WO2022127654A1 - 一种抗纤维化药物组合物及其应用 - Google Patents

一种抗纤维化药物组合物及其应用 Download PDF

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WO2022127654A1
WO2022127654A1 PCT/CN2021/136240 CN2021136240W WO2022127654A1 WO 2022127654 A1 WO2022127654 A1 WO 2022127654A1 CN 2021136240 W CN2021136240 W CN 2021136240W WO 2022127654 A1 WO2022127654 A1 WO 2022127654A1
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fibrosis
mice
nrp1
epcr
hif2α
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English (en)
French (fr)
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丁楅森
曹中炜
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四川大学华西第二医院
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Priority to EP21905583.7A priority Critical patent/EP4282424A1/en
Publication of WO2022127654A1 publication Critical patent/WO2022127654A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • 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
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biomedicine, in particular to an anti-fibrosis pharmaceutical composition and application thereof.
  • Fibrosis can occur in multiple organs and is the ultimate pathological outcome of many common chronic inflammatory and immune-mediated metabolic diseases and the main cause of morbidity and mortality in these diseases.
  • a variety of noxious stimuli including toxins, infectious pathogens, autoimmune responses, and mechanical stress, can induce fibrotic cellular responses. Fibrosis affects all tissues in the body and, if left unchecked, can lead to organ failure and death.
  • Fibrosis is a repair response to protect the relative integrity of tissues and organs after tissue damage.
  • myofibroblasts derived from multiple sources including resident fibroblasts, mesenchymal cells, circulating fibroblasts, and transdifferentiation of other cell types
  • myofibroblasts derived from multiple sources can initiate wounds by remodeling the extracellular milieu Healing responses to restore tissue integrity and promote parenchymal cell replacement.
  • this pro-fibrotic program is turned off when the tissue heals.
  • ECM extracellular matrix
  • Mammalian organs such as liver, lung and kidney can undergo compensatory regeneration to replace damaged tissue.
  • the regenerative capacity of most mammals is limited by the aging process. Damaged tissue in aging organs is often replaced by excessive scarring, leading to fibrosis and organ dysfunction.
  • the regeneration capacity of aging organs appears to be similarly inhibited, and these aging organs are all prone to fibrosis after injury.
  • TGF- ⁇ transforming growth factor- ⁇
  • TGF- ⁇ transforming growth factor- ⁇
  • Integrins are thought to enhance signaling from soluble pro-fibrotic growth factors such as TGF- ⁇ 1. Therefore, targeting integrins and targeting TGF- ⁇ are hot research directions.
  • continuous systemic inhibition of TGF- ⁇ 1 and targeted integrins has strong side effects or safety problems, and the actual treatment effect is not good; the effect of other drugs for the treatment of fibrosis is also not ideal.
  • Fibrosis has long been thought to be irreversible.
  • the existing drugs for the treatment of fibrosis are often only able to inhibit fibrosis to a certain extent, but it is difficult to prevent or reverse fibrosis. Therefore, there is still a need to develop new means to promote regeneration of damaged organs while resisting fibrosis.
  • the present invention provides an anti-fibrotic pharmaceutical composition
  • an anti-fibrotic pharmaceutical composition comprising: (a) an NRP1 inhibitor and/or a HIF2 ⁇ inhibitor; (b) an EPCR pathway activator, the EPCR pathway activator promotes the EPCR pathway activity.
  • the NRP1 inhibitor includes one or more of a substance that inhibits NRP1 protein activity, a substance that degrades NRP1 protein activity, and a genetic tool that reduces NRP1 protein level;
  • the HIF2 ⁇ inhibitor includes a substance that inhibits HIF2 ⁇ protein activity One or more of a substance that degrades HIF2 ⁇ protein activity, and a genetic tool that reduces HIF2 ⁇ protein levels.
  • the NRP1 inhibitor includes EG00229 or a derivative thereof;
  • the HIF2 ⁇ inhibitor includes HIF-2 ⁇ -IN-1 or a derivative thereof.
  • the EPCR pathway activator includes activated protein C.
  • the fibrosis includes organ fibrosis, optionally age-related organ fibrosis.
  • the organ fibrosis includes one or more of liver fibrosis, renal fibrosis, pulmonary fibrosis and cardiac fibrosis, and the pulmonary fibrosis is optionally fibrosis caused by immunotherapy-related pneumonia.
  • the gene tool for reducing NRP1 protein level includes one or more of RNA interference, microRNA, gene editing and gene knockout;
  • the gene tool for reducing HIF2 ⁇ protein level includes RNA interference, microRNA, gene editing and One or more of the gene knockouts.
  • composition also includes a pharmaceutically acceptable carrier.
  • the present invention also provides the use of a pharmaceutical composition in the preparation of an anti-fibrotic agent, the pharmaceutical composition comprising: (a) NRP1 inhibitor and/or HIF2 ⁇ inhibitor; (b) EPCR pathway An activator, the EPCR pathway activator promotes the activity of the EPCR pathway.
  • the application includes reducing the degree of fibrosis of the damaged organ and/or promoting the regeneration capacity of the damaged organ, the regeneration capacity including one or more of cell proliferation capacity, tissue repair capacity and functional recovery capacity, so
  • the tissue repair capability is optionally fibrotic-free repair
  • the organ optionally includes one or more of liver, kidney, lung, and heart.
  • the application includes reducing platelet-macrophage colony formation; and/or reducing platelet expression of one or more of IL-1 ⁇ , SDF1, and TIMP1.
  • EPCR pathway activator is used in combination with NRP1 inhibitor and/or HIF2 ⁇ inhibitor to prepare the anti-fibrotic agent, and the NRP1 inhibitor and/or HIF2 ⁇ inhibitor is used to promote the expression of EPCR.
  • the application includes reducing the activity of the Rho pathway; and/or reducing the deposition of inflammatory monocytes; and/or promoting the pro-regenerative function of the endothelial microenvironment.
  • the present invention provides an anti-fibrosis pharmaceutical composition and its application.
  • the EPCR (endothelial cell protein C receptor) targeted by the technical solution of the present invention is a transmembrane glycoprotein located on the cell membrane specifically expressed by endothelial cells, and vascular endothelial cells are distributed in almost all organs and have a huge number in vivo. .
  • Experimenters of the present invention have found through a series of experiments that the inhibition of the EPCR pathway is an important node in the transition from organ regeneration to fibrosis.
  • the experimenter of the present invention utilizes the interaction between multiple cell types including vascular endothelial cells, platelets and macrophages, and simultaneously intervenes in the upstream of EPCR inhibition (ie, inhibits NRP1 and/or HIF2 ⁇ ) and EPCR, and upregulates EPCR to a certain extent.
  • EPCR inhibition ie, inhibits NRP1 and/or HIF2 ⁇
  • the downstream of EPCR inhibition ie, IL-1 ⁇ , TIMP1, etc.
  • the existing technologies often only target a single target, and the therapeutic effect is not good; while directly targeting integrin or targeting TGF- ⁇ , there are problems such as safety.
  • the anti-fibrotic drug composition provided by the present invention can regulate the production of multiple cell types (ie, vascular endothelial cells, platelets, and macrophages in the circulatory system).
  • a variety of molecules can regulate the fibrosis of damaged organs from multiple angles and promote the regeneration of damaged cells.
  • the technical solution of the present invention can inhibit the pro-fibrotic platelet IL-1 ⁇ and macrophage TIMP1, and the macrophage TIMP1 has been proved to be able to stimulate the activation of fibroblasts through integrins (it can also be understood that integrins are downstream of EPCR).
  • the technical solution of the present invention has obvious therapeutic effect on anti-fibrosis, which is embodied as: reducing the expression of IL-1 ⁇ , SDF1, TIMP1 and reducing the "platelet-macrophage" colony that promotes fibrosis, inhibiting endothelial cells Rho activates and reduces the deposition of inflammatory monocytes to reduce the degree of fibrosis in damaged organs and promote regeneration (including parenchymal cell proliferation, tissue repair, functional recovery, etc. fibrosis-free) repair, etc. It should be emphasized that the technical solution of the present invention can further inhibit the activation of fibroblasts by reducing the expression of TIMP1 in macrophages. The above therapeutic effects have been verified in lung, liver, kidney regeneration and fibrosis models and their corresponding aging models.
  • Fibrosis has long been thought to be irreversible.
  • the existing technologies are often only able to inhibit fibrosis to a certain extent, and it is difficult to "reverse fibrosis”.
  • the present invention regulates the expression of EPCR by targeting EPCR (ie, EPCR pathway activator + NRP1 inhibitor and/or HIF2 ⁇ inhibitor, and then targeting EPCR), thereby affecting the expression of platelet IL1 ⁇ and macrophage TIMP1 in the circulation system, namely Normalize the abnormal interaction between platelets-macrophages-vascular endothelial cells, inhibit fibrosis to a certain extent and restore the regeneration capacity of damaged organs, and achieve "fibrosis reversal" (ie, fibrosis-free repair of damaged organs) , the effect of transitioning damaged organs from fibrosis to regeneration).
  • the pharmaceutical composition provided by the present invention can also alleviate immunotherapy-induced pneumonia (Immunotherapy-induced pneumonitis) to a certain extent.
  • the anti-fibrotic pharmaceutical composition and its application provided by the technical solution of the present invention are helpful for systematically alleviating or treating or even reversing organ fibrosis to a certain extent.
  • the present invention provides an application of an EPCR pathway activator in the preparation of an anti-fibrotic agent, wherein the EPCR pathway activator promotes the activity of the EPCR pathway.
  • the EPCR pathway activator includes activated protein C.
  • the fibrosis includes organ fibrosis, optionally age-related organ fibrosis.
  • the organ fibrosis includes one or more of liver fibrosis, renal fibrosis, pulmonary fibrosis and cardiac fibrosis, and the pulmonary fibrosis is optionally fibrosis caused by immunotherapy-related pneumonia.
  • the application includes reducing the degree of fibrosis of the damaged organ and/or promoting the regeneration capacity of the damaged organ, the regeneration capacity including one or more of cell proliferation capacity, tissue repair capacity and functional recovery capacity.
  • the application includes reducing platelet-macrophage colony formation; and/or reducing platelet expression of one or more of IL-1 ⁇ , SDF1, and TIMP1.
  • EPCR pathway activator is used in combination with NRP1 inhibitor and/or HIF2 ⁇ inhibitor to prepare the anti-fibrotic agent, and the NRP1 inhibitor and/or HIF2 ⁇ inhibitor is used to promote the expression of EPCR.
  • the NRP1 inhibitor includes one or more of a substance that inhibits the activity of the NRP1 protein, a substance that degrades the activity of the NRP1 protein, and a genetic tool that reduces the level of the NRP1 protein;
  • the HIF2 ⁇ inhibitor includes a substance that inhibits the activity of the HIF2 ⁇ protein.
  • the NRP1 inhibitor includes EG00229 or a derivative thereof;
  • the HIF2 ⁇ inhibitor includes HIF-2 ⁇ -IN-1 or a derivative thereof.
  • the substances that inhibit the activity of NRP1 protein include small molecule drugs and/or antibodies; the substances that inhibit the activity of HIF2 ⁇ protein include small molecule drugs and/or antibodies.
  • the gene tool for reducing NRP1 protein level includes one or more of RNA interference, microRNA, gene editing and gene knockout;
  • the gene tool for reducing HIF2 ⁇ protein level includes RNA interference, microRNA, gene editing and One or more of the gene knockouts.
  • the present invention also provides a marker group for evaluating organ fibrosis, characterized in that, the marker group includes one or more of NRP1, HIF2 ⁇ and EPCR;
  • the expression level can be used to assess the degree of organ fibrosis and/or the impairment of organ function.
  • the marker group further includes one or more of PlGF, SDF1, IL-1 ⁇ , CXCR4, and TIMP.
  • the organ fibrosis includes one or more of liver fibrosis, renal fibrosis, pulmonary fibrosis and cardiac fibrosis.
  • organ fibrosis is aging-related organ fibrosis.
  • the present invention also provides a kit for evaluating organ fibrosis, which is characterized by comprising a marker group, wherein the marker group includes one or more of NRP1, HIF2 ⁇ and EPCR.
  • the marker group further includes one or more of PlGF, SDF1, IL-1 ⁇ , CXCR4, and TIMP.
  • the kit is used to detect the gene expression level of the marker in biological samples
  • the biological samples include one or more of blood samples, serum samples, plasma samples, tissue samples, organ samples and cell samples. kind.
  • the organ fibrosis includes one or more of liver fibrosis, renal fibrosis, pulmonary fibrosis and cardiac fibrosis.
  • organ fibrosis is aging-related organ fibrosis.
  • the invention provides the application of the EPCR pathway activator in the preparation of an anti-fibrotic agent.
  • the EPCR (endothelial cell protein C receptor) targeted by the technical solution of the present invention is a transmembrane glycoprotein located on the cell membrane specifically expressed by endothelial cells, and vascular endothelial cells are distributed in almost all organs and have a huge number in vivo. .
  • Experimenters of the present invention have found through a series of experiments that the inhibition of the EPCR pathway is an important node in the transition from organ regeneration to fibrosis.
  • the experimenters of the present invention utilize the interaction between multiple cell types including vascular endothelial cells, platelets and macrophages, and intervene in the upstream of EPCR inhibition (ie, inhibit NRP1 and/or HIF2 ⁇ ) to help up-regulate EPCR, which in turn has a negative effect on EPCR inhibition.
  • Downstream ie, IL-1 ⁇ , TIMP1, etc.
  • EPCR inhibition ie, inhibit NRP1 and/or HIF2 ⁇
  • Downstream ie, IL-1 ⁇ , TIMP1, etc.
  • the existing technologies often only target a single target, and the therapeutic effect is not good; while directly targeting integrin or targeting TGF- ⁇ , there are problems such as safety.
  • the application of the EPCR pathway activator provided by the present invention in the preparation of an anti-fibrotic agent, combined with the feature that the circulatory system is readily accessible, can regulate a variety of cell types (ie, vascular endothelial cells, platelets, A variety of molecules (such as EPCR, IL-1 ⁇ , TIMP1, etc.) produced by macrophages regulate the fibrosis of damaged organs from multiple perspectives and promote the regeneration of damaged cells.
  • the technical solution of the present invention can inhibit the pro-fibrotic platelet IL-1 ⁇ and macrophage TIMP1, and the macrophage TIMP1 has been proved to be able to stimulate the activation of fibroblasts through integrins (it can also be understood that integrins are downstream of EPCR).
  • the technical solution of the present invention has obvious therapeutic effect on anti-fibrosis, which is embodied as: reducing the expression of IL-1 ⁇ , SDF1, TIMP1 and reducing the "platelet-macrophage" colony that promotes fibrosis, inhibiting endothelial cells Rho pathway activation, reducing the deposition of inflammatory monocytes to reduce the degree of fibrosis in damaged organs and promote regeneration (including parenchymal cell proliferation, tissue repair, functional recovery, etc. of damaged organs), and promote the non-fibrosis of damaged organs (fibrosis-free) repair, etc.
  • the technical solution of the present invention can further inhibit the activation of fibroblasts by reducing the expression of TIMP1 in macrophages. The above therapeutic effects have been verified in lung, liver, kidney regeneration and fibrosis models and their corresponding aging models.
  • Fibrosis has long been thought to be irreversible.
  • the existing technologies are often only able to inhibit fibrosis to a certain extent, and it is difficult to "reverse fibrosis".
  • the present invention regulates the expression of EPCR by targeting EPCR (or in combination with NRP1 inhibitor and/or HIF2 ⁇ inhibitor, and then targeting EPCR), thereby affecting the expression of platelet IL1 ⁇ and macrophage TIMP1 in the circulation system, that is, normalization
  • the abnormal interaction between platelets-macrophages-vascular endothelial cells inhibits fibrosis to a certain extent and restores the regeneration ability of damaged organs, and achieves "reversal of fibrosis" (ie, fibrosis-free repair of damaged organs, enabling transition from fibrosis to regeneration in damaged organs).
  • the application provided by the present invention can also alleviate immunotherapy-induced pneumonia (Immunotherapy-induced pneumonitis) to a certain extent.
  • the application of the EPCR pathway activator provided by the technical solution of the present invention in the preparation of an anti-fibrotic agent is helpful for systematically alleviating or treating or even reversing organ fibrosis to a certain extent.
  • EPCR Endothelial protein C receptor
  • HIF2 ⁇ Hypoxia-inducible-factor2 ⁇ , hypoxia-inducible factor 2 ⁇
  • NRP1 Neuropilin1, neuropilin 1
  • TIMP1 Tissue inhibitor of metalloproteinases1, tissue inhibitor of metalloproteinase 1
  • IL-1 ⁇ Interleukin-1 ⁇ , interleukin-1 ⁇
  • ECs Endothelial cells, endothelial cells
  • PCECs Pulmonary capillary endothelial cells, pulmonary capillary endothelial cells
  • PlGF Placental growth factor, placental growth factor
  • SDF1 stromal-derived factor 1, Matrix-derived factor 1
  • HDAC Histone deacetylase, histone deacetylase
  • VPA Valproic acid, valproic acid
  • DNMT DNA methyltransferase, DNA methyltransferase
  • AZA 5-azacitidine, 5-azacytidine
  • PNX
  • promoting refers to the further improvement of the described subject at an existing level, including one or more of a quantitative level, an expression level, a functional level, and an ability level.
  • reversing fibrosis refers to shifting an organ in a fibrotic state toward regeneration, including reducing the level of fibrosis, reducing the severity of symptoms caused by fibrosis, and enhancing regenerative capacity.
  • promoting the activity of the EPCR pathway refers to enhancing EPCR signaling and/or increasing the expression level of the EPCR, allowing the EPCR to interact with its downstream molecules.
  • Fig. 1 (Fig. 1A-Fig. 1I) is an experimental graph showing that abnormal induction of NRP1 in senescent endothelial cells in the lung inhibits regeneration to promote fibrosis;
  • Fig. 2 (Fig. 2A-Fig. 2R) is an experimental graph showing that reprogrammed EPCR signaling inhibits regeneration and stimulates fibrosis in endothelial cells of senescent liver and kidney;
  • Fig. 3 (Fig. 3A-Fig. 3H) is an experimental graph showing that EPCR inhibited by endothelial cell NRP1 upregulates stromal-derived factor 1 (SDF1) to recruit pro-fibrotic macrophages in organs of aged mice;
  • SDF1 stromal-derived factor 1
  • Fig. 4 (Fig. 4A-Fig. 4K) is an experimental graph showing that macrophage/monocyte-derived tissue inhibitor of metalloproteinase promotes fibroblast activation and enhances fibrosis;
  • FIG. 5 is an experimental diagram of platelet-derived IL-1 ⁇ interacting with macrophages to form pro-fibrotic hematopoietic colonies;
  • Fig. 6 is an experimental graph of genetic targeting of endothelial cell NRP1 or platelet IL-1 ⁇ to reduce fibrosis in aging lung after bleomycin injury;
  • Fig. 7 (Fig. 7A-Fig. 7P) is an experimental graph showing that blockade of endothelial cell HIF2 ⁇ or platelet IL-1 ⁇ reduces immunotherapy-induced pneumonia, liver fibrosis and renal fibrosis in aging organs;
  • Figure 8 shows impaired lung regeneration, aberrant induction of neuropilin 1 (NRP1)-HIF2 ⁇ pathway and inhibited endothelial protein in endothelial cells of aged mouse lungs after pneumonectomy (PNX)
  • PNX neuropilin 1
  • EPCR EPCR
  • Fig. 9 (Fig. 9A-Fig. 9K) is an experimental graph showing that activation of NRP1-HIF2 ⁇ pathway reduces the expression of EPCR in aging liver and kidney EC;
  • FIG. 10 is an experimental graph of the recruitment of pro-fibrotic macrophages by endothelial cells NRP1 in aged organs after injury;
  • FIG. 11 is an experimental graph of the phenotype of macrophages in the mouse group
  • Fig. 12 (Fig. 12A-Fig. 12H) is an experimental graph of the effects of EPCR neutralizing antibody and macrophage TIMP1 on lung regeneration and fibrosis in young mice;
  • Fig. 13 (Fig. 13A-Fig. 13E) is an experimental graph showing that abnormal NRP1 signaling in endothelial cells promotes pulmonary fibrosis and aging by recruiting TIMP1-expressing pro-fibrotic macrophages;
  • Fig. 14 (Fig. 14A-Fig. 14E) is an experimental graph of the abnormal NRP1-HIF2 ⁇ signal of endothelial cells and the pro-fibrotic function of TIMP1 in aged liver and kidney after injury;
  • Figure 15 is a schematic diagram of the role model of the APC-EPCR pathway in promoting the repair of non-fibrotic organs
  • Fig. 16 (Fig. 16A-Fig. 16I) is an experimental graph showing that the inhibition of EPCR in endothelial cells and the induction of NRP1 are related to pulmonary fibrosis after repeated inhalation of hydrochloric acid in mice;
  • Fig. 17 (Fig. 17A-Fig. 17G) is an experimental graph showing that blockade of Nrp1 restores the expression of EPCR in the endothelial microenvironment, which cooperates with APC to promote the repair of fibrosis-free lung after acid injury;
  • FIG. 18 is an experimental diagram of targeting Nrp1/HIF2 ⁇ and EPCR pathways to alleviate liver and kidney fibrosis;
  • FIG. 19 is an experimental graph of NRP1-HIF2 ⁇ inhibition and APC treatment promoting adaptive repair of liver and kidney after long-term injury.
