生物或功能类似物，例如米索前列醇；PEG2受体EP4或EP2及其功能性片段；降解或下调PGE2和/或EP4或EP2受体的分子例如降解酶的抑制剂，例如PGE2降解酶15-PDGH的抑制剂，例如SW033291；受体EP4或EP2激动剂，例如布他前列腺素、AE1-329或KMN-80；上调cAMP-PKA信号通路信号传导活性的物质，例如cAMP类似物如双丁酰环磷酸腺苷、cAMP激动剂如腺苷酸环化酶(AC)激活物，例如佛司可林，PGI2受体激动剂，如伊洛前列腺素；和能够增加PGE2或其功能性片段和/或内皮细胞PGE2受体EP4和/或EP2或其功能性片段表达的物质。
  3. 权利要求1或2的方法，其中所述上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质选自米索前列醇、布他前列腺素、AE1-329、双丁酰环磷酸腺苷和佛司可林。
  4. 权利要求2的方法，其中所述能够增加PGE2或其功能性片段和/或血管内皮细胞PGE2受体EP4和/或EP2或其功能性片段表达的物质是能够表达PGE2或其功能性片段和/或PGE2受体EP4和/或EP2或其功能性片段的表达或基因治疗载体。
  5. 权利要求4的方法，其中所述表达或基因治疗载体包含与启动子可操纵地连接的编码PGE2或其功能性片段和/或PGE2受体EP4和/或EP2或其功能性片段的核苷酸序列。
  6. 权利要求1-5任一项的方法，其中血管重构是与血管相关疾病或状况相关的血管重构。
  7. 权利要求6的方法，其中所述血管相关疾病或状况选自血管壁损伤(如介入支架导致的物理性损伤、动脉粥样硬化导致的血管损伤、高血脂导致的血管损伤、高血压导致的血管损伤、糖尿病造成的血管损伤、自身 免疫病造成的血管损伤)、损伤后血管狭窄、损伤后血流功能障碍、血栓、PCI和Bypass术后血管再狭窄、冠心病、心肌缺血、心肌梗死、心梗后心力衰竭、心梗后心律失常、动脉粥样硬化、脑梗及其任意组合。
  8. 上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质在制备用于抑制对象中血管重构的药物中的应用。
  9. 权利要求8的应用，其中所述上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质选自PGE2或其功能性片段；PGE2的功能衍生物和功能类似物，例如米索前列醇；PEG2受体EP4和/或EP2及其功能性片段；降解或下调PGE2和/或EP4和/或EP2受体的分子例如降解酶的抑制剂，例如PGE2降解酶15-PDGH的抑制剂，例如SW033291；受体EP4和/或EP2激动剂，例如布他前列腺素、AE1-329或KMN-80；上调cAMP-PKA信号通路信号传导活性的物质，例如cAMP类似物如双丁酰环磷酸腺苷、cAMP激动剂如腺苷酸环化酶(AC)激活物，例如佛司可林，PGI2受体激动剂，如伊洛前列腺素；和能够增加内皮细胞PGE2或其功能性片段和/或PGE2受体EP4或/和EP2或其功能性片段表达的物质。
  10. 权利要求8或9的应用，其中血管重构是与血管相关疾病或状况相关的血管重构，优选所述血管相关疾病或状况选自血管壁损伤(如介入支架导致的物理性损伤、动脉粥样硬化导致的血管损伤、高血脂导致的血管损伤、高血压导致的血管损伤、糖尿病造成的血管损伤、自身免疫病造成的血管损伤)、损伤后血管狭窄、损伤后血流功能障碍、血栓、PCI和Bypass术后血管再狭窄、冠心病、心肌缺血、心肌梗死、心梗后心力衰竭、心梗后心律失常、动脉粥样硬化、脑梗及其任意组合。
  11. 权利要求8-10任一项的应用，其中所述药物用于通过口服、含服、吸入、静脉注射、动脉注射、肌肉注射、皮下注射、腹腔注射或局部施用的方式给对象施用。
  12. 用于抑制对象中血管重构的制品，其能够表达或释放上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质。
  13. 权利要求12的制品，其中所述上调PGE2受体EP4和/或EP2信号通路的物质选自：PGE2或其功能性片段；PGE2的功能衍生物和功能类似 物，例如米索前列醇；PEG2受体EP4和/或EP2及其功能性片段；降解或下调PGE2和/或EP4和/或EP2受体的分子例如降解酶的抑制剂，例如PGE2降解酶15-PDGH的抑制剂，例如SW033291；受体EP4和/或EP2激动剂，例如布他前列腺素、AE1-329或KMN-80；上调cAMP-PKA信号通路信号传导活性的物质，例如cAMP类似物如双丁酰环磷酸腺苷、cAMP激动剂如腺苷酸环化酶(AC)激活物，例如佛司可林，PGI2受体激动剂，如伊洛前列腺素；以及PGE2或其功能性片段和/或PGE2受体EP4和/或EP2或其功能性片段的表达或基因治疗载体。
  14. 权利要求12或13的制品，其中所述制品包含或涂覆有所述上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质。
  15. 权利要求14的制品，其中所述表达或基因治疗载体选自：
    (a)一种表达或基因治疗载体，其中编码PGE2多肽或其功能性片段的核苷酸序列可操纵地与启动子连接；
    (b)一种表达或基因治疗载体，其中编码PGE2受体EP4和/或EP2或其功能性片段的核苷酸序列与启动子可操纵地连接；和
    (c)一种表达或基因治疗载体，其中编码PGE2受体EP4和/或EP2的激动剂的核苷酸序列与启动子可操纵地连接。
  16. 权利要求12-15任一项的制品，其是血管支架或带有球囊的导管。
  17. 权利要求12-16任一项的制品，其中所述上调PGE2受体EP4和/或EP2信号通路信号传导活性的物质选自米索前列醇、布他前列腺素、AE1-329、双丁酰环磷酸腺苷和佛司可林。
  18. 权利要求12-17任一项的制品，其中所述制品用于调节与选自血管壁损伤(如介入支架导致的物理性损伤、动脉粥样硬化导致的血管损伤、高血脂导致的血管损伤、高血压导致的血管损伤、糖尿病造成的血管损伤、自身免疫病造成的血管损伤)、损伤后血管狭窄、损伤后血流功能障碍、血栓、PCI和Bypass术后血管再狭窄、冠心病、心肌缺血、心肌梗死、心梗后心力衰竭、心梗后心律失常、动脉粥样硬化、脑梗及其任意组合的血管相关疾病或状况相关的血管重构。
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