WO2020264156A1 - Diméthylarginine asymétrique (adma) en tant que marqueur de pathologies vasculaires - Google Patents

Diméthylarginine asymétrique (adma) en tant que marqueur de pathologies vasculaires Download PDF

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WO2020264156A1
WO2020264156A1 PCT/US2020/039613 US2020039613W WO2020264156A1 WO 2020264156 A1 WO2020264156 A1 WO 2020264156A1 US 2020039613 W US2020039613 W US 2020039613W WO 2020264156 A1 WO2020264156 A1 WO 2020264156A1
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adma
subject
gsno
mice
vascular
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Inderjit Singh
Avtar K. Singh
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Musc Foundation For Research Development
The U.S. Government As Represented By The Department Of Veterans Affairs
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • the present invention relates generally to the field of medicine. More particularly, it concerns methods of detecting asymmetric dimethylarginine (ADMA) as a marker for diseases, such as vascular diseases e.g., vascular disease of the nervous system.
  • ADMA asymmetric dimethylarginine
  • VCID is the second most common form of dementia behind Alzheimer’s disease (AD). It encompasses a wide spectrum of cerebrovascular-driven cognitive impairment, from mild cognitive impairment to fully developed dementia with multiple risk factors, such as hypertension, cardiac disease, obesity, and type 2 diabetes (Snyder et al, 2015). At present, there is no known common disease mechanism behind the relationship between these vascular and metabolic risk factors and onset and progression of VCID and AD and thus no therapeutics and prognostic biomarkers are available.
  • NO produced by endothelial NOS plays a key role in vascular hemodynamics via activating cGMP-dependent mechanisms (Kraehling et al, 2017; Lahteenvuo and Rosenzweig, 2012). NO also exerts its actions via formation of secondary redox metabolites, such as peroxynitrite (ONOO-) and S-nitrosoglutathione (GSNO) (Arora et al, 2016).
  • eNOS endothelial NOS
  • ONOO- is generated by reaction between NO and superoxide anion ( ⁇ 2-) under oxidative stress conditions and has been implicated in various pathological events (e.g. vasoconstriction and endothelial cell death) via irreversible modification of proteins (3- nitrotyrosine: N-Tyr) and other cellular components (Pacher et al, 2007).
  • GSNO the most abundant low-molecular-weight S-nitrosothiol synthesized by reaction between NO and glutathione (GSH), participates in various physiological cellular processes via reversible S-nitrosylation of protein thiols (S-NO) (He and Frost, 2016; Gaston et al, 2003).
  • GSNO has been implicated in endothelial cell survival (Kang-Decker et al, 2007), vascular hemodynamics (Haidar and Stamler, 2013), and modulation of vascular inflammation (Won et al 2013; Prasad et al, 2007).
  • GSNO treatment of thrombin activated BMVECs inhibited RhoA/Ca 2+ mediated endothelial F-actin stress fiber formation as well as barrier disruption.
  • GSNO vs. ONOO- redox dependent endothelial NO metabolome
  • ADMA is known to inhibit eNOS activity for NO synthesis (MacAllister et al, 1994) while inducing its activity for ⁇ 2- synthesis, a phenomenon known as‘eNOS uncoupling’ which induces ONOO- synthesis (Karbach et al, 2014).
  • methylarginines have been described as a prospective marker as well as risk factor for cardiovascular diseases and metabolic syndrome (Visser et al, 2010).
  • the methylarginines such as N-monomethylarginine (NMMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA), are generated by methylation of proteins on L-arginine (L-Arg) residues by protein L-Arg methyltransferases (PRMTs) and their subsequent proteolysis (Visser et al, 2010).
  • PRMTs protein L-Arg methyltransferases
  • PRMTs protein L-Arg methyltransferases
  • methylarginines are known to inhibit function of cationic amino acid transporter (CAT) leading to alteration of L-Arg bio distribution.
  • CAT cationic amino acid transporter
  • NMMA and ADMA are known to dysregulate nitric oxide synthase (NOS), leading to reduced nitric oxide (NO) and increased superoxide anion (O2 *- ) synthesis.