  • the term "about” is typically expressed as +/- 5% of the stated value, more typically +/- 4% of the stated value, and more typically + /-3%, more typically +/-2% of said value, even more typically +/-1% of said value, even more typically +/-0.5% of said value.
  • FIG. 1 (A) Schematic diagram illustrating a protocol for testing alveolar regeneration strategies in young versus aged mice after pneumonectomy (PNX).
  • the platelet marker CD41, the macrophage marker F4/80, and the vascular endothelial cell marker VE-cadherin were co-stained.
  • platelets and macrophages form cell colonies (arrows).
  • E Heatmap of altered paracrine/vascular secretion genes in pulmonary capillary endothelial cells (PCECs) of 2-month-old and 20-month-old "old" mice after PNX.
  • PCECs pulmonary capillary endothelial cells
  • EEU 5-Ethynyl-2'-deoxyuridine incorporation was co-stained with surfactant protein C (SPC). Senescence was detected by staining with ⁇ -galactosidase ( ⁇ -Gal) activity. Immunostaining of LY6g assessed neutrophil accumulation. Vascular permeability was measured by injection of high molecular weight dextran (F).
  • F high molecular weight dextran
  • I In senescent endothelial cells, upregulation of NRP1 stimulates HIF2 ⁇ and inhibits anti-inflammatory and antithrombotic EPCR. Inhibition of EPCR activates platelets and macrophages to form profibrotic hematopoietic colonies. Data are presented as mean ⁇ standard error of the mean (SEM). Statistical differences were determined by one-way analysis of variance (ANOVA) followed by Tukey's test as a post hoc analysis. *P ⁇ 0.05.
  • FIG. 2 (A) Detection of liver regeneration in young and old mice after PH.
  • D HIF2 ⁇ protein in hepatic endothelial cells of the mouse group after PH. The expression of HIF2 ⁇ was detected by co-staining with the endothelial cell nuclear marker ETS-related gene (ERG) (E).
  • EI Accumulation of "platelet-macrophage" colonies, hepatocyte proliferation, and fibrosis in the liver of aged Nrp1 i ⁇ EC/i ⁇ EC mice and control mice after PH.
  • N/A indicates that monocytes were not transferred.
  • GH Sirius red staining of lung (G) and liver and kidney (H) of the indicated mouse groups.
  • K NRP1 is induced to upregulate HIF2 ⁇ and inhibit EPCR induction in aged endothelial cells following lung, liver and kidney injury. An endothelial microenvironment expressing SDF1 was formed to enhance the deposition of CXCR4 + TIMP1-high macrophages.
  • macrophages may contribute to the pro-fibrotic function of "platelet-macrophage” colonies, reversing regenerative responses to fibrosis in aging lung, liver and kidney.
  • Statistical differences were determined by one-way analysis of variance (ANOVA) followed by Tukey's test as post hoc analysis. Data are presented as mean ⁇ SEM.
  • FIG. 6 (A) Experimental procedure for the induction of lung injury and fibrosis by intratracheal injection of bleomycin (Bleo) in aged mice.
  • C Protein expression of HIF2 ⁇ and EPCR in the indicated mouse groups after bleomycin injury.
  • D Distribution of alveolar epithelial cells, SMA expression, accumulation of CXCR4 + macrophages, and senescence-associated ⁇ -galactosidase in the lung after bleomycin injury.
  • EF Protein levels of collagen I
  • F recovery of lung function
  • FIG. 1 Schematic of the adoptive transfer of platelets and monocytes to mice following bleomycin injury.
  • HI Deposition of "platelet-macrophage” aggregates (H) and lung TIMP1 expression (I) in the mice.
  • I lung TIMP1 expression
  • FIG. 7 (A) Mouse model of immunotherapy-induced pneumonia. Young and old mice were treated with anti-programmed cell death protein 1 (PD1) antibody and radiation. Animal morbidity and pulmonary fibrosis were measured.
  • (DF) Lung collagen deposition (D), IL-1 ⁇ concentration in bronchoalveolar lavage fluid (BALF) (E), and animal survival (F) in the indicated groups. Aged mice were transplanted with platelets and received the indicated treatments (n 8-10 mice per group).
  • G A method to detect the effect of platelet Il1 ⁇ on fibrosis and tumor progression in the lung after immunotherapy.
  • K Liver and kidney fibrosis induced by carbon tetrachloride (CCl 4 ) and folic acid injection, respectively.
  • M The amount of collagen type I protein in the liver of the mice.
  • N Sirius red staining of liver and kidney in the indicated mouse groups.
  • Figure 8 is a supplement to and related to the experiment of Figure 1.
  • AC Recovery of lung function, deposition of collagen and "platelet-macrophage" colonies in mice of the indicated ages after PNX. Blood oxygenation and inspiratory volume were measured. Sirius red staining and collagen I immunoblotting were performed and the formation of "platelet-macrophage" aggregates was examined. Twenty-month-old (senior) mice exhibited lower alveolar regeneration and higher fibrosis. In all figures, each point in the dot plot represents data from a single animal. Sh represents the sham operation group; PNX represents the pneumonectomy group.
  • Figure 9 is a supplement to the experiment of Figure 2 and is related to it.
  • A Examination of liver regeneration in young and old mice after partial hepatectomy (PH). The amount of hydroxyproline in the liver was measured in the indicated groups.
  • BC Distribution of perivascular neutrophils in the indicated groups after PH or renal ischemia-reperfusion (I/R).
  • E PlGF protein amounts in lung and liver of the indicated mouse groups after PNX and PH.
  • N 4 mice per group.
  • H EPCR inhibition by PlGF in liver ECs of the indicated mice.
  • N 4 per group.
  • HDAC histone deacetylase
  • DNMT DNA methyltransferase
  • AZA+AZA azacytidine
  • veh means excipient.
  • N 5.
  • K Epigenetically upregulated PlGF-NRP1 pathway after injury inhibits endothelial cell EPCR induction in aged mouse organs.
  • Figure 10 Supplement to the experiment of Figure 3, in relation to it.
  • E Lung cell proliferation in the group of mice.
  • F The amount of hydroxyproline in the liver of the indicated mice after PH.
  • NRP1-HIF2 ⁇ signaling in PCECs of aged mice inhibits EPCR to induce SDF1 expression.
  • Figure 11 is a supplement to the experiment of Figure 4 and is related to it.
  • A Expression of individual markers in the macrophages. Macrophages were isolated from lungs, livers and kidneys of 3-month-old (young) and aged mice following PNX, PH or renal I/R.
  • B Expression of the indicated genes in macrophages from aged control and Hif2a i ⁇ EC/i ⁇ EC mice from aged organs.
  • C Co-staining of F4/80 and CD86 in lung, liver and kidney of the indicated mouse groups. Scale bar is 50 ⁇ m.
  • Figure 12 is a supplement to the experiment of Figure 5 and is related to it.
  • (CD) Collagen deposition and hydroxyproline levels in young lungs after PNX and 1560 injection. Young mice were 3 months old. N 6, scale bar is 50 ⁇ m.
  • E Increased accumulation of "platelet-macrophage” aggregates in the lungs of young mice following EPCR antibody injection relative to mice treated with isotype-matched IgG.
  • monocytes were isolated from Timp1-null (Timp1 -/- ) and control Timp1 +/+ mice and transferred into young mice.
  • Figure 13 is a supplement to the experiment of Figure 6 and is related to it.
  • A Recruitment of CXCR4 + macrophages and treatment following intratracheal injection of bleomycin (Bleo) in groups of mice of the indicated genotypes.
  • DE Hydroxyproline amount, alveolar epithelial regeneration, and type I collagen deposition in aged Timp1 -/- mice and in young and aged Timp1 +/+ mice after Bleo injury. Young mice are 3-month-old mice, and the scale bar is 50 ⁇ m.
  • Figure 14 is a supplement to and related to the experiment of Figure 7.
  • A Expression of HIF2 ⁇ and EPCR in liver and kidney of groups of mice of the indicated genotypes after CCl4 or folic acid injury.
  • B The amount of hydroxyproline in the liver of the indicated mouse groups after CCl4 injury.
  • C Expression of pro-fibrotic TIMP1 in aged host/subject Thpo -/- mice transplanted with Il1a -/- or Il1a +/+ platelets after CCl 4 or folic acid treatment.
  • FIG. 16 (A) Experimental protocol for single or multiple intratracheal instillation of hydrochloric acid (acid) to induce lung injury.
  • B-C Recovery of respiratory function (inspiratory volume) and fibrosis (hydroxyproline) in damaged lungs were assessed after acid instillation. Each point represents data from a single mouse.
  • D Collagen deposition in the lungs of the indicated mice was detected by Sirius Red staining.
  • FIG. 17 (A) Expression of EPCR and HIF2 protein in VE-cadherin + (VE-cadherin + ) lung endothelial cells from endothelial cell-specific knockout of Nrp1 mice.
  • Floxed Nrp1 mice were crossed with mice expressing tamoxifen-responsive endothelial cell specific VE-cadherin-Cre ERT2 (VE-cadherin-Cre ERT2 ).
  • Treatment of mice with tamoxifen induces specific deletion of Nrp1 in endothelial cells.
  • Nrp1 endothelial haplodeficiency mice were used as controls.
  • Expression of HIF2 and EPCR in mice at the indicated times after acid injury was detected by immunostaining.
  • Nrp1 inhibitor + APC Activated Protein C
  • D Nrp1 inhibitor + APC treatment promotes lung repair and bypasses fibrosis. Recovery of respiratory function and deposition of hydroxyproline in mouse lungs were examined at the indicated time points. Notably, the therapeutic effect of Nrp1 inhibitor + APC was attenuated by the EPCR blocking antibody 1560.
  • FIG. 18 (A) Induction of liver and kidney fibrosis by repeated injections of CCl 4 and folic acid injections, respectively. Liver fibrosis in mice was induced by 8 intraperitoneal injections of CCl 4 every 3 days, and renal fibrosis in mice was induced by folic acid. (B) EPCR inhibition and Nrp1-induced expression in endothelial cells are associated with liver and kidney fibrosis. (C) The combination of HIF2 ⁇ inhibitor HY and APC blocks liver and kidney fibrosis. The therapeutic effect of a "two-pronged" strategy targeting Nrp1 (or HIF2 ⁇ ) and EPCR was tested in mouse models of liver and kidney fibrosis.
  • mice were treated with APC, Nrp1 inhibitor EG, HIF2 ⁇ inhibitor HY or EPCR neutralizing antibody 1560.
  • D Expression of HIF2, EPCR, Nrp1 in liver and kidney at indicated time points after injury and the treatment.
  • the EC markers VE-cadherin and ERG were also stained to detect the vascular distribution of the tested molecules.
  • EF Recovery of liver and kidney function after injection of CCl4 and folic acid and the indicated treatments.
  • (CD) Hepatic hydroxyproline amounts in injured livers treated with the indicated on day 20 after the 8th CCl4. N 8-9 mice per group.
  • HY HIF2 ⁇ inhibitor, HIF-2 ⁇ -IN-1 (HY-19949).
  • E Serum creatinine levels in kidneys at day 100 after folic acid injection and the treatment.
  • N 8 mice per group.
  • G Distribution of CXCR4 + macrophages in liver and kidney of injured mice treated with this. Following the described treatment of CCl4 and folic acid injection, liver and kidney sections were stained for CXCR4, F4/80 and VE-cadherin.
  • Nrp1 mice and HIF2 ⁇ mice were obtained from the Jackson Laboratory.
  • Mice expressing EC-specific Cdh5-(PAC) -Cre ERT2 /VE-cadherin- Cre ERT2 were provided by Dr. Ralf Adams; this mouse strain was crossed with floxed Nrp1 and Hif2a mice to generate Nrp1 i ⁇ EC/i ⁇ EC , Hif2a i ⁇ EC/i ⁇ EC and control mice.
  • mice 4-week-old mice were treated with an intraperitoneal (ip) injection of tamoxifen at a dose of 250 mg/kg for 10 days and discontinued for one week after the 3rd and 6th doses (or 150 mg/kg for 6 days). , and a 3-day break after the 3rd).
  • Quantitative polymerase chain reaction (PCR) confirmed deletion of target genes in ECs. Nrp1 i ⁇ EC/i ⁇ EC mice and Hif2a i ⁇ EC/i ⁇ EC mice and age-matched littermates were used in mouse lung, liver and kidney injury models.
  • Floxed Il1a mice were generated using the CRISPR/Cas9 system by GemPhamatech in Nanjing, China.
  • mice were injected intraperitoneally (ip) with 0.1 mg/kg APC every 3 days. Meanwhile, in order to verify the specificity of the EPCR pathway, the EPCR signal was blocked by intraperitoneal injection (ip) of 1.5 mg/kg neutralizing antibody 1560 into mice every 3 days.
  • mice were injected intraperitoneally (ip) with 50 mg/kg EG00229 or 25 mg/kg HIF-2 ⁇ -IN-1 (HY-19949), respectively, every 3 days.
  • EG00229 and its derivatives (EG00229(N 2 -[[3-[(2,1,3-Benzothiadiazol-4-ylsulfonyl)amino]-2-thienyl]carbonyl]-L-arginine) and EG 00229trifluoroacetate(N 2 -[[3-[(2,1,3-Benzothiadiazol-4-ylsulfonyl)amino]-2-thienyl]carbonyl]-L-arginine trifluoroacetate) and HIF-2 ⁇ -IN-1(3- [[(3R)-4-(Difluoromethyl)-2,2-difluoro-3-hydroxy-1,1-dioxo-3H-1-benzothiophen-5-yl]oxy]-5-fluorobenzonitrile) is mainly due to the above
  • the inhibitor can effectively inhibit NRP1 and HIF2 ⁇ in vivo, so that it can be used to prove the technical
  • a mouse model of lung regeneration and repair To examine alveolar regeneration and repair in mice, left lung PNX (ref. 19) and lung injury (ref. 7) models were adapted. Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). PNX was performed when the left lobe was excised and sutured around the hilum. PCECs were purified from 20-month-old (designated as old) and 3-month-old (designated as young) mice after PNX, and total RNA was isolated and subjected to transcriptomic analysis as described. Lung injury was induced by intratracheal injection of 2 U/kg of bleomycin.
  • mice were injected intraperitoneally with 1.5 mg/kg of EPCR neutralizing antibody 1560 after PNX and Bleo injections (ref. 46).
  • Young Nrp1 i ⁇ EC/i ⁇ EC , Hif2a i ⁇ EC/i ⁇ EC and control mice after PNX and PH were also administered 20 ⁇ g/kg of mouse PlGF or the same molar amounts of HGF and PDGF-BB (Peprotech).
  • lung injury was also induced by repeated intratracheal hydrochloric acid injection model (ref. 76).
  • mice were administered 0.1 mg/kg APC, 50 mg/kg NRP1 inhibitor, 25 mg/kg HIF2 ⁇ inhibitor and 1.5 mg/kg 1560 every 3 days after PNX and 1st acid injection. Lung function and oxygen tension in the arterial blood of treated mice were measured at the time points described previously (ref. 7).
  • mice were injected intraperitoneally with 100 mg/kg of 5-ethynyl-2'-deoxyuridine (EDU) 1 hour before sacrifice, and EDU incorporation was detected by the EDU cell proliferation kit. After all surgical procedures, mice were supplemented with 1 mL of PBS by intraperitoneal injection.
  • EDU 5-ethynyl-2'-deoxyuridine
  • mice were injected with PD1 antibody at a dose of 10 mg/kg every 4 days. Mice were monitored for survival and lung tissue from mice was obtained 3 months after irradiation. BALF was collected as previously described (ref. 19).
  • LLC Lewis lung cancer
  • To detect immunotherapy-induced pneumonia in tumor-bearing mice 2 million Lewis lung cancer (LLC) cells were transplanted into aged Thpo -/- mice through the jugular vein.
  • LLC Lewis lung cancer
  • I11a +/+ or I11a ⁇ / ⁇ platelets were transplanted into test mice after LLC cells were injected. Mice also received PD1 antibody and radiation treatment. Following platelet transplantation, test mice were analyzed for pulmonary fibrosis, tumor burden, and animal survival at the times indicated.
  • TIMP1 treatment of lung fibroblasts and hepatic stellate cells Lung and liver fibroblasts were obtained from ScienceCell Research Laboratories and cultured as previously described (ref. 8). Lung and liver fibroblasts were treated with 20 ng/ml of TIMPl, 10 ng/ml of TGF[beta]l or 20 ng/ml of TIMPl and 10 ng/ml of TGF[beta]l. To determine the role of ⁇ 1 integrin in TIMP1-mediated activation of fibroblasts, ⁇ 1 integrin was silenced in fibroblasts by shRNA. After treatment, lung and liver fibroblasts were recovered for analysis of Smad2 phosphorylation and SMA protein expression by immunoblotting. Smad2 phosphorylation and SMA protein expression were quantified by densitometry of immunoblotting.
  • Mouse liver regeneration and repair model Induction of liver regeneration in mice by PH. A midline laparotomy was performed on anesthetized mice, and the three anterior most lobes (right medial, left medial, and left lateral lobes) were excised. After opening the abdomen and exposing the liver, lift the left lobe that will be resected. A 5-0 silk suture was placed under the lobe and positioned to the origin of the lobe. Cut off the knotted lobe distal to the suture and repeat the process for the other lobes to complete the PH. After surgical removal of 70% of the liver mass, the skin was closed. Injection of CCl4 was used to induce liver injury as previously described (ref. 8).
  • CCl4 was diluted in oil (Sigma-Aldrich) to yield a concentration of 40% (0.64 mg/ml) and injected intraperitoneally into mice at a dose of 1.6 g/kg.
  • Mice were injected with 0.1 mg/kg APC, 50 mg/kg NRP1 inhibitor, 25 mg/kg HIF2 ⁇ inhibitor and 1.5 mg/kg 1560 every 3 days after PH and the 1st CCl4 injection.
  • the regeneration of liver function was assessed by measuring serum levels of bilirubin and AST at the indicated time points after PH (ref. 8).
  • mice Mouse kidney regeneration and repair model.
  • the mouse kidney I/R model described (ref. 72) was used. After the mice were anesthetized, ischemia was induced by a 30-minute retroperitoneal approach over both kidneys. Sham surgery was performed with exposed kidneys without inducing ischemia.
  • To generate renal fibrosis mice were given a single intraperitoneal injection of folic acid at a dose of 250 mg/kg as previously described (ref. 74). Mice were injected with 0.1 mg/kg APC, 50 mg/kg NRP1 inhibitor, 25 mg/kg HIF2 ⁇ inhibitor and 1.5 mg/kg 1560 every 3 days after renal I/R and folic acid injection after day 40. At the times indicated, serum creatinine concentrations were measured by the picric acid method, and tissue morphology and fibrosis were analyzed by co-staining with Sirius red and hematoxylin.
  • mice were treated with 10 mg/kg of the HDAC inhibitor VPA, 0.5 mg/kg of the DNMT1 inhibitor azacytidine (AZA), or a combination therapy of VPA and AZA (VPA+AZA).
  • VPA HDAC inhibitor
  • AZA DNMT1 inhibitor azacytidine
  • VPA+AZA a combination therapy of VPA and AZA
  • IF Immunofluorescence
  • Mouse lung, liver and kidney tissues were obtained for histological analysis. 10 ⁇ m thick tissue cryosections were blocked (5% donkey serum/0.3% Triton X-100) and incubated in primary antibody overnight at 4°C: anti-VE-cadherin polyclonal antibody (pAb, 2 ⁇ g/ml, R&D Systems, MN), anti-SMA antibody (pAb, 2 ⁇ g/ml, Abcam, CA), anti-EPCR monoclonal antibody (mAb 1560, 5 ⁇ g/ml), anti-pingoprotein (mAb, 5 ⁇ g/ml, R&D, MN), anti-water Channel protein 5 (pAb, 5 ⁇ g/ml, Abcam, CA), anti-CD41 (mAb, 5 ⁇ g/ml, BD Bioscience).
  • Nuclear staining was performed using Prolong Gold mounting medium (Invitrogen) with DAPI after incubation in fluorophore-conjugated secondary antibody (2.5 ⁇ g/ml, Jackson ImmunoResearch, PA). To determine the immunofluorescent staining signal in prepared tissue sections, the fluorescent cells in each slide were independently assessed and quantified on five different high-power fields, which are representative of results from a single specimen.
  • Tissue Fibrosis Assay Lung, liver and kidney fibrotic responses were determined at the indicated times after injury. Tissue was homogenized in tissue lysis buffer. Collagen I immunoblotting was performed on the obtained tissue lysates. The protein levels of collagen I were compared between the different groups. Sirius red and hematoxylin staining was performed on paraffin-embedded tissue sections to determine tissue morphology and collagen deposition and distribution (ref. 8). Sirius red positive fibrotic parenchyma was determined and quantified from five random fields per section. The amount of hydroxyproline in the liver and lung was quantified to determine the degree of fibrosis.
  • monocytes were isolated from the bone marrow of WT or Timp1 -/- mice.
  • the adoptive transfer of the monocytes was repeated after the first transplantation until the test mice were sacrificed.
  • Lung, hepatic, renal function and fibrosis were compared between different groups receiving WT or Timpl -/- monocytes. Collagen deposition and morphological characteristics were detected by Sirius red and hematoxylin staining.
  • Mouse platelet adoptive transfer model The strategy of infusing platelets through the jugular vein as previously described (ref. 24) was used. Platelets from Il1a (Il1a ⁇ Plt/ ⁇ Plt ) mice were isolated and concentrated to obtain Il1a ⁇ / ⁇ platelets. Wild-type platelets were used as control platelets. Briefly, blood was collected from anesthetized mice with a syringe containing 0.5 mL of ACD (12.5 g/L sodium citrate, 10.0 g/L D-glucose, and 6.85 g/L citric acid).
  • ACD 12.5 g/L sodium citrate, 10.0 g/L D-glucose, and 6.85 g/L citric acid.
  • the collected blood was transferred to 5 mL of buffer containing 150 mM NaCl and 20 mM PIPES, and then centrifuged at 100 g for 15 minutes. After centrifugation, the platelet-rich supernatant was collected. The supernatant was centrifuged again at 1000g for 10 minutes. The obtained platelet pellet was resuspended and counted. Over 15 minutes, 2 x 109 platelets were injected through the exposed jugular vein of the test mice.
  • Example 2 The transition from "regeneration to fibrosis" in the lungs of aged mice is associated with the formation of "platelet-macrophage” colonies and the reprogramming of endothelial cells
  • the present inventors used pneumonectomy (PNX) to compare alveolar regeneration and fibrosis in mice of different ages (Fig. 1A), including the recovery of lung function, epithelial structure and hydroxyproline content (Fig. 1B-C, 8A-B).
  • PNX pneumonectomy
  • 20-month-old mice after PNX showed significant inhibition of alveolar regeneration and increased fibrosis compared to 2-, 3-, and 6-month-old mice. Therefore, the experimenters of the present invention defined 20-month-old mice as "old” mice, and compared the regenerative ability of aged mice with 2-month-old (2-mon) or 3-month-old (3-mon) mice.
  • Endothelial cells on the inner side of the vascular lumen form an anti-inflammatory and antithrombotic interface to maintain organ homeostasis. Therefore, the present experimenters hypothesized that the reprogramming of key node molecules in ECs activates platelets and macrophages to promote fibrosis.
  • PCECs pulmonary capillary endothelial cells
  • EPCR is selectively expressed in ECs, and both neuropilin 1 (NRP1) and HIF2 ⁇ are expressed in ECs by pathological conditions induced (FIG. 8D).
  • EPCR expression was lower in PCECs of aged mice after PNX compared to 2-, 3-, and 6-month-old mice.
  • NRP1 and HIF2 ⁇ was elevated in PCECs of aged mice after PNX.
  • aged lungs after PNX appear to display altered vasosecreted factor signaling.
  • Example 3 Normalizing the NRP1-HIF2 ⁇ -EPCR circuit in endothelial cells reduces the 'regeneration to fibrosis' transition in the aged lung
  • NRP1 is a co-receptor for various cytokines.
  • the present inventors constructed mice specifically deficient in NRP1 in endothelial cells (Nrp1 i ⁇ EC/i ⁇ EC ).
  • Floxed NRP1 mice were crossed with mice expressing the EC-specific vascular endothelial cell-cadherin (VE-cadherin) (Cdh5) -Cre ERT2 .
  • Tamoxifen treatment of the offspring deleted NRP1 in endothelial cells of the offspring (Figure 8E).
  • Nrp1 i ⁇ EC/i ⁇ EC mice assessed regenerative and fibrotic responses in the lungs of aged Nrp1 i ⁇ EC/i ⁇ EC mice.
  • Aged Nrp1 i ⁇ EC/i ⁇ EC mice after PNX exhibited less collagen deposition, enhanced alveolar regeneration capacity, decreased senescence and fibroblast activation, and increased proliferation of type II alveolar epithelial progenitors compared to control aged mice and lower numbers of perivascular neutrophils (Fig. 1H, Fig. 8H-I).
  • Antibody 1560 inhibited the beneficial effect observed in aged Nrp1 i ⁇ EC/i ⁇ EC mice.
  • Example Four Reprogrammed hematopoietic-vascular microenvironment in aging liver and kidney reverses regeneration to fibrosis
  • Nrp1 i ⁇ EC/i ⁇ EC mice Compared with aged control mice, aged Nrp1 i ⁇ EC/i ⁇ EC mice showed improved kidney repair with a higher degree of cell proliferation, less fibrosis and a lower number of "platelet-macrophage" colonies (Fig. 2O-R, Figure 9C).
  • Antibody 1560 reversed the phenotype of aged Nrp1 i ⁇ EC/i ⁇ EC mice after renal I/R, suggesting that the beneficial effects of Nrp1 deletion depend on EPCR.
  • Example 5 Epigenetic upregulation of the placental growth factor (PlGF)-NRP1 axis in ECs to stimulate HIF2 ⁇ to inhibit EPCR
  • NRP1 binds different ligands, including placental growth factor (PlGF), hepatocyte growth factor (HGF), platelet-derived growth factor BB (PDGF-BB), and transforming growth factor ⁇ 1 (TGF- ⁇ 1).
  • PlGF protein was upregulated in aged lung and liver following PNX and PH ( Figure 9E).
  • PlGF did not inhibit the upregulation of EPCR (FIG. 9H). Therefore, the PlGF-NRP1-Hif2 ⁇ axis may be responsible for the inhibition of EPCR in aging organs after injury.
  • VPA+AZA treatment inhibited endothelial NRP1 upregulation in liver and kidney ECs in aged mice ( Figure 9I).
  • VPA+AZA also inhibited the upregulation of PlGF in aged lung, liver and kidney following PNX, PH or renal I/R ( Figure 9J). Therefore, the expression of NRP1 and PlGF may be epigenetically regulated in aging organs (Fig. 9K).
  • Example 6 The NRP1-HIF2 ⁇ axis in PCECs stimulates stromal-derived factor 1 (SDF1) to recruit pro-fibrotic macrophages
  • PCECs produce paracrine or vasosecretory factors to regulate alveolar repair. Therefore, the present inventors analyzed the expression of factors in lung endothelial cells and fibroblasts in control group and Nrp1 i ⁇ EC/i ⁇ EC mice (Fig. 10A-B). The chemokine SDF1 showed the most inhibition in PCECs from Nrp1 i ⁇ EC/i ⁇ EC mice. Since SDF1-CXCR4 signaling regulates hematopoietic cell function and organ repair, the present inventors investigated the expression pattern of SDF1 in young and old Sdf1 reporter mice (FIG. 3A). After PNX, SDF1 was induced in PCECs of aged mice (but not the young group), and deletion of Nrp1 in ECs suppressed SDF1 expression (Fig. 3A).
  • HIF2 ⁇ mediates the production of SDF1 in aged endothelial cells.
  • Aged Hif2a i ⁇ EC/i ⁇ EC mice after PNX showed lower levels of Sdf1, Par1, E-selectin and VCAM1 (Fig. 3B). Since SDF1, E-selectin, and VCAM1 promote endothelial-macrophage crosstalk, we stained for SDF1 receptor CXCR4 and macrophage markers.
  • Example 7 CXCR4 + macrophages inhibit the regeneration of aged organs and cause fibrosis
  • TIMP1 Tissue inhibitor of metalloproteinase 1
  • TIMP1 stimulates fibroblast activation through integrin ⁇ 1.
  • TIMP1 The profibrotic effect of TIMP1 was examined in aged Timp1-deficient mice (Timp1 -/- ). After PNX, aged Timp1 -/- mice exhibited reduced pulmonary fibrosis and enhanced recovery of alveolar epithelial coverage compared with control mice (Fig. 4A). TIMP1 expression was higher in macrophages from aged mouse organs after PNX, PH or kidney I/R compared to young organs (Fig. 4B, Fig. 11A-C). Treatment with the CXCR4 antagonist AMD3100 reduced TIMP1 upregulation in macrophages from aged organs of mice.
  • FIG. 4C In order to reveal the role of macrophages with high expression of CXCR4 + TIMP1 (CXCR4 + TIMP1 high ), the present inventors adopted an adoptive macrophage transfer method ( FIG. 4C ). Transplantation of Timp1-null monocytes promoted the recovery of lung function and reduced collagen deposition in aged pneumonectomized mice (FIG. 4D-G). Following PH and renal I/R, transfer of Timp1 -/- monocytes (but not Timp1 +/+ ) to aged mice enhanced parenchymal functional recovery and blocked fibrosis (Figure 4H-J).
  • Example 8 Platelets produce interleukin 1 ⁇ (IL-1 ⁇ ) to activate profibrotic TIMP1 + macrophages
  • Platelets produce pro-inflammatory cytokines, such as interleukins, to interact with macrophages.
  • the present inventors constructed mice with platelet-specific deletion of IL-1 ⁇ (Il1a ⁇ Plt/ ⁇ Plt ) ( FIG. 5A-B ). Because of the fibrosis-like phenotype in young mice after EPCR-neutralizing antibody 1560 induced PNX (FIG. 12A-E), the inventors treated 3-month-old Il1a ⁇ Plt/ ⁇ Plt mice with antibody 1560 to establish EPCR and platelet Correlation between IL-1 ⁇ .
  • FIG. 5H A platelet adoptive transfer model (ref. 52) was used (Fig. 5H).
  • Aged Thpo -/- mice were injected with wild-type (Il1a +/+ ) or Il1a knockout (Il1a -/- ) platelets, followed by PNX, liver resection or renal I/R.
  • the expression and fibrosis of TIMP1 in the lung, liver, and kidney of the test mice were assessed.
  • Aged mice injected with Il1a -/- platelets showed reduced TIMP1 expression and less collagen deposition in the tested organs compared to aged mice transferred with Il1a +/+ platelets (FIG. 5I-K).
  • Example 9 Abnormal endothelial cell-platelet crosstalk stimulates regeneration but not fibrosis in aging organs
  • Experimenters of the present invention used the bleomycin (Bleo) model as a model of pulmonary fibrosis.
  • Aged Nrp1 i ⁇ EC/i ⁇ EC mice and control mice were treated with intratracheal injection of bleomycin (FIG. 6A).
  • PlGF was up-regulated in the lungs of aged mice (Fig. 6B), and aged Nrp1 i ⁇ EC/i ⁇ EC mice showed higher EPCR up-regulation and HIF2 ⁇ inhibition than aged control mice (Fig. 6C), which was correlated with reduced fibrosis Metabolism, enhanced recovery of alveolar regeneration, decreased cellular senescence, and decreased recruitment of CXCR4 + macrophages around the blood vessels ( Figure 6D-F, Figure 13A-C).
  • Il1a +/+ and Il1a ⁇ / ⁇ platelets were transplanted into aged mice after Bleo injury ( FIG. 6G ).
  • I11a -/- platelets reduced hematopoietic colony formation and TIMP1 expression (FIG. 6H).
  • Antibody 1560 did not alter the effect of Il1a -/- platelets.
  • the differential effect of EPCR antibodies on aged Nrp1 i ⁇ EC/i ⁇ EC mice and aged Il1a ⁇ PLT/ ⁇ PLT mice suggests that NRP1 in senescent endothelial cells causes platelet IL-1 ⁇ production.
  • Immune checkpoint blockade such as anti-programmed cell death protein 1 (PD1) antibodies
  • PD1 antibodies has shown significant clinical benefit in multiple cancer types.
  • immunotherapy may have potential risks in some patients, resulting in immunotherapy-related adverse events (IrAEs).
  • IrAEs immunotherapy-related adverse events
  • Pneumonia is one of the common IrAEs after immune checkpoint blockade therapy.
  • Experimenters of the present invention tested whether aged mice were more prone to pulmonary fibrosis after PD1 antibody treatment, especially when combined with radiotherapy. Aged and young mice were exposed to thoracic radiation for 1 month to stimulate immunotherapy-induced pneumonitis (with or without PD1 antibody treatment after radiation) (Figure 7A).
  • PD1 antibody treatment after thoracic irradiation caused a higher degree of pulmonary fibrosis in aged Hif2a +/+ mice than in 3-month-old Hif2a +/+ mice or aged Hif2a i ⁇ EC/i ⁇ EC mice (Fig. 7B-C).
  • Aged Hif2a i ⁇ EC/i ⁇ EC mice had reduced mortality and pulmonary fibrosis, which was reversed by the EPCR antibody (Fig. 7B-C).
  • Aged mice transplanted with Il1a ⁇ / ⁇ (but not Il1a +/+ ) platelets were protected from death and pulmonary fibrosis ( FIG. 7D-F ).
  • EPCR antibodies did not affect this protective effect of Il1a ⁇ / ⁇ platelets, suggesting that the profibrotic function of platelet IL-1 ⁇ after checkpoint blockade is downstream of EPCR inhibition. Therefore, the abnormal crosstalk between endothelial NRP1 and platelet IL-1 ⁇ may contribute to immunotherapy-induced pneumonia in elderly patients.
  • mice were used to examine the profibrotic role of EC-platelet aberrant crosstalk in immunotherapy-induced pneumonia.
  • LCC lewis lung cancer
  • aged mice were transplanted with Il1a +/+ or Il1a ⁇ / ⁇ platelets, followed by PD1 antibody treatment ( FIG. 7G ).
  • Mice injected with Il1a -/- platelets exhibited reduced tumor cell burden and pulmonary fibrosis compared to mice injected with Il1a +/+ platelets (FIG. 7H-I).
  • Mice injected with Il1a ⁇ / ⁇ platelets had improved survival compared to mice injected with Il1a +/+ platelets ( FIG. 7J ). Therefore, in the immunotherapy of lung tumors, platelet-produced Il-1 ⁇ may cause pulmonary fibrosis and exacerbate mortality.
  • FIG. 7K Young and old mice received hepatotoxin injections of carbon tetrachloride (CCl 4 ) or folic acid (ref. 2) to induce liver or kidney fibrosis ( FIG. 7K ). Following injury, PlGF expression was upregulated in aged injured livers (Fig. 7L). Inhibition of EPCR and induction of HIF2 ⁇ in EC of aged control mice, restored in aged Nrp1 i ⁇ EC/i ⁇ EC mice (Fig. 14A), attenuated parenchymal damage and fibrosis in injured liver and kidney (Fig. 7M-N , Figure 14B).
  • lung endothelial cells form a microenvironment to initiate lung regeneration and prevent fibrosis (refs 7, 17, 19, 51).
  • Experimenters of the present invention found that in damaged lungs, livers and kidneys, endothelial cells abnormally induce NRP1, abnormally induce transcription factor HIF2 and inhibit EPCR. Disruption of the EPCR protective pathway in the endothelial microenvironment leads to persistent fibrosis in the damaged lung, liver and kidney. Genetic ablation or pharmacological blockage of NRP1 restored EPCR expression in damaged endothelial cells in lung, liver and kidney.
  • the experimenters of the present invention adopted a "two-hit” strategy, namely inducing EPCR and injecting its molecular chaperone, activated protein C (APC).
  • APC activated protein C
  • Co-administration of NRP1/HIF2 ⁇ inhibitors synergizes with APC to: 1) inhibit Rho activation in endothelial cells, 2) reduce inflammatory monocyte deposition, and 3) promote lung, liver and Fibrosis-free repair of the kidney. This actually increases the expression of EPCR through upstream pathways, including but not limited to NRP1 and HIF2 ⁇ .
  • This example presents a model for the role of the APC-EPCR pathway in promoting the repair of non-fibrotic organs (Figure 15).
  • the model outlines that EPCR on lung endothelial cells promotes lung regeneration and avoids pulmonary fibrosis after lung injury.
  • Persistent lung injury activates the NRP1 pathway in endothelial cells to upregulate HIF2, which inhibits EPCR and upregulates PAR1.
  • Disruption of the EPCR pathway in the endothelial microenvironment reverses the regenerative response to pulmonary fibrosis.
  • Therapeutic, NRP1 inhibitor or HIF2 ⁇ inhibitor restores the expression of EPCR, which cooperates with the administration of activated protein C (APC) to restore the protective APC-EPCR pathway.
  • APC activated protein C
  • Nrp1 i ⁇ EC/i ⁇ EC mice showed higher EPCR expression and lower HIF2 ⁇ and PAR1 expression in PCECs compared to control mice ( Figure 17A-B).
  • repeated lung injury resulted in reprogramming of NRP1-HIF2 ⁇ -EPCR signaling in PCECs.
  • FIG. 17C It was then tested whether targeting reprogrammed PCECs by injection of APC and EG (APC+EG) restored lung repair and reduced fibrosis (FIG. 17C).
  • APC+EG most effectively restored lung function and reduced fibrosis among all tested groups after repeated acid insults (Figure 17D-G).
  • Perivascular macrophage recruitment was inhibited in APC+EG-treated PCECs following repeated lung injury (FIG. 17F-G). It was previously shown that APC inhibits the activation of the Rho pathway in ECs. Indeed, the phosphorylated myosin light chain (p-MLC) of PCECs was blocked after APC+EG treatment (Fig. 17F-G).
  • a two-pronged strategy namely co-injection of APC and NRP1 inhibitor, stimulates normalized EPCR signaling in chronically damaged PCECs and reduces the recruitment of profibrotic perivascular macrophages, i.e. stimulates normalized EPCR pathway in PCECs, Promotes fibrosis-free lung repair.
  • FIG. 18A The therapeutic effect of the above two-pronged strategy was then tested in mouse models of chronic injury in liver and kidney (FIG. 18A).
  • Mice were repeatedly injected with the liver toxin carbon tetrachloride (CCl4) or folic acid (ref. 77).
  • CCl4 liver toxin carbon tetrachloride
  • folic acid ref. 77
  • EPCR was induced in ECs after the first injection of CCl4 or on day 10 after folic acid injection.
  • NRP1 and HIF2 ⁇ were stimulated in ECs after the 8th CCl4 injection and 100 days after folic acid injection (FIG. 19A).
  • Nrp1 i ⁇ EC/i ⁇ EC mice with APC inhibited HIF2 ⁇ induction and enhanced EPCR expression in chronically injured liver and kidney ECs ( Figure 19B), suggesting that EPCR has a pro-regenerative function in liver and kidney repair.
  • mice were then treated with APC, EG or the HIF2 ⁇ inhibitor HIF-2 ⁇ -IN-1 (HY-19949) and assessed after the 8th CCl4 injection or 100 days after folic acid injection.
  • APC+EG inhibited the expression of Sdf1, Par1, Vcam1 and Hif2a in liver and kidney ECs (FIG. 18B) and reduced liver fibrosis (FIG. 19C).
  • Co-injection of APC and HY-19949 (APC+HY) blocked perivascular CXCR4 + macrophage recruitment and inhibited parenchymal damage and fibrosis in injured liver and kidney ( Figure 18C, Figure 19D-G). Furthermore, after fibrotic liver and kidney injury, APC+EG blocked
  • SDF1 reported the expression of SDF1 in mouse ECs (Fig. 18D) and enhanced the recovery of parenchymal function (Fig. 18E-F). All these beneficial effects were blocked by injection of EPCR neutralizing antibody 1560. Therefore, through a two-pronged strategy (APC+NRP1/HIF2 ⁇ ), targeting reprogrammed NRP1-HIF2 ⁇ -EPCR (which can also be understood as targeting reprogrammed ECs in the liver and kidney) enables long-term damage to the liver and kidney to achieve fiber-free chemical repair.
  • APC+NRP1/HIF2 ⁇ targeting reprogrammed NRP1-HIF2 ⁇ -EPCR
  • EPCRs have pleiotropic functions in regulating thrombosis, inflammation and development.
  • the present data demonstrate that EPCR induction in senescent ECs is inhibited by aberrantly activated NRP1-HIF2 ⁇ pathway.
  • Platelets can be activated in stages to exert different pathophysiological effects. Following lung or liver resection, activated platelets provide various mediators to initiate lung and liver regeneration.
  • the present inventors have previously demonstrated the function of SDF1 in promoting lung and liver regeneration using pneumonectomy and liver resection.
  • pneumonectomy and liver resection models One of the important features of the pneumonectomy and liver resection models is that these procedures induce physiological regeneration that is not involved in the marked inflammation and thrombosis in normal young mice.
  • SDF1 may primarily stimulate its receptors on the vascular endothelium or epithelial cells/hepatocytes, promoting the proliferation of epithelial and endothelial cells.
  • SDF1 causes regeneration of blood vessels and epithelial/hepatocytes.
  • the procedure of lung resection or liver resection was associated with thrombosis and inflammation.
  • SDF1 enhanced the recruitment and accumulation of profibrotic TIMP1-producing CXCR4 + macrophages. Therefore, in the absence of overt inflammation and thrombosis, SDF1 may act mainly on epithelial and endothelial cells to promote regeneration.
  • SDF1 activates CXCR4 in inflammatory cells such as macrophages, producing pro-fibrotic factors such as TIMP1.
  • the cellular mechanisms revealed by the present invention may help to normalize abnormal activation of immune cells and circumvent the pro-fibrotic function of immune cells in clinical settings.
  • the mechanism can be used to deal with IrAEs.
  • the present invention found that in aged mice treated with PD1 blockade, targeting platelet IL-1 ⁇ or endothelial cell HIF2 ⁇ improved the survival rate of aged mice and alleviated pulmonary fibrosis. These data suggest that pneumonia and fibrosis may be risk factors in elderly patients after immunotherapy, especially in combination with chemotherapy or radiotherapy.
  • the present invention demonstrates that senescence-associated aberrant reprogramming of crosstalk between platelets, macrophages and vascular cells in aging organs to reverse regeneration into fibrosis.
  • the mechanism of organ fibrosis revealed by the present invention is not only specific to aging.
  • Normalizing the profibrotic hematopoietic-vascular microenvironment may contribute to restoring regenerative capacity under pathological conditions.
  • Genetic and pharmacological targeting of Neuropilin-1 or HIF2 ⁇ normalizes EPCR signaling in reprogrammed ECs, which synergizes with the EPCR agonist (agonist) activating protein C (APC) to suppress senescent and chronically impaired Recruitment of pro-fibrotic macrophages in organs.
  • Normalization of dysregulated vascular and immune microenvironments may restore the regenerative capacity of aging or fibrotic organs to some extent.
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  • Vascular endothelial growth factor ⁇ B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation. 2010, Circulation 122, 1725 ⁇ 1733.
  • Endothelial ⁇ derived angiocrine signals induce and sustain regenerative lung alveolarization. 2011, Cell 147, 539 ⁇ 553.
  • Migrating Platelets are Mechano ⁇ scavengers that Collect and Bundle Bacteria.2017, Cell 171, 1368 ⁇ 1382.
  • Semaphorin 3E and plexin ⁇ D1 control vascular pattern independently of neuropilins. 2005, Science 307, 265 ⁇ 268.
  • alphav integrin identifies a core molecular pathway that regulates fibrosis in several organs. 2013, Nature medicine 19, 1617 ⁇ 1624.
  • Activated protein C protects again diabetic nephropathy by inhibiting endothelial and podocyte apoptosis. 2007, Nat Med 13, 1349 ⁇ 1358.
  • HIF1alpha and HIF2alpha arebling rivalry in hypoxic tumour growth and progression.2011, Nat Rev Cancer 12,9 ⁇ 22.
  • Perivascular Gli1+progenitors are key contributors to injury ⁇ induced organ fibrosis.2015, Cell stem cell 16,51 ⁇ 66.
  • the lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. 2017, Nature 544, 105 ⁇ 109.
  • Platelet ⁇ derived serotonin mediates liver regeneration.2006, Science 312, 104 ⁇ 107.
  • WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature. 2005, Nature 437, 417-421.
  • Peptidylarginine deiminase 4 promotes age ⁇ related organ fibrosis. 2017, The Journal of experimental medicine 214, 439 ⁇ 458.
  • Pulmonary fibrosis patterns and perpetrators. 2012, The Journal of clinical investigation 122, 2756 ⁇ 2762.
  • Platelet ⁇ derived SDF ⁇ 1 primes the pulmonary capillary vascular niche to drive lung alveolar regeneration.2015, Nature cell biology 17, 123 ⁇ 136.
  • Platelet ⁇ derived nucleotides promote tumor ⁇ cell transendothelial migration and metastasis via P2Y2 receptor. 2013, Cancer cell 24, 130 ⁇ 137.
  • VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis. 2006, Nature medicine 12, 793 ⁇ 800.
  • 61.Timp1 interacts with beta ⁇ 1 integrin and CD63 along melanoma genesis and confers anoikis resistance by activating PI3 ⁇ K signaling pathway independently of Akt phosphorylation.2013,Molecular cancer 12,22.
  • Hepatocyte TAZ/WWTR1 Promotes Inflammation and Fibrosis in Nonalcoholic Steatohepatitis. 2016, Cell Metab 24, 848 ⁇ 862.
  • Ephrin ⁇ B2 controls VEGF ⁇ induced angiogenesis and lymphangiogenesis. 2010, Nature 465, 483 ⁇ 486.
  • hypoxia-inducible factor 1 protects the murine heart and aorta from pressure overload by suppression of TGF-beta signaling.2012, Proceedings of the National Academy of Sciences of the United States of America 109, E841-850.
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  • Endothelial ⁇ to ⁇ mesenchymal transition contributes to cardiac fibrosis. 2007, Nature medicine 13, 952 ⁇ 961.

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Abstract

一种抗纤维化药物组合物及其应用，所述抗纤维化药物组合物具体包括：(a)NRP1抑制剂和/或HIF2α抑制剂；(b)EPCR通路活化剂，所述EPCR通路活化剂促进EPCR通路的活性。所述抗纤维化药物组合物有助于一定程度上减轻器官纤维化并促进相应器官修复和再生。

Description

一种抗纤维化药物组合物及其应用 技术领域
本发明涉及生物医药领域，具体涉及一种抗纤维化药物组合物及其应用。
背景技术
纤维化(Fibrosis)可以发生于多种器官，是许多常见的慢性炎症性、免疫介导的代谢性疾病的最终病理结果以及这些疾病发病率和死亡率的主要原因。多种有害刺激(包括毒素、传染性病原体、自身免疫反应和机械应激)能够诱导纤维化细胞反应。纤维化会影响身体的所有组织，如果不加以控制，会导致器官衰竭和死亡。
纤维化是组织遭受损伤后，以保护组织器官的相对完整性的修复反应。为了响应组织损伤，源自多种来源的肌成纤维细胞(包括常驻成纤维细胞、间充质细胞、循环成纤维细胞以及其他细胞类型的转分化)可通过重塑细胞外环境来启动伤口愈合反应，以恢复组织完整性并促进实质细胞的替换。通常，当组织愈合时，这种促纤维化程序被关闭。然而，持续的损伤和损害会导致这一过程的失调，导致细胞外基质(ECM)蛋白(包括胶原、层粘连蛋白和纤维连接蛋白)在病理上的过度沉积，并伴随着肌成纤维细胞活性的上调，造成巨噬细胞(和单核细胞)和免疫细胞浸润的慢性炎症环境。而这种“过度沉积”，虽然修复了损伤，但不具备器官实质细胞的结构和功能，反而会引起器官的纤维化和功能障碍。
哺乳动物的器官(例如肝、肺和肾)能够进行代偿性再生来替代受损的组织。然而，大多数哺乳动物的再生能力受到衰老过程的限制。衰老器官中受损的组织常被过度瘢痕(excessive scar)替代，导致纤维化和器官功能障碍。鉴于不同的哺乳动物器官具有不同的自我修复能力，而衰老器官的再生能力似乎受到类似地抑制，并且这些衰老器官在损伤后都容易发生纤维化。
目前对纤维化机制的研究有很多，其中，转化生长因子-β(TGF-β)家族成员被认为在纤维化过程中起主要作用，TGF-β是主要的促纤维化细胞因子。研究认为，许多纤维化疾病都是通过TGF通路介导，促进成纤维细胞分化为肌成纤维细胞，而肌成纤维细胞产生和分泌ECM蛋白增多，造成ECM沉积，致使器官的结构发生变化并硬化。而整合素被认为可以增强来自可溶性促纤维化生长因子(例如TGF-β1)的信号。因此，靶向整合素和靶向TGF-β是热门研究方向。然而，持续地全身性抑制TGF-β1和靶向整合素，具有强烈副作用或存在安全性问题，实际治疗效果不佳；其他治疗纤维化的药物的效果，也并不理想。
长期以来，人们一直认为纤维化不可逆。目前存在的治疗纤维化的药物，往往仅能够在一定程度上抑制纤维化，而难以阻止或逆转纤维化。因此，仍需要开发能够抗纤维化的同时，促进受损器官的再生的新手段。
发明内容
本发明提供了一种抗纤维化药物组合物，所述药物组合物包括：(a)NRP1抑制剂和/或HIF2α抑制剂；(b)EPCR通路活化剂，所述EPCR通路活化剂促进EPCR通路的活性。
进一步地，所述NRP1抑制剂包括抑制NRP1蛋白活性的物质、降解NRP1蛋白活性的物质和降低NRP1蛋白水平的基因工具中的一种或多种；所述HIF2α抑制剂包括抑制HIF2α蛋白活性的物质、降解HIF2α蛋白活性的物质和降低HIF2α蛋白水平的基因工具中的一种或多种。
进一步地，所述NRP1抑制剂包括EG00229或其衍生物；所述HIF2α抑制剂包括HIF-2α-IN-1或其衍生物。
进一步地，所述EPCR通路活化剂包括活化蛋白C。
进一步地，所述纤维化包括器官纤维化，所述器官纤维化可选地为与衰老相关的器官 纤维化。
进一步地，所述器官纤维化包括肝纤维化、肾纤维化、肺纤维化和心纤维化中的一种或多种，所述肺纤维化可选地为免疫治疗相关肺炎引起的纤维化。
进一步地，所述降低NRP1蛋白水平的基因工具包括RNA干扰、microRNA、基因编辑和基因敲除中的一种或多种；所述降低HIF2α蛋白水平的基因工具包括RNA干扰、microRNA、基因编辑和基因敲除中的一种或多种。
进一步地，所述组合物还包括药学上可接受的载体。
另一方面，本发明还提供了一种药物组合物在在制备抗纤维化剂中的应用，所述药物组合物包括：(a)NRP1抑制剂和/或HIF2α抑制剂；(b)EPCR通路活化剂，所述EPCR通路活化剂促进EPCR通路的活性。
进一步地，所述应用包括减轻受损器官纤维化程度和/或促进受损器官的再生能力，所述再生能力包括细胞增殖能力、组织修复能力和功能恢复能力中的一种或多种，所述组织修复能力可选地为无纤维化修复，所述器官可选地包括肝、肾、肺和心中的一种或多种。
进一步地，所述应用包括减轻血小板-巨噬细胞集落的形成；和/或降低血小板IL-1α、SDF1、TIMP1中的一种或多种的表达。
进一步地，所述EPCR通路活化剂用于与NRP1抑制剂和/或HIF2α抑制剂联用制备所述抗纤维化剂，所述NRP1抑制剂和/或HIF2α抑制剂用于促进EPCR的表达。
进一步地，所述应用包括降低Rho通路活性；和/或减少炎症单核细胞的沉积；和/或促进内皮微环境的促再生功能。
有益技术效果
本发明提供一种抗纤维化药物组合物及其应用。本发明技术方案所靶向的EPCR(内皮细胞蛋白C受体)，是内皮细胞特异性表达的位于细胞膜的跨膜糖蛋白，而血管内皮细胞分布在几乎所有的器官中，在生物体内数量庞大。本发明实验人员经一系列实验发现，EPCR通路的抑制是器官再生向纤维化转变的一个重要节点。本发明实验人员利用血管内皮细胞、血小板以及巨噬细胞在内的多细胞类型之间相互作用，同时干预EPCR抑制的上游(即抑制NRP1和/或HIF2α)和EPCR，在一定程度上上调EPCR，进而对EPCR抑制的下游(即IL-1α、TIMP1等)进行抑制，在一定程度上促进无纤维化的器官修复。现有技术往往仅针对单一靶点，治疗效果不佳；而直接靶向整合素或靶向TGF-β，存在安全性等问题。而本发明提供的抗纤维化药物组合物，结合循环系统易于干预(readily accessible)这一特点，能够调节多种细胞类型(即循环系统中的血管内皮细胞、血小板、巨噬细胞)产生的多种分子(例如EPCR、IL-1α、TIMP1等)，以多角度来调节受损器官的纤维化，并促进受损细胞的再生。本发明的技术方案能够抑制促纤维化的血小板IL-1α和巨噬细胞TIMP1，而巨噬细胞TIMP1又经证明，其能够通过整合素刺激成纤维细胞的活化(也可以理解为，整合素为EPCR的下游)。因此，本发明技术方案在抗纤维化方面的治疗效果明显，具体体现为：降低IL-1α、SDF1、TIMP1的表达并减轻促纤维化的“血小板-巨噬细胞”集落，抑制内皮细胞中的Rho激活、减少炎性单核细胞的沉积，以减轻受损器官纤维化程度并促进再生(包括受损器官的实质细胞增殖、组织修复、功能恢复等)、促进受损器官的无纤维化(fibrosis-free)修复等。需要强调的是，本发明的技术方案还能通过降低巨噬细胞TIMP1表达等方式，进一步抑制成纤维细胞的活化。上述治疗效果在肺、肝、肾的再生和纤维化模型及其相应衰老模型中均得到了验证。
长期以来，人们一直认为纤维化不可逆。现有技术往往仅能够在一定程度上抑制纤维化，而难以“逆转纤维化”。本发明通过靶向EPCR(即EPCR通路活化剂+NRP1抑制剂和/或HIF2α抑制剂，进而靶向EPCR)，调控EPCR的表达，进而影响循环系统中血小板IL1α和巨噬细胞TIMP1的表达，即正常化血小板-巨噬细胞-血管内皮细胞之间的异常相互作用，在一定程度上抑制纤维化并恢复受损器官的再生能力，实现“逆转纤维化”(即受损器官的 无纤维化修复，使受损器官从纤维化向再生转变)的效果。此外，本发明提供的药物组合物，还可以在一定程度上缓解免疫治疗诱导的肺炎(Immunotherapy-induced pneumonitis)。
综上所述，本发明技术方案提供的抗纤维化药物组合物及其应用，有助于在一定程度上系统地缓解或治疗，甚至逆转器官纤维化。
另一方面，本发明提供了一种EPCR通路活化剂在制备抗纤维化剂中的应用，所述EPCR通路活化剂促进EPCR通路的活性。
进一步地，所述EPCR通路活化剂包括活化蛋白C。
进一步地，所述纤维化包括器官纤维化，所述器官纤维化可选地为与衰老相关的器官纤维化。
进一步地，所述器官纤维化包括肝纤维化、肾纤维化、肺纤维化和心纤维化中的一种或多种，所述肺纤维化可选地为免疫治疗相关肺炎引起的纤维化。
进一步地，所述应用包括减轻受损器官的纤维化程度和/或促进受损器官的再生能力，所述再生能力包括细胞增殖能力、组织修复能力和功能恢复能力中的一种或多种。
进一步地，所述应用包括减轻血小板-巨噬细胞集落的形成；和/或降低血小板IL-1α、SDF1、TIMP1中的一种或多种的表达。
进一步地，所述EPCR通路活化剂用于与NRP1抑制剂和/或HIF2α抑制剂联用制备所述抗纤维化剂，所述NRP1抑制剂和/或HIF2α抑制剂用于促进EPCR的表达。
进一步地，所述NRP1抑制剂包括抑制NRP1蛋白活性的物质、降解NRP1蛋白活性的物质和降低NRP1蛋白水平的基因工具中的一种或多种；所述HIF2α抑制剂包括抑制HIF2α蛋白活性的物质、降解HIF2α蛋白活性的物质和降低HIF2α蛋白水平的基因工具中的一种或多种。
进一步地，所述NRP1抑制剂包括EG00229或其衍生物；所述HIF2α抑制剂包括HIF-2α-IN-1或其衍生物。
进一步地，所述抑制NRP1蛋白活性的物质包括小分子药物和/或抗体；所述抑制HIF2α蛋白活性的物质包括小分子药物和/或抗体。
进一步地，所述降低NRP1蛋白水平的基因工具包括RNA干扰、microRNA、基因编辑和基因敲除中的一种或多种；所述降低HIF2α蛋白水平的基因工具包括RNA干扰、microRNA、基因编辑和基因敲除中的一种或多种。
另一方面，本发明还提供一种用于评估器官纤维化的标记物组，其特征在于，所述标记物组包括NRP1、HIF2α和EPCR中的一种或多种；所述标记物组的表达量可用于评估器官纤维化程度和/或器官功能受损情况。
进一步地，所述标记物组进一步包括PlGF、SDF1、IL-1α、CXCR4、TIMP中的一种或多种。
进一步地，所述器官纤维化包括肝纤维化、肾纤维化、肺纤维化和心纤维化中的一种或多种。
进一步地，所述器官纤维化为与衰老相关的器官纤维化。
另一方面，本发明还提供一种用于评估器官纤维化的试剂盒，其特征在于，包括一种标记物组，所述标记物组包括NRP1、HIF2α和EPCR中的一种或多种。
进一步地，所述标记物组进一步包括PlGF、SDF1、IL-1α、CXCR4、TIMP中的一种或多种。
进一步地，所述试剂盒用于检测生物样本中所述标记物的基因表达水平，所述生物样本包括血液样本、血清样本、血浆样本、组织样本、器官样本和细胞样本中的一种或多种。
进一步地，所述器官纤维化包括肝纤维化、肾纤维化、肺纤维化和心纤维化中的一种或多种。
进一步地，所述器官纤维化为与衰老相关的器官纤维化。
有益技术效果
本发明提供EPCR通路活化剂在制备抗纤维化剂中的应用。本发明技术方案所靶向的EPCR(内皮细胞蛋白C受体)，是内皮细胞特异性表达的位于细胞膜的跨膜糖蛋白，而血管内皮细胞分布在几乎所有的器官中，在生物体内数量庞大。本发明实验人员经一系列实验发现，EPCR通路的抑制是器官再生向纤维化转变的一个重要节点。本发明实验人员利用血管内皮细胞、血小板以及巨噬细胞在内的多细胞类型之间相互作用，干预EPCR抑制的上游(即抑制NRP1和/或HIF2α)有助于上调EPCR，进而对EPCR抑制的下游(即IL-1α、TIMP1等)进行抑制；同理，还可以直接干预EPCR或在直接干预EPCR的同时干预EPCR抑制的上游，在一定程度上促进无纤维化的器官修复。现有技术往往仅针对单一靶点，治疗效果不佳；而直接靶向整合素或靶向TGF-β，存在安全性等问题。而本发明提供的EPCR通路活化剂在制备抗纤维化剂中的应用，结合循环系统易于干预(readily accessible)这一特点，能够调节多种细胞类型(即循环系统中的血管内皮细胞、血小板、巨噬细胞)产生的多种分子(例如EPCR、IL-1α、TIMP1等)，以多角度来调节受损器官的纤维化，并促进受损细胞的再生。本发明的技术方案能够抑制促纤维化的血小板IL-1α和巨噬细胞TIMP1，而巨噬细胞TIMP1又经证明，其能够通过整合素刺激成纤维细胞的活化(也可以理解为，整合素为EPCR的下游)。因此，本发明技术方案在抗纤维化方面的治疗效果明显，具体体现为：降低IL-1α、SDF1、TIMP1的表达并减轻促纤维化的“血小板-巨噬细胞”集落，抑制内皮细胞中的Rho通路激活、减少炎性单核细胞的沉积，以减轻受损器官纤维化程度并促进再生(包括受损器官的实质细胞增殖、组织修复、功能恢复等)、促进受损器官的无纤维化(fibrosis-free)修复等。需要强调的是，本发明的技术方案还能通过降低巨噬细胞TIMP1表达等方式，进一步抑制成纤维细胞的活化。上述治疗效果在肺、肝、肾的再生和纤维化模型及其相应衰老模型中均得到了验证。
长期以来，人们一直认为纤维化不可逆。现有技术往往仅能够在一定程度上抑制纤维化，而难以“逆转纤维化”。本发明通过靶向EPCR(或与NRP1抑制剂和/或HIF2α抑制剂联用，进而靶向EPCR)，调控EPCR的表达，进而影响循环系统中血小板IL1α和巨噬细胞TIMP1的表达，即正常化血小板-巨噬细胞-血管内皮细胞之间的异常相互作用，在一定程度上抑制纤维化并恢复受损器官的再生能力，实现“逆转纤维化”(即受损器官的无纤维化修复，使受损器官从纤维化向再生转变)的效果。此外，本发明提供的应用，还可以在一定程度上缓解免疫治疗诱导的肺炎(Immunotherapy-induced pneumonitis)。
综上所述，本发明技术方案提供的EPCR通路活化剂在制备抗纤维化剂中的应用，有助于在一定程度上系统地缓解或治疗，甚至逆转器官纤维化。
本发明涉及的主要英文缩写汇总
EPCR：Endothelial protein C receptor，内皮蛋白C受体；HIF2α：Hypoxia-inducible-factor2α，缺氧诱导因子2α；NRP1：Neuropilin1，神经纤毛蛋白1；TIMP1：Tissue inhibitor of metalloproteinases1，金属蛋白酶组织抑制剂1；IL-1α：Interleukin-1α，白介素1α；ECs：Endothelial cells，内皮细胞；PCECs：Pulmonary capillary endothelial cells，肺毛细血管内皮细胞；PlGF：Placental growth factor，胎盘生长因子；SDF1：stromal-derived factor 1，基质衍生因子1；HDAC：Histone deacetylase，组蛋白脱乙酰基酶；VPA：Valproic acid，丙戊酸；DNMT：DNA methyltransferase，DNA甲基转移酶；AZA：5-azacitidine，5-氮杂胞苷；PNX：Pheumonectomy，肺切除手术；PH：Partial hepatectomy，肝部分切除术；I/R：Ischemia-reperfusion，缺血再灌注。
如本文所使用，“促进”是指所描述对象在既有水平的进一步提高，所述既有水平包括数量水平、表达水平、功能水平、能力水平中的一种或多种。
如本文所使用，“逆转纤维化”是指将呈纤维化状态的器官向再生方向转变，包括降低纤维化的水平、降低纤维化引起的症状的严重程度、增强再生能力。
如本文所使用，“促进EPCR通路的活性”是指增强EPCR信号和/或增加EPCR的表达水平，使得EPCR与其下游分子产生作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍。显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。
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图1（图1A-图1I）为肺中衰老的内皮细胞中NRP1的异常诱导抑制再生以促进纤维化的实验图；
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图2（图2A-图2R）为衰老的肝和肾的内皮细胞中重编程的EPCR信号抑制再生并刺激纤维化的实验图；
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图3（图3A-图3H）为老年小鼠器官中受内皮细胞NRP1抑制的EPCR上调基质衍生因子1(SDF1)以募集促纤维化的巨噬细胞的实验图；
[根据细则91更正 27.12.2021]　
图4（图4A-图4K）为巨噬细胞/单核细胞来源的金属蛋白酶组织抑制剂促进成纤维细胞的活化并增强纤维化的实验图；
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图5（图5A-图5L）为血小板源性IL-1α与巨噬细胞相互作用以形成促纤维化的造血集落的实验图；
[根据细则91更正 27.12.2021]　
图6（图6A-图6L）为内皮细胞NRP1或血小板IL-1α的遗传靶向，减少博来霉素损伤后衰老的肺的纤维化的实验图；
[根据细则91更正 27.12.2021]　
图7（图7A-图7P）为内皮细胞HIF2α或血小板IL-1α的阻断减少衰老器官中免疫治疗诱导的肺炎、肝纤维化和肾纤维化的实验图；
[根据细则91更正 27.12.2021]　
图8（图8A-图8I）为肺切除术(PNX)后衰老小鼠肺的内皮细胞中受损的肺再生、神经纤毛蛋白1(NRP1)-HIF2α通路的异常诱导和受抑制的内皮蛋白C受体(EPCR)的实验图；
[根据细则91更正 27.12.2021]　
图9（图9A-图9K）为NRP1-HIF2α通路的活化，降低衰老的肝和肾EC中EPCR的表达的实验图；
[根据细则91更正 27.12.2021]　
图10（图10A-图10I）为损伤后老年器官中内皮细胞NRP1募集促纤维化的巨噬细胞的实验图；
[根据细则91更正 27.12.2021]　
图11（图11A-图11C）为所述小鼠组中巨噬细胞的表型的实验图；
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图12（图12A-图12H）为年轻小鼠中EPCR中和抗体与巨噬细胞TIMP1对肺再生和纤维化的影响的实验图；
[根据细则91更正 27.12.2021]　
图13（图13A-图13E）为内皮细胞中异常的NRP1信号，通过募集表达TIMP1的促纤维化的巨噬细胞，促进肺纤维化和衰老的实验图；
[根据细则91更正 27.12.2021]　
图14（图14A-图14E）为损伤后的老年肝和肾中，内皮细胞的异常的NRP1-HIF2α信号及TIMP1的促纤维化功能的实验图；
[根据细则91更正 27.12.2021]　
图15为APC-EPCR通路在促进无纤维化器官修复中的作用模型的示意图；
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图16（图16A-图16I）为小鼠反复吸入盐酸后，内皮细胞中EPCR的抑制和NRP1的诱导与肺纤维化有关的实验图；
[根据细则91更正 27.12.2021]　
图17（图17A-图17G）为Nrp1的阻断恢复内皮微环境中EPCR的表达，其与APC协同作用以促进酸损伤后无纤维化肺修复的实验图；
[根据细则91更正 27.12.2021]　
图18（图18A-图18F）为靶向Nrp1/HIF2α和EPCR通路，减轻肝和肾的纤维化的实验图；
[根据细则91更正 27.12.2021]　
图19（图19A-图19G）为NRP1-HIF2α抑制和APC治疗，促进长期损伤后的肝脏和肾脏的适应性修复的实验图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的 附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。
如在本说明书中使用的，术语“大约”，典型地表示为所述值的+/-5％，更典型的是所述值的+/-4％，更典型的是所述值的+/-3％，更典型的是所述值的+/-2％，甚至更典型的是所述值的+/-1％，甚至更典型的是所述值的+/-0.5％。
在本说明书中，某些实施方式可能以一种处于某个范围的格式公开。应该理解，这种“处于某个范围”的描述仅仅是为了方便和简洁，且不应该被解释为对所公开范围的僵化限制。因此，范围的描述应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如，范围1～6的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。
附图详细说明
图1：(A)表明检测肺切除手术(PNX)后年轻小鼠与老年小鼠肺泡再生策略的方案的示意图。(B-C)所示年龄的小鼠的肺泡上皮结构的恢复(B)以及羟脯氨酸量(C)。进行I型肺泡上皮细胞(AEC)标记物水通道蛋白5(AQP5)和平足蛋白(PDPN)的免疫染色和天狼星红染色。20月龄小鼠(定义为“老年”小鼠)显示出更低程度的肺泡再生和更高程度的纤维化；每组n＝8只小鼠。比例尺皆为50μm(除非另有指明)。(D)PNX后，小鼠中血小板和巨噬细胞的肺部沉积。血小板标记物CD41、巨噬细胞标记物F4/80和血管内皮细胞标记物VE-钙黏蛋白共染色。老年小鼠肺中，血小板与巨噬细胞形成细胞集落(箭头所示)。(E)PNX后，2月龄以及20月龄“老年”小鼠的肺毛细血管内皮细胞(PCECs)中改变的旁分泌/血管分泌基因的热图。(F)PNX后，老年对照组小鼠以及诱导性EC特异性删除Nrp1的小鼠(Nrp1 iΔEC/iΔEC)中内皮蛋白C受体(EPCR)和缺氧诱导因子2α(HIF2α)的表达。(G)与对照组小鼠相比，PNX后的老年Nrp1 iΔEC/iΔEC小鼠中“血小板-巨噬细胞”集落的形成减少，这种现象被EPCR中和抗体1560逆转。(H)在所示年龄的Nrp1 iΔEC/iΔEC小鼠以及Nrp1 +/+小鼠中再生和纤维化应答。评估PNX后II型肺泡上皮祖细胞的增殖。5-乙炔基-2'-脱氧尿苷(EDU)掺入(incorporation)与表面活性蛋白C(surfactant protein C，SPC)共染色。用β-半乳糖苷酶(β-Gal)活性染色检测衰老(senescence)。LY6g的免疫染色评估中性粒细胞的积聚(accumulation)。通过注射高分子量右旋糖酐(F)检测血管通透性。(I)在衰老内皮细胞中，NRP1的上调刺激HIF2α并抑制抗炎症且抗血栓的EPCR。EPCR的抑制活化血小板和巨噬细胞以形成促纤维化的造血集落。数据表示为平均值±平均值标准误差(SEM)。统计学差异由单因素方差分析(ANOVA)及随后作为事后分析的Tukey检验确定。*P<0.05。
图2：(A)检测PH后的年轻小鼠和老年小鼠的肝再生。(B-C)评估造血集落的沉积(B)以及内皮细胞中所示基因的转录水平(C)(每组n＝8-9只小鼠)。(D)PH后，所述小鼠组的肝内皮细胞中HIF2α蛋白。HIF2α的表达由与内皮细胞核标记物ETS相关基因(ERG)(E)共染色检测。(E-I)PH后，老年Nrp1 iΔEC/iΔEC小鼠以及对照组小鼠的肝脏中“血小板-巨噬细胞”集落的积聚、肝细胞增殖以及纤维化。进行天狼星红染色(E)。检测EDU掺入来评估肝细胞增殖(F)。所示小鼠中天冬氨酸转氨酶(AST)的血清浓度(G)(每组n＝7只小鼠)，血小板-巨噬细胞聚集体(aggregate)的分布(H)，和血浆胆红素浓度(I) (每组n＝9只小鼠)。(J)检测缺血再灌注(I/R)后年轻小鼠和老年小鼠的肾修复。(K-N)I/R后2月龄和20月龄“老年”小鼠在肾内皮细胞中“血小板-巨噬细胞”集落的形成(K)、所示基因的转录水平(L和M)和EPCR和NRP1蛋白的表达(N)(每组n＝6只小鼠)。(O-R)I/R后老年Nrp1 iΔEC/iΔEC小鼠以及对照组小鼠中造血集落的分布、肾纤维化、细胞增殖和肾功能的恢复(O)。评估EDU掺入来测量肾细胞的增殖(P)。共染色血小板、巨噬细胞和内皮细胞于肾载玻片(Q)，测定所述小鼠的血清肌酐(R)(每组n＝9只小鼠)。数据表示为平均值±SEM。统计学差异以单因素方差分析(ANOVA)及随后的Tukey事后检验确定。
图3：(A)SDF1报告小鼠或缺乏内皮细胞NRP1的报告小鼠的PCECs中SDF1的表达。在肺切片中SDF1表达(红色荧光蛋白)与VE-钙黏蛋白共染色。(B)PNX后的老年对照组小鼠以及EC特异性HIF2α敲除(Hif2a iΔEC/iΔEC)小鼠的PCECs中所示基因的表达(每组n＝6只小鼠)。*P<0.05，与老年对照组相对； #P<0.05，与用1560抗体处理的老年Hif2a iΔEC/iΔEC小鼠相比较。(C-D)所示小鼠中CXCR4 +巨噬细胞的募集以及II型肺泡祖细胞的增殖。巨噬细胞的定量显示为(C)(每组n＝6只小鼠)。CXCR4与F4/80共染色并且测量EDU掺入来评估细胞增殖(每组n＝6只小鼠)(D)。(E)所示小鼠中肺部巨噬细胞中细胞因子的表达(每组n＝6只动物)。(F-H)小鼠成纤维细胞或星状细胞中，Smad2磷酸化(p-Smad2)和α-SMA蛋白表达。用shRNA(sh整合素β1)沉默成纤维细胞中的整合素β1。代表性印迹图像显示为(F)，并且蛋白定量显示为(G)和(H)(每组n＝5个单个样本)。数据表示为平均值±SEM。统计学差异以单因素方差分析(ANOVA)及随后的Tukey事后检验评估。
图4：(A)PNX后缺乏Timp1(Timp1 -/-)老年小鼠以及年轻和老年对照组小鼠的肺泡上皮细胞的恢复以及胶原沉积。(B)所述小鼠的巨噬细胞中Timp1的表达。老年小鼠还用CXCR4拮抗剂AMD3100或者赋形剂(vehicle)(每组n＝5只小鼠)。(C)描述检验巨噬细胞TIMP1促进衰老相关的纤维化的作用的方法的方案的示意图。(D-F)有或没有转移所述单核细胞的年轻和老年的经肺切除的小鼠中，肺部功能的恢复(D)、动脉氧合(E)和羟脯氨酸量(F)的恢复(每组n＝9只小鼠)。N/A表示未转移单核细胞。(G-H)所示小鼠组的肺(G)以及肝脏和肾脏(H)的天狼星红染色。(I-J)经所示处理的小鼠中血浆胆红素(I)和血清肌酐(J)的浓度(每组n＝9只小鼠)。N/A表示未转移单核细胞。(K)在肺、肝和肾损伤后，老年内皮细胞中NRP1受到诱导以上调HIF2α并且抑制EPCR的诱导。形成表达SDF1的内皮微环境，以增强CXCR4 +TIMP1高表达的巨噬细胞的沉积。这些巨噬细胞可能有助于“血小板-巨噬细胞”集落的促纤维化功能，在衰老的肺、肝和肾中将再生性应答逆转成纤维化。统计学差异以单因素方差分析(ANOVA)及随后作为事后分析的Tukey检验确定。数据表示为平均值±SEM。
图5：(A-B)血小板特异性删除IL-1α小鼠(Il1a ΔPlt/ΔPlt)。Floxed IL-1α(Il1a loxP/loxP)小鼠与表达血小板特异性Pf4-cre小鼠杂交，产生Il1a ΔPlt/ΔPlt小鼠(A)。比较所述组在PNX、PH或肾I/R后，TIMP1的表达以及“血小板-巨噬细胞”集落的形成(B)。(C-E)年轻对照组以及Il1a ΔPlt/ΔPlt小鼠在所示处理后，“血小板-巨噬细胞”集落在肺(C)、肝(D)、肾(E)的沉积(每组n＝4只小鼠)。(F-G)检测(F)和定量(G)所示小鼠组中TIMP蛋白的表达(每组n＝4只小鼠)。(H)检验衰老的器官中血小板源性IL-1α对纤维化的影响的策略。Il1a +/+和Il1a -/-血小板注入到老年Thpo -/-小鼠中。评估受试小鼠中TIMP1的表达和胶原沉积。(I-K)在受试小鼠的衰老器官中，Il1a -/-(而不是Il1a +/+)抑制TIMP1的表达(I)，并减少再生到纤维化的转变。定量不同组的TIMP1表达(每组n＝4只小鼠)。(L)衰老器官中重编程的内皮细胞EPCR信号导致血小板活化。由血小板提供的IL-1α刺激募集的巨噬细胞，促进促纤维化的“血小板-巨噬细胞”集落的形成，并且在损伤后抑制再生至促进纤维化。数据表示为平均值±SEM。统计学差异以单因素方差分析(ANOVA)及随后作为事后分析的 Tukey检验。
图6：(A)通过向老年小鼠气管内注射博来霉素(Bleo)来诱导肺损伤和纤维化的实验流程。(B)博来霉素损伤后，老年小鼠肺的PlGF蛋白表达(每组n＝6只小鼠)。(C)博来霉素损伤后，所示小鼠组中HIF2α和EPCR的蛋白表达。(D)博来霉素损伤后，肺的肺泡上皮细胞的分布、SMA的表达、CXCR4 +巨噬细胞的积聚和衰老相关的β-半乳糖苷酶。(E-F)检测博来霉素损伤后的所示小鼠的I型胶原的蛋白水平(E)以及肺部功能的恢复(F)(每组n＝10只小鼠)。(G)博来霉素损伤后，过继转移血小板和单核细胞到小鼠的示意图。(H-I)所述小鼠中“血小板-巨噬细胞”聚集体的沉积(H)及肺TIMP1的表达(I)。博来霉素损伤的小鼠中，血小板Il1a -/-的移植抑制了造血血管集落(hematopoietic-vascular rosette)的形成以及TIMP1的表达(每组n＝4只小鼠)。(J-K)博来霉素损伤后，移植所示单核细胞的老年受试小鼠进行羟脯氨酸量的测量(J)和天狼星红染色(K)。N/A表示未转移单核细胞。(每组n＝6只小鼠)。(L)衰老内皮细胞中抗血栓的EPCR信号的抑制，导致血小板来源的IL-1α的产生，其与表达促纤维化的TIMP1的血管周围的巨噬细胞相互作用。数据表示为平均值±SEM，统计学差异以单因素方差分析(ANOVA)及随后的Tukey事后检验确定。
图7：(A)免疫治疗诱导的肺炎的小鼠模型。用抗程序性细胞死亡蛋白1(PD1)抗体和幅射处理年轻和老年小鼠。测量动物发病率以及肺纤维化。(B-C)所示小鼠的肺胶原沉积(B)以及动物生存率(C)(每组n＝10只小鼠)。(D-F)所示组中肺胶原沉积(D)、支气管肺泡灌洗液(bronchioalveolar lavage fluid，BALF)中IL-1α的浓度(E)、和动物生存率(F)。老年小鼠被移植血小板并接受所示处理(每组n＝8-10只小鼠)。(G)检测免疫治疗后肺中血小板Il1α对纤维化的影响和肿瘤进展的方法。向老年小鼠注射lewis肺癌(LLC)细胞、移植所示血小板，并接受所述处理。(H-I)所示处理后，评估肺肿瘤负荷、胶原沉积(H)和肺羟脯氨酸量(I)(每组n＝10-12只小鼠)。(J)已移植血小板的小鼠的存活曲线(每组n＝10只小鼠)。(K)通过分别注射四氯化碳(CCl 4)和注射叶酸诱导出的肝和肾纤维化。(L)CCl 4损伤后小鼠肝脏的PlGF蛋白量(每组n＝8只小鼠)。(M)所述小鼠的肝中I型胶原的蛋白量。(N)所示小鼠组中肝、肾的天狼星红染色。(O)CCl 4或叶酸处理后，移植所示血小板的老年受试小鼠中造血集落的形成。(P)CCl 4损伤后，注射所述单核细胞的老年受试小鼠的肝羟脯氨酸(每组n＝6只小鼠)。数据表示为平均值±SEM，统计学差异以单因素方差分析(ANOVA)及随后的Tukey事后检验确定。
图8：是对图1实验的补充，与其相关。(A-C)PNX后的所示年龄小鼠的肺部功能的恢复、胶原和“血小板-巨噬细胞”集落的沉积。测量血液氧合和吸气量。进行天狼星红染色和I型胶原免疫印迹，并检测“血小板-巨噬细胞”聚集体的形成。20月龄(老年)小鼠展示出更低的肺泡再生和更高的纤维化。在所有附图中，点图中的每个点表示来自单个动物的数据。Sh表示假手术组；PNX表示肺切除术组。(D)与2月龄小鼠肺相比，PNX后的衰老的小鼠肺的肺毛细血管内皮细胞(PCECs)中，改变的旁分泌相关基因。每组N＝5-6只。图8中比例尺为50μm。**，与老年组相对，P<0.05。统计学差异由单因素方差分析(ANOVA)及随后的Tukey事后检验确定。(E)比较老年对照组小鼠与诱导性内皮细胞特异性删除Nrp1(Nrp1 iΔEC/iΔEC)小鼠之间，肺和肝内皮细胞中神经纤毛蛋白1的表达。(F)PNX后，对照组小鼠与老年Nrp1 iΔEC/iΔEC小鼠中EPCR和HIF2α的表达。分别比较3月龄小鼠(年轻)与20月龄小鼠(老年)。每组N＝5只小鼠。(G)与对照组小鼠相比，老年Nrp1 iΔEC/iΔEC小鼠的“血小板-巨噬细胞”集落的形成减少，而这种现象被EPCR中和抗体1560逆转。每组N＝6只小鼠。(H)所示小鼠组肺中羟脯氨酸量、β-半乳糖苷酶(β-gal)活性以及p16的表达。*P<0.05，统计学差异由单因素方差分析(ANOVA)及随后的Tukey事后检验确定。(I)PNX后，所示小鼠组的血管生长和灌注、血管周围中性粒细胞的分布、血管渗漏(vascular leak)和肺II型肺泡祖细胞的增殖。向小鼠静脉注射B4同工凝集素(B4-isolectin)和伊文思蓝(Evans  blue)。对肺中同工凝集素 +血管和残留的伊文思蓝定量。比例尺为50μm。
图9：是对图2实验的补充，与其相关。(A)检测部分肝切除术(PH)后年轻和老年小鼠的肝再生。测量所示组中肝中羟脯氨酸量。(B-C)PH或肾缺血再灌注(I/R)后，所示组的血管周围中性粒细胞的分布。(D)假手术(sham)以及PNX后，年轻Nrp1 +/+、老年Nrp1 +/+，以及老年Nrp1 iΔEC/iΔEC小鼠的肺ECs中EPCR蛋白水平。EPCR蛋白定量以柱状图显示。每组N＝4-5只小鼠。(E)PNX和PH后，所示小鼠组的肺和肝中PlGF蛋白量。每组N＝4只小鼠。(F)注射PlGF和等量的血小板源性生长因子BB(PDGF)、肝细胞生长因子(HGF)、血管内皮细胞生长因子165(VEGF)、肿瘤生长因子β1(TGFβ1)后，所示小鼠组肺内皮细胞的EPCR蛋白水平。N＝4。(G)3月龄对照组、Nrp1 iΔEC/iΔEC小鼠以及内皮细胞特异性诱导性敲除HIF2α(Hif2a iΔEC/iΔEC)小鼠的肺ECs中PlGF介导的EPCR蛋白的抑制。每组N＝4。(H)所示小鼠的肝ECs中通过PlGF的EPCR抑制。每组N＝4只。(I)PNX、PH或肾I/R后，所示小鼠的内皮细胞中NRP1蛋白的表达。用组蛋白脱乙酰基酶(HDAC)抑制剂(VPA)、DNA甲基转移酶(DNMT)抑制剂氮杂胞苷(AZA)或VPA与AZA的联合注射(VPA+AZA)处理老年小鼠。veh表示赋形剂。N＝5。(J)所示小鼠组中PlGF的蛋白量。veh表示赋形剂；每组N＝5-6只小鼠。(K)在老年小鼠器官中，损伤后表观遗传上调的PlGF-NRP1通路，抑制内皮细胞EPCR的诱导。
图10：对图3实验的补充，与其相关。(A)PNX后，老年Nrp1 iΔEC/iΔEC小鼠以及对照组小鼠的肺毛细血管内皮细胞中所示基因的表达。N＝5。(B)PNX后，SDF1在所示小鼠的肺中成纤维细胞中的转录表达。N＝6。(C-D)PNX后，小鼠肺中CXCR4 +巨噬细胞的募集。通过CXCR4与F4/80的共染色，检测表达CXCR4的巨噬细胞。N＝6。(E)所述小鼠组中肺细胞增殖。(F)PH后，所示小鼠肝中的羟脯氨酸量。统计学差异由单因素方差分析(ANOVA)及随后的Tukey事后检验确定。(G)老年Nrp1 iΔEC/iΔEC小鼠以及年轻和老年对照组小鼠的内皮细胞中所示基因的表达。N＝5。*P<0.05，与老年Nrp1 +/+组相对；#P<0.05，与用1560处理的老年Nrp1 iΔEC/iΔEC小鼠相比。(H)所示小鼠的CXCR4 +巨噬细胞的数量。PH和肾I/R后的肝脏和肾脏切片，用于CXCR4和F4/80染色。N＝5。(I)正常化老年PCECs中EPCR信号，阻断血管微环境的促纤维化功能。PNX后，老年小鼠PCECs中NRP1-HIF2α信号，抑制EPCR以诱导SDF1表达。在老年、经肺切除的肺中PCECs中SDF1的表达，募集促纤维化的TIMP1 +血管周围巨噬细胞，以阻碍肺泡再生，并且促进纤维化。
图11：是对图4实验的补充，与其相关。(A)单个标记物在所述巨噬细胞中的表达。从PNX、PH或肾脏I/R后的3月龄(年轻)和老年小鼠的肺、肝和肾中分离出巨噬细胞。(B)来自衰老器官的老年对照组以及Hif2a iΔEC/iΔEC小鼠的巨噬细胞中所示基因的表达。(C)所示小鼠组的肺、肝和肾中F4/80与CD86的共染色。比例尺为50μm。
图12：是对图5实验的补充，与其相关。(A-B)向PNX后的3月龄小鼠(年轻)注射EPCR中和抗体1560。检测I型肺泡上皮细胞的恢复以及血液氧合。(C-D)PNX以及注射1560后，年轻的肺的胶原沉积以及羟脯氨酸水平。年轻小鼠为3月龄。N＝6，比例尺为50μm。(E)相对于用同型匹配的(isotype-matched)IgG处理的小鼠，注射EPCR抗体后的年轻小鼠肺中“血小板-巨噬细胞”聚集体的增加的积聚。(F)检测EPCR阻断后巨噬细胞TIMP1对肺纤维化的作用的策略。注射EPCR抗体后，从Timp1-无效(Timp1 -/-)以及对照组Timp1 +/+小鼠中分离出单核细胞，并转移到年轻小鼠中。(G-H)EPCR抗体的阻断后，Timp1 -/-(而不是Timp1 +/+)单核细胞的移植，减少了肺纤维化的增加。分析小鼠肺中胶原沉积和羟脯氨酸量。N＝5，比例尺为50μm。
图13：是对图6实验的补充，与其相关。(A)所示基因型的小鼠组中，CXCR4 +巨噬细胞的募集及气管内注射博来霉素(Bleo)后的处理。(B-C)Bleo损伤后，所示小鼠组中β-半乳糖苷酶和p16的表达水平。每组N＝5-6只小鼠。(D-E)Bleo损伤后，老年Timp1 -/-小鼠以及年轻和老年Timp1 +/+小鼠的羟脯氨酸量、肺泡上皮细胞再生以及I型胶原的沉积。年 轻小鼠为3月龄小鼠，比例尺为50μm。
图14：是对图7实验的补充，与其相关。(A)CCl 4或叶酸损伤后，所示基因型的小鼠组的肝和肾中HIF2α和EPCR的表达。(B)CCl 4损伤后，所示小鼠组的肝中羟脯氨酸量。(C)CCl 4或者叶酸处理后，已移植Il1a -/-或者Il1a +/+血小板的老年宿主/受试Thpo -/-小鼠中的促纤维化的TIMP1的表达。在肝或者肾损伤后，已转移Il1a -/-血小板的Thpo -/-小鼠中，TIMP1在肝或肾中的表达不受EPCR抗体的影响，这意味着血小板产生的Il1a是EPCR抑制的下游。(D)CCl 4或叶酸处理后，已移植Timp1 -/-或Timp1 +/+单核细胞的老年WT小鼠中胶原沉积。(E)胆管结扎(BDL)(一种胆汁淤积性肝损伤模型)后，已转移所示单核细胞的老年WT小鼠的肝羟脯氨酸和天狼星红染色。比例尺为50μm。
图16：(A)气管内单次或多次滴注盐酸(酸)诱导肺损伤的实验方案。(B-C)酸滴注后，评估受损肺的呼吸功能(吸气量)的恢复和纤维化(羟脯氨酸)。每个点表示来自单个小鼠的数据。(D)用天狼星红染色法检测所示小鼠肺的胶原蛋白沉积。(E-H)在每次酸注射后，肺内皮细胞中EPCR、PAR1、NRP1和HIF2的表达。按先前方法分离肺内皮细胞，并通过定量PCR检测基因表达。每组N＝6-8只小鼠。(I)免疫染色检测所示小鼠组的肺中EPCR和NRP1蛋白。比例尺＝50μm。
图17：(A)内皮细胞特异性敲除Nrp1的小鼠的VE-钙黏蛋白 +(VE-cadherin +)肺内皮细胞中EPCR和HIF2蛋白的表达。将Floxed Nrp1小鼠与表达他莫昔芬反应性(tamoxifen-responsive)内皮细胞特异性VE-钙黏蛋白-Cre ERT2(VE-cadherin-Cre ERT2)的小鼠杂交。用他莫昔芬处理小鼠，诱导内皮细胞中Nrp1的特异性缺失。以Nrp1内皮单链缺失(haplodeficiency)小鼠为对照。用免疫染色法检测，小鼠在酸损伤后所示时间内HIF2和EPCR的表达。(B)HIF2和EPCR在对照组小鼠和内皮特异性敲除Nrp1基因的小鼠肺内皮细胞中的转录表达。每组N＝6只小鼠。(C)描述检测Nrp1抑制剂、活化蛋白C(APC)和Nrp1抑制剂和APC的联合(Nrp1抑制剂+APC)的治疗效果的策略的方案。(D)Nrp1抑制剂+APC治疗促进肺修复并绕过纤维化。在所示时间点检测小鼠肺中呼吸功能的恢复和羟脯氨酸的沉积。值得注意的是，Nrp1抑制剂+APC的治疗效果被EPCR阻断抗体1560减弱。(E)用Nrp1抑制剂、APC或APC+Nrp1抑制剂治疗后，酸损伤小鼠的肺功能(吸气量)的恢复。(F-G)Nrp1抑制剂+APC联合治疗，减少酸损伤小鼠肺中I型胶原沉积(F)和减少F4/80 +巨噬细胞/单核细胞(G)的募集。同时，如磷酸化肌球蛋白轻链(phosphorylated myosin light chain，p-MLC)和VE-钙黏蛋白共染色显示，Nrp1抑制剂+APC降低了肺内皮细胞中Rho激活。比例尺＝50μm。
图18：(A)分别通过重复注射CCl 4和叶酸注射液诱导肝、肾纤维化。通过每3天腹腔注射8次CCl 4诱导小鼠肝纤维化，通过叶酸诱导小鼠肾纤维化。(B)内皮细胞中EPCR抑制和Nrp1诱导的表达与肝、肾纤维化有关。(C)HIF2α抑制剂HY和APC的联合，阻断肝、肾纤维化。小鼠肝和肾纤维化模型中检测了靶向Nrp1(或HIF2α)和EPCR的“双管齐下”策略的治疗效果。用APC、Nrp1抑制剂EG、HIF2α抑制剂HY或EPCR中和抗体1560处理小鼠。(D)HIF2、EPCR、Nrp1在损伤和所述处理后所示时间点的肝脏和肾脏中的表达。EC标记物VE-钙黏蛋白和ERG也被染色以检测被测分子的血管分布。(E-F)在注射CCl 4和叶酸及所示处理后，肝和肾功能的恢复。
图19：(A)ECs中EPCR的抑制和NRP1-HIF2α的激活，与肝脏和肾脏纤维化有关。EC标记物VE-钙黏蛋白被染色以确定被检测的分子的血管分布。在图19中，比例尺＝50um。(B)在损伤和所述处理后，Nrp1 iΔEC/iΔEC小鼠的肝脏和肾脏中HIF2α和EPCR的表达。(C-D)在第8次CCl4后的第20天，用所示处理的损伤的肝脏中肝脏羟脯氨酸量。每组N＝8-9只小鼠。HY，HIF2α抑制剂，HIF-2α-IN-1(HY-19949)。(E)叶酸注射和所述处理后第100天的肾脏中血清肌酐量。每组N＝8只小鼠。(F)叶酸注射后第100天，用所述处理的受损小鼠肾脏中纤维化面积的百分比。每组N＝6只小鼠。(G)用所述处理的受损小鼠肝脏和肾脏 中CXCR4 +巨噬细胞的分布。CCl4和叶酸注射的所述处理后，肝脏和肾脏切片被CXCR4、F4/80和VE-钙黏蛋白染色。
实施例一：材料和方法
表1关键资源
Figure PCTCN2021136240-appb-000001
Figure PCTCN2021136240-appb-000002
动物养护和所用小鼠品系。Floxed神经纤毛蛋白1(Nrp1)小鼠和HIF2α(Hif2a)小鼠以及缺乏金属蛋白酶组织抑制剂1(Timp1)的小鼠、血小板因子-Cre(Pf4-Cre)小鼠获自杰克逊实验室。表达EC特异性Cdh5-(PAC)-Cre ERT2/VE-钙黏蛋白-Cre ERT2的小鼠由Ralf Adams博士提供；该小鼠品系与floxed Nrp1和Hif2a小鼠杂交以产生Nrp1 iΔEC/iΔEC、Hif2a iΔEC/iΔEC和对照小鼠。腹腔内(i.p.)注射剂量为250mg/kg的他莫昔芬处理4周龄小鼠10天，并在第3次和第6次给药后中断一周(或以150mg/kg为剂量处理6天，并在第3次后中断3天)。定量聚合酶链反应(PCR)证实ECs中目标基因的删除。Nrp1 iΔEC/iΔEC小鼠和Hif2a iΔEC/iΔEC小鼠以及年龄一致 的同窝小鼠用于小鼠肺、肝和肾的损伤模型。Floxed Il1a小鼠由中国南京的集萃药康生物科技股份有限公司(GemPhamatech)用CRISPR/Cas9系统生成。共同注射Cas9mRNA、sgRNA和供体到受精卵中。sgRNA引导在内含子2-3和4-5中的Cas9核酸内切酶切割，并产生双链断裂。这种断裂通过同源重组导致loxP位点，并且外显子3和外显子4的区域被两侧插入loxP位点(floxed)。Il1a floxed小鼠与表达血小板因子4-Cre的小鼠交配。由此，血小板中两个loxP位点之间的序列被特异性删除，且Il1a基因被移码突变破坏。为了刺激EPCR信号，每3天对小鼠腹腔内注射(i.p.)0.1mg/kg APC。同时为了验证EPCR通路的特异性，通过每3天对小鼠腹腔内注射(i.p.)1.5mg/kg中和抗体1560阻断EPCR信号。为了抑制NRP1或HIF2α，每3天分别对小鼠腹腔内注射(i.p.)50mg/kg EG00229或25mg/kg HIF-2α-IN-1(HY-19949)。
本发明实施例中选择EG00229及其衍生物(EG00229(N 2-[[3-[(2,1,3-Benzothiadiazol-4-ylsulfonyl)amino]-2-thienyl]carbonyl]-L-arginine)和EG 00229trifluoroacetate(N 2-[[3-[(2,1,3-Benzothiadiazol-4-ylsulfonyl)amino]-2-thienyl]carbonyl]-L-arginine trifluoroacetate)和HIF-2α-IN-1(3-[[(3R)-4-(Difluoromethyl)-2,2-difluoro-3-hydroxy-1,1-dioxo-3H-1-benzothiophen-5-yl]oxy]-5-fluorobenzonitrile)的主要原因是上述抑制剂能够在体内有效抑制NRP1和HIF2α，从而可以用来证明抑制NRP1和HIF2α后所能产生的技术效果。但对本领域技术人员而言，本发明技术方案明显地并不局限于采用上述抑制剂。通过图1-3、6-11、13-14、17、19等敲除NRP1或HIF2α的实验结果可知，只要是能够有效抑制NRP1和HIF2α的物质(包括合适的小分子药物、抗体)或者可以通过对相应基因(例如，NRP1、HIF2α)进行编辑或敲除达到上述抑制效果的技术方案，都应在本发明的保护范围之内。同样地，本发明实施例中选择使用活化蛋白C(APC)的原因在于APC能够在体内有效刺激EPCR的信号并促进EPCR的表达。但通过EPCR中和抗体1560对EPCR信号进行阻断后，APC的有益效果被阻断的实验结果可以看出，本发明技术方案的关键是激活EPCR信号通路。因此，对本领域技术人员而言，任何能够刺激EPCR信号和/或促进EPCR的表达的技术方案，都应在本发明的保护范围之内，并不局限于采用APC。所有动物实验均按照四川大学的机构动物护理和使用委员会(Institutional Animal Care and Use Committee)批准的实验方案进行。
小鼠肺再生与修复模型。为了检测小鼠肺泡再生和修复，调整了左肺PNX(参考文献19)和肺损伤(参考文献7)模型。用氯胺酮(ketamine)(100mg/kg)和甲苯噻嗪(xylazine)(10mg/kg)的混合物麻醉小鼠。当左肺叶被切除并在肺门周围缝合时进行PNX。从PNX后的20月龄(认定为老年)和3月龄(认定为年轻)的小鼠中纯化得到PCECs，并分离总RNA和按描述对其进行转录组分析。采用气管内注射2U/kg的博来霉素来诱导肺损伤。为了阻断EPCR信号，在PNX和Bleo注射后，向小鼠腹腔内注射1.5mg/kg的EPCR中和抗体1560(参考文献46)。还向PNX和PH后的年轻Nrp1 iΔEC/iΔEC、Hif2a iΔEC/iΔEC和对照小鼠施用20μg/kg的小鼠PlGF或相同摩尔量的HGF和PDGF-BB(Peprotech)。另外，还通过反复的气管内盐酸注射模型来诱导肺损伤(参考文献76)。对麻醉后的小鼠进行经口气管滴注(Orotracheal instillation)，并滴注20μl 0.1M盐酸(pH 1.0)的等渗溶液(iso-osmolar solution)。每次注射后，观察小鼠以确保其从麻醉中完全恢复，并使用外部热源保持其体温。恢复后，小鼠被转移到可以获得食物和水的通风的笼子里。在PNX和第1次酸注射后每3天，对小鼠施用0.1mg/kg APC、50mg/kg NRP1抑制剂、25mg/kg HIF2α抑制剂和1.5mg/kg 1560。在所述的时间点，如前所述(参考文献7)检测受处理的小鼠动脉血中的肺功能和氧张力。为了测量细胞增殖，在处死前1小时，向小鼠腹腔内注射100mg/kg的5-乙炔基-2'-脱氧尿苷(EDU)，并且通过EDU细胞增殖试剂盒检测EDU掺入。在所有手术流程后，通过向小鼠腹腔内注射来补充1mL PBS。
免疫治疗诱导的肺炎的小鼠模型。在幅射后1个月，每4天、以10mg/kg的剂量向小鼠注射PD1抗体。监测小鼠的存活，并在幅射后3个月得到小鼠的肺组织。如前所述(参考文献19)，收集BALF。为了检测荷瘤小鼠中免疫治疗诱导的肺炎，通过颈静脉血管、将200 万个Lewis肺癌(LLC)细胞移植到老年Thpo -/-小鼠。为了检测血小板IL1α的作用，在注射LLC细胞后，将Il1a +/+或Il1a -/-血小板移植到受试小鼠中。小鼠同样接受PD1抗体和幅射处理。血小板移植后，在所示时间分析受试小鼠中肺纤维化、肿瘤负荷和动物存活率。
肺成纤维细胞和肝星状细胞的TIMP1处理。肺和肝成纤维细胞来自ScienceCell研究实验室(ScienceCell Research Laboratories)，并如前所述培养(参考文献8)。肺和肝成纤维细胞用20ng/ml的TIMP1、10ng/ml的TGFβ1或20ng/ml的TIMP1和10ng/ml的TGFβ1处理。为了确定β1整合素在TIMP1介导的成纤维细胞的活化中的作用，通过shRNA使成纤维细胞中β1整合素沉默。处理后，回收肺和肝成纤维细胞用于通过免疫印迹分析Smad2磷酸化和SMA蛋白表达。Smad2磷酸化和SMA蛋白表达通过免疫印迹的光密度法定量。
小鼠肝脏再生与修复模型。通过PH诱导小鼠肝再生。对麻醉的小鼠进行中线剖腹手术，切除三个最前叶(右内侧叶、左内侧叶和左外侧叶)。打开腹部并暴露肝脏后，抬起将被切除的左叶。将5-0丝缝合线放置在所述叶下并定位到所述叶的起点。切断缝合处远端的打结叶，并对其他叶重复此流程以完成PH。手术切除70％的肝脏块(liver mass)后，封闭皮肤。如前所述(参考文献8)，注射CCl 4用于诱导肝损伤。CCl 4在油(Sigma-Aldrich)中稀释以生成40％(0.64mg/ml)的浓度，并以1.6g/kg的剂量向小鼠腹腔注射。在PH和第1次CCl 4注射后每3天，对小鼠注射0.1mg/kg APC、50mg/kg NRP1抑制剂、25mg/kg HIF2α抑制剂和1.5mg/kg 1560。PH后的所示时点，通过测量胆红素和AST的血清水平来评估肝功能的再生(参考文献8)。
小鼠肾脏再生与修复模型。采用描述的(参考文献72)小鼠肾I/R模型。麻醉小鼠后，通过在双肾上、30分钟的腹膜后途径(retroperitoneal approach)诱导缺血。假手术是在不诱导缺血下暴露肾脏进行的。为了产生肾纤维化，如前所述(参考文献74)，以250mg/kg的剂量向小鼠单次腹腔注射叶酸。在肾I/R和叶酸注射后第40天后每3天，对小鼠注射0.1mg/kg APC、50mg/kg NRP1抑制剂、25mg/kg HIF2α抑制剂和1.5mg/kg 1560。在所示时间，通过苦味酸法测量血清肌酐浓度，并通过天狼星红和苏木精共染色分析组织形态和纤维化。
生长因子的注射与HDAC和DNMT抑制剂的治疗。3月龄(年轻)小鼠接受PNX、PH或肾脏I/R，并每三天注射20μg/kg的PlGF或等摩尔量的PDGF-BB、HGF、VEGF165或TGFβ1。3月龄Nrp1 +/+、Nrp iΔEC/iΔEC或Hif2a iΔEC/iΔEC小鼠同样用PlGF处理。为了检测PlGF和NRP1的调节，用10mg/kg的HDAC抑制剂VPA、0.5mg/kg的DNMT1抑制剂氮杂胞苷(AZA)或VPA和AZA的联合治疗(VPA+AZA)处理老年小鼠。通过免疫印迹法检测EPCR和NRP1蛋白在分离出的肺、肝和肾ECs中的表达，并通过ELISA法检测已匀浆的器官中PlGF量。
免疫荧光(IF)和形态计量分析。得到用于组织学分析的小鼠肺、肝和肾组织。封闭(5％驴血清/0.3％Triton X-100)10μm厚的组织冰冻切片并孵育在一抗中，4℃过夜：抗VE-钙黏蛋白多克隆抗体(pAb，2μg/ml，R&D Systems,MN)、抗SMA抗体(pAb，2μg/ml，Abcam，CA)、抗EPCR单克隆抗体(mAb 1560，5μg/ml)、抗平足蛋白(mAb，5μg/ml，R&D，MN)、抗水通道蛋白5(pAb，5μg/ml，Abcam，CA)、抗CD41(mAb，5μg/ml，BD Bioscience)。在荧光团缀合的(fluorophore-conjugated)二抗(2.5μg/ml，Jackson ImmunoResearch，PA)中孵育后，使用Prolong Gold封片剂(Invitrogen)与DAPI进行核染色。为了确定准备好的组织切片中免疫荧光染色信号，在五个不同的高倍视野上独立评估并定量每张载玻片中的荧光细胞，其代表单个标本的结果。
组织纤维化测定。在损伤后的所示时间测定肺、肝和肾的纤维化应答。组织在组织裂解缓冲液中均质。对获得的组织裂解物进行胶原蛋白I的免疫印迹。比较不同组之间胶原蛋白I的蛋白水平。对石蜡包埋的组织切片进行天狼星红和苏木精染色，来确定组织形态和胶原沉积及分布(参考文献8)。天狼星红阳性的纤维化的实质(parenchyma)是由每个切片的五个随机视野中确定并定量的。定量肝和肺中羟脯氨酸量，以确定纤维化的程度。
巨噬细胞/单核细胞的消耗(depletion)、分离和过继转移。用氯膦酸盐脂质体法选择性消耗小鼠的巨噬细胞/单核细胞(参考文献7)。PNX、PH或肾I/R的两天前及其后的每十天，将30μL氯膦酸盐包裹在脂质体(阴离子氯氟松，Clophosome-A)或空对照脂质体(Formu Max，Palo Alto，CA)中，静脉注射到小鼠体内，在慢性肺损伤期间消耗巨噬细胞/单核细胞。PNX、PH或肾I/R后，通过抗体包被的磁珠分离，分离出所示小鼠组织的巨噬细胞/单核细胞。通过定量PCR检测基因的表达。为了检测表达TIMP1的巨噬细胞/单核细胞在器官修复中的作用，从WT或Timp1 -/-小鼠的骨髓中分离出单核细胞。PNX、PH、肾I/R或注射CCl 4、博来霉素或叶酸的后1天，分别将3×10 6个WT和Timp1 -/-单核细胞以静脉注射注入已消耗巨噬细胞的WT小鼠。在第一次移植后重复所述单核细胞的过继转移，直到处死受试小鼠。在接受WT或Timp1 -/-单核细胞的不同组之间比较肺的、肝的、肾的功能与纤维化。通过天狼星红和苏木精染色检测胶原沉积和形态学特征。
小鼠血小板过继转移模型。采用先前描述(参考文献24)中的通过颈静脉注入血小板的策略。分离并浓缩血小板特异性删除Il1a(Il1a ΔPlt/ΔPlt)小鼠的血小板以获得Il1a -/-血小板。野生型的血小板用作对照血小板。简而言之，用含有0.5mL ACD(12.5g/L柠檬酸钠、10.0g/L D-葡萄糖和6.85g/L柠檬酸)的注射器从麻醉的小鼠处采集血液。将收集的血液转移到含有150mM NaCl和20mM PIPES的5mL缓冲液中，并然后以100g离心15分钟。离心后，收集富含血小板的上清液。上清液再以1000g离心10分钟。将获得的血小板沉淀重悬并计数。在15分钟内，通过受试小鼠暴露的颈静脉注入2×10 9个血小板。
图像获得与分析。荧光图像记录在AxioVert LSM710共聚焦显微镜(蔡司)上。切片的分析记录在奥林巴斯BX51显微镜(Olympus America，NY)上。使用带有校准标准曲线的ImageJ软件进行免疫印迹图像的光密度分析。
定量和统计分析。用Prism 8软件包(GraphPad)进行计算。对于具有2个以上的组的数据集，采用单因素方差分析(one-way ANOVA)及随后的Tukey事后检验来确定显著性差异。实施例的统计学细节可以在图例中找到。所有数据均以平均值±平均值标准误差(SEM)表示。误差条(error bar)显示SEM，并且中心显示平均值。将p值<0.05视为统计学显著。
实施例二：老年小鼠肺中“再生向纤维化”的转变与“血小板-巨噬细胞”集落的形成以及内皮细胞的重编程有关
本发明实验人员用肺切除手术(PNX)来比较不同年龄的小鼠的肺泡再生和纤维化情况(图1A)，包括肺功能的恢复、上皮结构和羟脯氨酸量(图1B-C，图8A-B)。与2、3以及6月龄小鼠相比，PNX后的20月龄小鼠显示肺泡再生的显著抑制和增加的纤维化。因此，本发明实验人员将20月龄小鼠定义为“老年”小鼠，并比较老年小鼠与2月龄(2-mon)或3月龄(3-mon)小鼠的再生能力。
血小板和巨噬细胞在器官修复中起着重要作用。基于此，本发明实验人员将PNX后的小鼠肺中的CD41 +血小板和F4/80 +巨噬细胞的沉积染色(图1D)，发现PNX后的2月龄和3月龄小鼠肺中血小板的沉积极少；相反，老年小鼠肺中血小板与巨噬细胞联合，形成血管周围细胞集落(perivascular cellular rosettes)(图8C)。这些数据表明，血小板和巨噬细胞之间失调的相互作用可能有助于衰老的肺中“再生向纤维化”的转变。
在血管腔内侧的内皮细胞(ECs)形成抗炎症和抗血栓的界面，以维持器官的稳态。因此，本发明实验人员假设：ECs中的关键节点分子的重编程，活化血小板和巨噬细胞以促进纤维化。为了确定血管ECs在衰老相关的纤维化中的作用，本发明实验人员分离了PNX后2月龄和20月龄的小鼠的肺毛细血管内皮细胞(PCECs)。在转录谱(transcription profiles)的基础上(图1E)，比较了EPCR的表达，EPCR在ECs中是选择性表达的，神经纤毛蛋白1(NRP1)和HIF2α二者都是通过病理条件在ECs中诱导出(图8D)。相比于2月龄、3月龄与6月龄的小鼠，EPCR的表达在PNX后的老年小鼠的PCECs中更低。相反，NRP1和HIF2α的表达在PNX后的老年小鼠的PCECs中升高。因此，PNX后的衰老的肺似乎展示改 变的血管分泌因子信号。
实施例三：老年肺中，正常化内皮细胞中的NRP1-HIF2α-EPCR回路减少“再生向纤维化”的转变
NRP1是多种细胞因子的共受体。为了探究NRP1在衰老相关的纤维化中的作用，本发明实验人员构建了内皮细胞中NRP1特异性缺乏的小鼠(Nrp1 iΔEC/iΔEC)。Floxed NRP1小鼠与表达EC特异性的血管内皮细胞-钙黏蛋白(VE-cadherin)(Cdh5)-Cre ERT2的小鼠杂交。后代的他莫昔芬(Tamoxifen)处理，将后代的内皮细胞中NRP1删除(图8E)。老年Nrp1 iΔEC/iΔEC小鼠接受PNX，并且3月龄(年轻)小鼠与老年Nrp1 +/+小鼠作为对照。与老年对照小鼠相比，PNX后的老年Nrp1 iΔEC/iΔEC小鼠的PCECs中显示更低的HIF2α表达和更高的EPCR水平(图1F，图8F)。经肺切除的老年肺中，内皮细胞NRP1的删除抑制了“血小板-巨噬细胞”集落的形成(图1G，图8G)。由于老年Nrp1 iΔEC/iΔEC小鼠中EPCR表达上调，实验人员假定：老年Nrp1 iΔEC/iΔEC小鼠中血管周围造血集落的形成的减少，依赖于EPCR的恢复。实际上，EPCR中和抗体1560阻断EPCR，恢复了老年Nrp1 iΔEC/iΔEC小鼠的细胞集落的形成，这说明在EPCR的抑制以及随后血小板和巨噬细胞的活化中，NRP1的诱导的上位作用。
为了检验所述假设，本发明实验人员评估了老年Nrp1 iΔEC/iΔEC小鼠肺中再生的和纤维化的应答。与对照老年小鼠相比，PNX后的老年Nrp1 iΔEC/iΔEC小鼠展示出更少的胶原沉积、增强的肺泡再生能力、降低的衰老和成纤维细胞活化、增加的II型肺泡上皮祖细胞增殖和更低数量的血管周围中性粒细胞(图1H，图8H-I)。抗体1560抑制了在老年Nrp1 iΔEC/iΔEC小鼠中观察到的所述有益效果。这些数据表明，PNX后的老年Nrp1 iΔEC/iΔEC小鼠中EPCR的正常化有利于再生而非纤维化(图1I)。
实施例四：衰老的肝和肾中重编程的造血-血管微环境将再生逆转成纤维化
评估不同年龄的小鼠的肝再生(图2A)。部分肝切除手术(PH)后，老年小鼠肝脏表现出纤维化样的症状(图9A)。PH后，在老年小鼠肝脏(而非3月龄小鼠)中还观察到“血小板-巨噬细胞”集落(图2B)，在肝内皮细胞中NRP1、HIF2α和EPCR的表达有相似的变化(图2C-D)。与对照老年小鼠相比，PH后，老年Nrp1 iΔEC/iΔEC小鼠展示出增加的肝细胞增殖和更低的纤维化，这与降低的“血小板-巨噬细胞”集落的形成有关(图2E-I，图9B)。PH后，注射1560抗体的老年Nrp1 iΔEC/iΔEC小鼠逆转了其表型，这说明PH后的老年小鼠肝脏中NRP1依赖性EPCR的抑制，活化血小板和巨噬细胞。
通过缺血再灌注(I/R)，诱导老年对照组和老年Nrp1 iΔEC/iΔEC小鼠的急性肾损伤(图2J)。I/R后，老年小鼠的肾功能因纤维化而受损，其在老年小鼠(而非2月龄小鼠)肾中伴有“血小板-巨噬细胞”集落的形成(图2K)。在老年肾内皮细胞中，I/R相似地增加NRP1和HIF2α的表达并下调EPCR(图2L-N)。与老年对照组小鼠相比，老年Nrp1 iΔEC/iΔEC小鼠显示，提高的肾脏修复与更高的细胞增殖程度、更少的纤维化和更少数量的“血小板-巨噬细胞”集落(图2O-R，图9C)。抗体1560逆转了肾I/R后的老年Nrp1 iΔEC/iΔEC小鼠的表型，这说明Nrp1的删除的有益效果取决于EPCR。
实施例五：ECs中胎盘生长因子(PlGF)-NRP1轴的表观遗传上调，刺激HIF2α以抑制EPCR
PNX后，EPCR在年轻小鼠肺内皮细胞中被诱导出，而其在衰老的肺内皮细胞中被抑制(图9D)。内皮细胞中NRP1的删除，恢复了经肺切除的老年肺中EPCR的诱导。NRP1结合不同的配体，包括胎盘生长因子(PlGF)、肝细胞生长因子(HGF)、血小板源性生长因子BB(PDGF-BB)和转化生长因子β1(TGF-β1)。PlGF蛋白在PNX和PH后的衰老的肺和肝脏中上调(图9E)。向PNX后的年轻小鼠注射PlGF而非其他NRP1配体，抑制了EPCR表达并增强了HIF2α，这与老年小鼠的表型相似(图9F)。而所述PlGF的效果在Nrp1 iΔEC/iΔEC小鼠中被抑制(图9G)。PNX后的老年肺中，内皮细胞中删除Hif2α(Hif2a iΔEC/iΔEC)，相似地提 高了EPCR的表达，并且注射PlGF没有抑制年轻Hif2a iΔEC/iΔEC小鼠的EPCR(图9G)。PH后的缺乏内皮细胞NRP1或者HIF2α的肝脏中，PlGF没有抑制EPCR的上调(图9H)。因此，PlGF-NRP1-Hif2α轴可能是造成损伤后的衰老器官中EPCR的抑制的原因。
因为表观遗传的改变有助于器官纤维化，本发明实验人员检测了组蛋白修饰和DNA甲基化对内皮细胞NRP1和PlGF表达的影响。PNX后，用组蛋白脱乙酰基酶(HDAC)抑制剂丙戊酸(VPA)、DNA甲基转移酶(DNMT)抑制剂5-氮杂胞苷(AZA)或VPA和AZA的联合治疗处理老年动物。对PNX后的老年小鼠的VPA的治疗，降低了肺内皮细胞中NRP1的表达，而其被AZA的联合治疗(VPA+AZA)进一步降低(图9I)。PH和肾脏I/R后，在老年小鼠中，VPA+AZA治疗抑制了肝和肾ECs中内皮细胞NRP1的上调(图9I)。VPA+AZA还抑制了PNX、PH或肾脏I/R后的老年肺、肝和肾中PlGF的上调(图9J)。因此，NRP1和PlGF的表达可能在衰老器官中受到表观遗传调控(图9K)。
实施例六：PCECs中NRP1-HIF2α轴刺激基质衍生因子1(SDF1)以募集促纤维化的巨噬细胞
PCECs产生旁分泌或血管分泌因子来调节肺泡的修复。因此，本发明实验人员分析了对照组和Nrp1 iΔEC/iΔEC小鼠中肺内皮细胞和成纤维细胞中因子的表达(图10A-B)。Nrp1 iΔEC/iΔEC小鼠的PCECs中趋化因子SDF1显示受到最多的抑制。由于SDF1-CXCR4信号调节造血细胞功能和器官修复，本发明实验人员研究了在年轻和老年Sdf1报告小鼠(reporter mice)中SDF1的表达模式(图3A)。PNX后，SDF1在老年小鼠(而不是年轻组)的PCECs中诱导出，并且ECs中Nrp1的删除抑制了SDF1表达(图3A)。
接下来，本发明实验人员检测在老年内皮细胞中HIF2α是否介导SDF1的产生。PNX后的老年Hif2a iΔEC/iΔEC小鼠，显示出较低水平的Sdf1、Par1、E-选择素和VCAM1(图3B)。由于SDF1、E-选择素和VCAM1促进内皮细胞-巨噬细胞串扰，实验人员对SDF1受体CXCR4和巨噬细胞标记物染色。与老年对照小鼠的肺相比，PNX后的老年Hif2a iΔEC/iΔEC小鼠和老年Nrp1 iΔEC/iΔEC小鼠肺中CXCR4 +巨噬细胞的募集降低，这种现象被抗体1560逆转(图3C-D，图10C-D)。PNX后，老年Hif2a iΔEC/iΔEC小鼠肺泡上皮祖细胞的增殖增强，且抗体1560阻断了细胞增殖的增加(图3D，图10E)。PH和肾I/R后，老年Nrp1 iΔEC/iΔEC小鼠显示出较低的纤维化(图10F)，其伴有内皮细胞中SDF1和炎症基因的更低表达(图10G)。结果是，PH和肾I/R后，老年Hif2a iΔEC/iΔEC小鼠中血管周围CXCR4 +巨噬细胞的富集减少(图10H)。因此，这些血管周围的CXCR4 +巨噬细胞的募集，可能有助于老年器官中促纤维化的“血小板-巨噬细胞”集落的形成。
实施例七：CXCR4 +巨噬细胞抑制老年器官的再生并引起纤维化
分离出PNX后的老年对照组和老年Hif2a iΔEC/iΔEC小鼠的肺巨噬细胞(图3E)。金属蛋白酶组织抑制剂1(TIMP1)在老年Hif2a iΔEC/iΔEC小鼠和对照组小鼠之间存在显著差异。接下来探究TIMP1的促纤维化作用。TIMP1被证明与参与活化成纤维细胞的整合素β1(β1integrin)相互作用。因此，本发明实验人员检验了TIMP1-整合素β1的相互作用对成纤维细胞活化的作用。用TIMP1孵育，增强了Smad2的磷酸化和平滑肌细胞肌动蛋白(SMA)的蛋白表达，而其通过用TGF-β1共处理被进一步增强(图3F-H)。通过沉默成纤维细胞中的整合素β1，减少了TIMP1引起的Smad2磷酸化和SMA上调。这些数据表明，TIMP1通过整合素β1刺激成纤维细胞的活化。
检测老年缺乏Timp1的小鼠(Timp1 -/-)中TIMP1的促纤维化作用。PNX后，与对照组小鼠相比，老年Timp1 -/-小鼠展示出降低的肺纤维化和增强的肺泡上皮覆盖的恢复(图4A)。与年轻器官相比，PNX、PH或肾脏I/R后的老年小鼠器官的巨噬细胞中，TIMP1的表达更高(图4B，图11A-C)。用CXCR4拮抗剂AMD3100处理，减少了小鼠老年器官的巨噬细胞中TIMP1的上调。这些发现提示，老年ECs中NRP1-HIF2α-EPCR通路的反常，刺激SDF1以募集表 达TIMP1的CXCR4 +巨噬细胞(图10I)。
为了揭示CXCR4 +TIMP1高表达(CXCR4 +TIMP1 high)的巨噬细胞的作用，本发明实验人员采用了一种过继(adoptive)巨噬细胞转移方法(图4C)。Timp1-无效(Timp1-null)单核细胞的移植，促进了已肺切除的(pneumonectomized)老年小鼠的肺功能恢复并减少了胶原沉积(图4D-G)。PH和肾脏I/R后，向老年小鼠转移Timp1 -/-单核细胞(而不是Timp1 +/+)，增强了实质功能的恢复并阻断了纤维化(图4H-J)。因此，老年器官的ECs中，NRP1-HIF2α-EPCR通路的重编程，诱导SDF1以募集促纤维化的CXCR4 +TIMP1高表达的巨噬细胞，阻碍再生并刺激纤维化(图4K)。
实施例八：血小板产生白细胞介素1α(IL-1α)来激活促纤维化的TIMP1 +巨噬细胞
血小板产生促炎细胞因子(如白介素)来与巨噬细胞相互作用。为了评估血小板IL-1α的作用，本发明实验人员构建了血小板特异性删除IL-1α的小鼠(Il1a ΔPlt/ΔPlt)(图5A-B)。因为EPCR中和抗体1560诱导PNX后的年轻小鼠中纤维化样的表型(图12A-E)，本发明实验人员使用抗体1560处理3月龄Il1a ΔPlt/ΔPlt小鼠，以建立EPCR与血小板IL-1α之间的相关性。删除血小板中的Il1a，阻断了“血小板-巨噬细胞”集落的形成，并且降低了EPCR单克隆抗体处理的小鼠中促纤维化的TIMP1的肺部表达(图5C-G)。将Timp1缺乏的巨噬细胞/单核细胞移植到已注射抗体1560的年轻小鼠，同样阻断了肺纤维化(图12F-H)。因此，老年ECs中EPCR的抑制，导致血小板IL-1α的产生和促纤维化的TIMP1 +巨噬细胞的活化，造成纤维化的表型。
使用血小板过继转移模型(参考文献52)(图5H)。向老年Thpo -/-小鼠注射野生型(Il1a +/+)或Il1a敲除(Il1a -/-)血小板，然后进行PNX、肝切除或肾I/R。评估受试小鼠的肺、肝、肾中TIMP1的表达和纤维化。与转移Il1a +/+血小板的老年小鼠相比，注射Il1a -/-血小板的老年小鼠的受试器官中，显示出降低的TIMP1表达和更少的胶原沉积(图5I-K)。这些数据提示，衰老器官中血小板IL-1α，促进促纤维化的“血小板-巨噬细胞”集落的形成和TIMP1的表达(图5L)。
实施例九：衰老器官中，正常化异常的内皮细胞-血小板串扰刺激再生而非纤维化
本发明实验人员使用博来霉素(Bleo)模型作为肺纤维化模型。用气管内注射博来霉素处理老年Nrp1 iΔEC/iΔEC小鼠和对照组小鼠(图6A)。Bleo注射后，老年小鼠肺中PlGF上调(图6B)，并且老年Nrp1 iΔEC/iΔEC小鼠显示出比老年对照组小鼠更高的EPCR上调和HIF2α抑制(图6C)，其与降低的纤维化、增强的肺泡再生的恢复、降低的细胞衰老和减少的血管周围CXCR4 +巨噬细胞的募集有关(图6D-F，图13A-C)。通过EPCR中和抗体阻断了老年Nrp1 iΔEC/iΔEC小鼠的表型提示，Bleo损伤后的老年小鼠中，ECs中重编程的NRP1-EPCR通路促进肺纤维化。
检测了所述Bleo模型中血小板和巨噬细胞对纤维化的作用。向Bleo损伤后的老年小鼠移植Il1a +/+和Il1a -/-血小板(图6G)。在损伤的受试肺中，移植Il1a -/-血小板减轻了造血集落的形成和TIMP1的表达(图6H)。抗体1560没有改变Il1a -/-血小板的效果。EPCR抗体对老年Nrp1 iΔEC/iΔEC小鼠和老年Il1a ΔPLT/ΔPLT小鼠中的差别效果提示，衰老的内皮细胞中的NRP1引起血小板IL-1α的产生。移植Timp1 -/-单核细胞或缺乏Timp1的老年小鼠，显示出恢复的肺泡再生和减轻的纤维化(图6I-K，图13D-E)。因此，衰老的ECs中重编程的NRP1-HIF2α-EPCR信号活化IL-1α +血小板，并募集TIMP1高表达的巨噬细胞，以形成促纤维化的造血集落(图6L)。
免疫检查点阻断(如抗程序性细胞死亡蛋白1(PD1)抗体)在多种癌症类型中显示出显著的临床效益。然而，免疫治疗可能在部分患者中有潜在的风险，导致免疫治疗相关的不良事件(IrAEs)。肺炎是免疫检查点阻断治疗后常见的IrAEs之一。本发明实验人员检测了老年小鼠在PD1抗体处理后是否更易肺纤维化，尤其是联同放疗时。将老年小鼠和年轻小鼠暴露于胸部幅射1个月，来刺激免疫治疗诱发的肺炎(幅射后用或不用PD1抗体治疗)(图7A)。胸部幅射后的PD1抗体处理，在老年Hif2a +/+小鼠引起的肺纤维化程度高于3月龄Hif2a +/+ 小鼠或老年Hif2a iΔEC/iΔEC小鼠(图7B-C)。老年Hif2a iΔEC/iΔEC小鼠的死亡率和肺纤维化程度减少，EPCR抗体逆转这种现象(图7B-C)。移植Il1a -/-(而不是Il1a +/+)血小板的老年小鼠免于死亡和肺纤维化(图7D-F)。相反，EPCR抗体没有影响Il1a -/-血小板的这种保护作用，这提示检查点阻断后血小板IL-1α的促纤维化功能是EPCR的抑制的下游。因此，所述内皮细胞NRP1与血小板IL-1α的异常串扰，可能有助于老年患者的免疫治疗诱导的肺炎。
用荷瘤小鼠来检验EC-血小板异常串扰在免疫治疗诱导的肺炎中的促纤维化作用。注射lewis肺癌(LCC)细胞后，向老年小鼠移植Il1a +/+或者Il1a -/-血小板，然后进行PD1抗体处理(图7G)。与注射Il1a +/+血小板的小鼠相比，注射Il1a -/-血小板的受试小鼠，表现出降低的肿瘤细胞负荷和肺部纤维化(图7H-I)。与注入Il1a +/+血小板的受试小鼠相比，注入Il1a -/-血小板的受试小鼠存活率提高(图7J)。因此，在肺肿瘤的免疫治疗中，血小板产生的Il-1α可能引起肺纤维化并加剧死亡率。
年轻小鼠和老年小鼠接受注射肝毒素的四氯化碳(CCl 4)或叶酸(参考文献2)，以诱导肝纤维化或肾纤维化(图7K)。损伤后，老年受损的肝脏中PlGF表达上调(图7L)。老年对照组小鼠的EC中EPCR的抑制和HIF2α的诱导，在老年Nrp1 iΔEC/iΔEC小鼠中恢复(图14A)，其减轻了受损肝脏和肾脏的实质损伤和纤维化(图7M-N，图14B)。移植Il1a -/-血小板相似地降低了，受损的肝脏和肾脏中促纤维化的造血-血管集落的形成和TIMP1的表达(图7O，图14C)。在肝或肾损伤后，老年Timp1 -/-小鼠或移植Timp1 -/-单核细胞的小鼠的纤维化低于老年对照小鼠(图7P，图14D-E)。因此，在老年肺、肝和肾中，正常化血小板-巨噬细胞-血管细胞之间的异常串扰，使进行无纤维化的修复成为可能。
实施例十：
现有研究已经证明，肺内皮细胞形成微环境来启动肺再生和阻止纤维化(参考文献7、17、19、51)。本发明实验人员发现，在受损的肺、肝和肾中，内皮细胞NRP1的异常诱导，异常诱导转录因子HIF2并抑制EPCR。内皮微环境中EPCR保护性(protective)通路的破坏，导致受损肺、肝和肾的持续纤维化。NRP1的遗传切除(genetic ablation)或药理学阻断(pharmacological blockage)恢复了肺、肝和肾中受损的内皮细胞中EPCR的表达。
为了恢复内皮微环境受损的促再生功能，本发明实验人员采用了一种“双管齐下(two-hit)”策略，即诱导EPCR并注射其分子伴侣—活化蛋白C(activated protein C，APC)。NRP1/HIF2α抑制剂的联合施用，与APC协同作用，以达到：1)抑制内皮细胞中的Rho激活(Rho activation)，2)减少炎性单核细胞的沉积，以及3)促进肺、肝和肾的无纤维化(fibrosis-free)修复。这实际上是通过上游通路增加了EPCR的表达，上游通路包括但不限于NRP1和HIF2α。实验数据表明，“微环境重编程疗法”对纤维化相关疾病的转化价值，包括临床应用的APC的给药和激活内皮微环境中其受体EPCR。通过本发明实验人员提出的“双管齐下”的技术方案，一方面通过活化EPCR并抑制NRP1和/或HIF2α来增加EPCR表达，加强其疗效，另一方面可以降低APC所需的使用剂量(与单独采用APC活化EPCR信号通路相比)，从而减少其副反应的发生。
本实施例提出一个APC-EPCR通路在促进无纤维化器官修复中的作用的模型(图15)。该模型概要为：肺损伤后，肺内皮细胞上的EPCR有促进肺再生和避开肺纤维化的作用。持续性肺损伤，激活内皮细胞中的NRP1通路来上调HIF2，这抑制EPCR和上调PAR1。内皮微环境中EPCR通路的破坏，将再生反应逆转成肺纤维化。在治疗上，NRP1抑制剂或HIF2α抑制剂恢复EPCR的表达，其与活化蛋白C(APC)的施用共同作用以恢复保护性APC-EPCR通路。此外，通过小分子与重组蛋白来激活EPCR，实验数据表明，重编程促再生的EPCR +内皮微环境，可能激活肺、肝和肾中的无纤维化修复。
在反复的肺损伤模型(repeated lung injury model)中检测EC重编程对肺部纤维化的作用(参考文献76)。将盐酸(Acid)滴注到小鼠气管里面，每10天注射1次，一共注射5 次(图16A)。注射3次酸后，受损的肺恢复了气体交换功能。相比之下，气体交换功能的恢复在第5次注射后被抑制(图16B)。被抑制的肺部功能恢复与持续的纤维化有关(图16C-D)。显著地，在PCECs中，EPCR的诱导与肺部功能的恢复有关；当纤维化发生时，NRP1、HIF2α和PAR1在PCECs中上调(图16E-I)。上述结果表明，小鼠反复吸入盐酸后，内皮细胞中EPCR的抑制和NRP1的诱导与肺纤维化有关。在第5次酸注射后，与对照组小鼠相比，Nrp1 iΔEC/iΔEC小鼠的PCECs中显示较高的EPCR表达和较低的HIF2α和PAR1表达(图17A-B)。因此，反复的肺损伤导致PCECs中NRP1-HIF2α-EPCR信号的重编程。
随后检测，通过注射APC和EG(APC+EG)来靶向重编程的PCECs是否会恢复肺部修复并减少纤维化(图17C)。在反复酸损伤后的所有检测的组中，APC+EG最有效地恢复了肺部功能并减少了纤维化(图17D-G)。在反复肺损伤后，经APC+EG治疗的PCECs中血管周围巨噬细胞的募集被抑制(图17F-G)。先前证明，APC抑制ECs中的Rho通路的激活。的确，在APC+EG治疗后，PCECs的磷酸化肌球蛋白轻链(phosphorylated myosin light chain，p-MLC)被阻断(图17F-G)。因此，双管齐下策略，即共同注射APC和NRP1抑制剂，刺激长期受损的PCECs中正常化的EPCR信号并减少促纤维化的血管周围巨噬细胞的募集，即刺激PCECs中正常化的EPCR通路，促进无纤维化的(fibrosis-free)肺修复。
然后在小鼠肝脏和肾脏长期损伤(chronic injury)模型中检测了上述双管齐下策略的治疗效果(图18A)。小鼠被反复注射肝脏毒素四氯化碳(CCl4)或注射叶酸(参考文献77)。在第1次注射CCl4后或注射叶酸后第10天，EPCR在ECs中被诱导出。相比之下，在第8次CCl4注射后和叶酸注射后100天，ECs中EPCR的表达被阻断且NRP1和HIF2α被刺激出(图19A)。用APC治疗Nrp1 iΔEC/iΔEC小鼠，抑制了HIF2α的诱导，增强了长期受损的肝脏和肾脏ECs中EPCR的表达(图19B)，表明EPCR在肝脏和肾脏修复中具有促进再生的功能。
然后用APC、EG或HIF2α抑制剂HIF-2α-IN-1(HY-19949)处理小鼠，并于第8次CCl4注射后或叶酸注射100天后进行评估。APC+EG抑制了肝脏和肾脏ECs中Sdf1、Par1、Vcam1和Hif2a的表达(图18B)，并减少了肝脏的纤维化(图19C)。共同注射APC和HY-19949(APC+HY)阻断了血管周围CXCR4 +巨噬细胞的募集，并抑制了受损肝脏和肾脏的实质损伤和纤维化(图18C，图19D-G)。此外，在纤维化的肝脏和肾脏损伤后，APC+EG阻断了
SDF1报告小鼠ECs中SDF1的表达(图18D)并增强了实质功能的恢复(图18E-F)。所有这些有益效果都被EPCR中和抗体1560的注射而阻断。因此，通过双管齐下策略(APC+NRP1/HIF2α)，靶向重编程的NRP1-HIF2α-EPCR(也可以理解为，靶向肝脏和肾脏中重编程的ECs)使长期损伤的肝脏和肾脏实现无纤维化修复。上述实验表明，在血管微环境中激活正常化的EPCR信号，促进多种器官的无纤维化修复。需要强调的是，通过图17-19等阻断EPCR信号的实验结果可知，EPCR中和抗体1560(阻断EPCR信号的一种方式)减弱了APC应达到的有益技术效果。因此，活化EPCR信号通路是本发明技术方案的关键，对本领域技术人员而言，任何能够刺激EPCR信号和/或促进EPCR的表达的物质都应在本发明的保护范围之内，并不局限于采用APC。
总结
衰老的器官在损伤后易发生纤维化。在上述实施例中，本发明实验人员展示了血小板、巨噬细胞和血管内皮细胞之间与衰老相关的重编程的串扰，导致再生能力的缺失以及纤维化。血小板释放的IL-1α可刺激巨噬细胞的促纤维化功能。血流动力学、血糖和血脂的变化都导致IL-1α的释放。本发明研究结果提示，在受损老年器官中，衰老的ECs中血小板的活化和IL-1α的产生，至少部分归因于抗血栓的EPCR信号的抑制。因此，血管微环境中多功能的EPCR信号的缺失，可能是衰老相关的再生向纤维化转变的因素之一。EPCR在调节血栓形成、炎症和发育方面具有多效性功能。本发明数据表明，衰老的ECs中EPCR的诱导受到异常激活的NRP1-HIF2α通路的抑制。
血小板可以被分级活化以发挥不同的病理生理学效果。在肺切除或肝切除后，活化的血小板提供各种介体以启动肺和肝的再生。本发明实验人员以前使用肺切除术和肝切除术证明SDF1在促进肺和肝再生的功能。肺切除和肝切除模型的重要特征之一是，这些流程诱导生理学再生，其不参与正常年轻小鼠中显著的炎症和血栓形成。在生理学再生中，SDF1可能主要刺激其在血管内皮或上皮细胞/肝细胞的受体，促进上皮和内皮细胞的增殖。因此，在肺切除或肝切除后的年轻小鼠中，SDF1引起血管和上皮/肝细胞的再生。相比之下，EPCR受到抑制的衰老动物中，肺切除或肝切除流程与血栓和炎症有关。在这些条件下，SDF1增强产生促纤维化的TIMP1的CXCR4 +巨噬细胞的募集和积聚。因此，在没有明显的炎症和血栓的情况下，SDF1可能主要作用于上皮细胞和内皮细胞来促进再生。在炎症和血栓水平较高的情况下，SDF1活化如巨噬细胞等炎症细胞中的CXCR4，产生如TIMP1等促纤维化的因子。
本发明揭示的细胞机制可能有助于使免疫细胞的异常激活正常化，并在临床情况下避开免疫细胞的促纤维化的功能。例如，所述机制可用来应对IrAEs。本发明发现，在用PD1阻断处理的老年小鼠中，靶向血小板IL-1α或内皮细胞HIF2α，提高了老年小鼠的生存率并减轻了肺纤维化。这些数据提示，肺炎和纤维化可能是免疫治疗(尤其是联同化疗或放疗)后老年患者的危险因素。
本发明表明，衰老器官中衰老相关的异常重编程血小板、巨噬细胞和血管细胞之间的串扰，以将再生逆转成纤维化。而本发明揭示的导致器官纤维化的机制，并非仅是衰老特异性的。正常化促纤维化的造血-血管微环境，可能有助于在病理条件下恢复再生能力。在重编程的ECs中，遗传学和药理学靶向Neuropilin-1或HIF2α，使EPCR信号正常化，这与EPCR活化剂(agonist)活化蛋白C(APC)协同作用，抑制衰老的和长期受损的器官中促纤维化的巨噬细胞的募集。失调的血管和免疫微环境的正常化，可能在一定程度上恢复衰老的或纤维化的器官的再生能力。
上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。
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Claims (23)

  1. 一种抗纤维化药物组合物，包括：
    (a)NRP1抑制剂和/或HIF2α抑制剂；
    (b)EPCR通路活化剂，所述EPCR通路活化剂促进EPCR通路的活性。
  2. 如权利要求1所述的药物组合物，其特征在于，所述NRP1抑制剂包括抑制NRP1蛋白活性的物质、降解NRP1蛋白活性的物质和降低NRP1蛋白水平的基因工具中的一种或多种；所述HIF2α抑制剂包括抑制HIF2α蛋白活性的物质、降解HIF2α蛋白活性的物质和降低HIF2α蛋白水平的基因工具中的一种或多种。
  3. 如权利要求1所述的药物组合物，其特征在于，所述NRP1抑制剂包括EG00229或其衍生物；所述HIF2α抑制剂包括HIF-2α-IN-1或其衍生物。
  4. 如权利要求1所述的药物组合物，其特征在于，所述EPCR通路活化剂包括活化蛋白C。
  5. 如权利要求1所述的药物组合物，其特征在于，所述纤维化包括器官纤维化，所述器官纤维化可选地为与衰老相关的器官纤维化。
  6. 如权利要求5所述的药物组合物，其特征在于，所述器官纤维化包括肝纤维化、肾纤维化、肺纤维化和心纤维化中的一种或多种，所述肺纤维化可选地为免疫治疗相关肺炎引起的纤维化。
  7. 如权利要求2所述的药物组合物，其特征在于，所述降低NRP1蛋白水平的基因工具包括RNA干扰、microRNA、基因编辑和基因敲除中的一种或多种；所述降低HIF2α蛋白水平的基因工具包括RNA干扰、microRNA、基因编辑和基因敲除中的一种或多种。
  8. 如权利要求1-7任一项所述的药物组合物，其特征在于，所述组合物还包括药学上可接受的载体。
  9. 如权利要求1-8任一项所述的药物组合物在制备抗纤维化剂中的应用。
  10. 如权利要求9所述的应用，所述应用包括减轻受损器官纤维化程度和/或促进受损器官的再生能力，所述再生能力包括细胞增殖能力、组织修复能力和功能恢复能力中的一种或多种，所述组织修复能力可选地为无纤维化修复，所述器官可选地包括肝、肾、肺和心中的一种或多种。
  11. 如权利要求9所述的应用，所述应用包括减轻血小板-巨噬细胞集落的形成；和/或降低血小板IL-1α、SDF1、TIMP1中的一种或多种的表达。
  12. 如权利要求9所述的应用，所述应用包括降低Rho通路活性；和/或减少炎症单核细胞的沉积；和/或促进内皮微环境的促再生功能。
  13. EPCR通路活化剂在制备抗纤维化剂中的应用，所述EPCR通路活化剂促进EPCR通路的活性。
  14. 如权利要求13所述的应用，其特征在于，所述EPCR通路活化剂包括活化蛋白C。
  15. 如权利要求13所述的应用，其特征在于，所述纤维化包括器官纤维化，所述器官纤维化可选地为与衰老相关的器官纤维化。
  16. 如权利要求15所述的应用，其特征在于，所述器官纤维化包括肝纤维化、肾纤维化、肺纤维化和心纤维化中的一种或多种，所述肺纤维化可选地为免疫治疗相关肺炎引起的纤维化。
  17. 如权利要求13所述的应用，其特征在于，所述应用包括减轻受损器官的纤维化程度和/或促进受损器官的再生能力，所述再生能力包括细胞增殖能力、组织修复能力和功能恢复能力中的一种或多种。
  18. 如权利要求13所述的应用，其特征在于，所述应用包括减轻血小板-巨噬细胞集落的形成；和/或降低血小板IL-1α、SDF1、TIMP1中的一种或多种的表达。
  19. 如权利要求13-18任一项所述的应用，其特征在于，所述EPCR通路活化剂用于与NRP1抑制剂和/或HIF2α抑制剂联用制备所述抗纤维化剂，所述NRP1抑制剂和/或HIF2α抑制剂用于促进EPCR的表达。
  20. 如权利要求19所述的应用，其特征在于，所述NRP1抑制剂包括抑制NRP1蛋白活性的物质、降解NRP1蛋白活性的物质和降低NRP1蛋白水平的基因工具中的一种或多种；所述HIF2α抑制剂包括抑制HIF2α蛋白活性的物质、降解HIF2α蛋白活性的物质和降低HIF2α蛋白水平的基因工具中的一种或多种。
  21. 如权利要求19所述的应用，其特征在于，所述NRP1抑制剂包括EG00229或其衍生物；所述HIF2α抑制剂包括HIF-2α-IN-1或其衍生物。
  22. 如权利要求20所述的应用，其特征在于，所述抑制NRP1蛋白活性的物质包括小分子药物和/或抗体；所述抑制HIF2α蛋白活性的物质包括小分子药物和/或抗体。
  23. 如权利要求20所述的应用，其特征在于，所述降低NRP1蛋白水平的基因工具包括RNA干扰、microRNA、基因编辑和基因敲除中的一种或多种；所述降低HIF2α蛋白水平的基因工具包括RNA干扰、microRNA、基因编辑和基因敲除中的一种或多种。
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