  • NOS nitric oxide synthase
  • O2 *- superoxide anion
  • NMMA and ADMA are present in cells and plasma under disease conditions and even under healthy conditions but at lower concentrations.
  • NMMA concentration is much lower due to its conversion into ADMA or SDMA (Visser et al, 2010), indicating that ADMA is the major endogenous methylated L-Arg that dysregulates NOS activity.
  • methylarginines may be used as biomarkers for VCID and AD.
  • Certain embodiments of the present disclosure concern an in vitro method for detecting a level of asymmetric dimethylarginine (ADMA) comprising obtaining a sample from a subject diagnosed with or at risk for a vascular and/or neurological pathology; and detecting the level of ADMA in said sample.
  • detecting comprises contacting said sample with an anti- ADMA antibody; and measuring the binding of the antibody to said ADMA, thereby detecting the level of said ADMA.
  • detecting comprises performing by mass spectrometry coupled with gas chromatography or liquid chromatography.
  • gas chromatography is further defined as gas chromatography-mass spectrometry or gas chromatography-mass spectrometry/mass spectrometry.
  • liquid chromatography is further defined as liquid chromatography-mass spectrometry or liquid chromatography-mass spectrometry /mass spectrometry.
  • said sample is a tissue biopsy, fine needle aspirate, saliva, urine, or plasma.
  • measuring comprises performing immunoblotting, immunohistochemistry, ELISA, or immunofluorescence.
  • the vascular and/or neurological pathology is associated with vascular cognitive impairment and dementia (VCID) or Alzheimer’s disease (AD).
  • the vascular and/or neurological pathology is associated with diabetes.
  • the vascular and/or neurological pathology is associated with Sickle Cell Disease.
  • the vascular and/or neurological pathology is dementia, multiple sclerosis (MS), Alzheimer’s, stroke, or traumatic brain injury.
  • an increased level of ADMA as compared to a control indicates a risk for or detects the presence of MS.
  • an increased level of ADMA as compared to a control indicates a risk or detects the presence of AD.
  • an increased level of ADMA as compared to a control indicates that initiation and progression of vascular disease associated dementia.
  • an increased level of ADMA as compared to a control identifies the subject as at risk for a brain microvascular pathology.
  • the method further comprises detecting decreased levels of tight junction proteins.
  • the tight junction proteins are ZO-1 and claudin- 1.
  • the method further comprises administering a therapy to a subject identified to have an increased level of ADMA as compared to a control sample.
  • the therapy is a GSNO and/or a GSNO reductase inhibitor.
  • a pharmaceutical composition comprising GSNO and/or a GSNO reductase inhibitor for use in a subject determined to have an increased level of ADMA according to the method of the embodiments.
  • a further embodiment provides a method of predicting response to a therapeutic in a subject having a vascular and/or neurological pathology comprising measuring the level of ADMA according to a method of the embodiments in a sample obtained from said subject, wherein if the sample has increased expression of ADMA, then the patient is predicted to have a favorable response to the therapeutic.
  • the therapeutic is GSNO and/or a GSNO reductase inhibitor.
  • a method of treating a subject having a vascular and/or neurological pathology comprising administering an effective amount of a therapeutic to the subject, wherein the subject has been determined to have an elevated level of ADMA.
  • the therapeutic is GSNO and/or a GSNO reductase inhibitor.
  • the therapeutic is an MS therapy, such as prednisone, methylprednisolone, glatiramer acetate, beta interferon, or dimethyl fumarate.
  • the subject is human.
  • the subject is determined to have an elevated level of ADMA by mass spectrometry coupled with gas chromatography or liquid chromatography or a quantitative immune-detection assay.
  • gas chromatography is further defined as gas chromatography-mass spectrometry or gas chromatography-mass spectrometry/mass spectrometry.
  • liquid chromatography is further defined as liquid chromatography-mass spectrometry or liquid chromatography-mass spectrometry/mass spectrometry.
  • the quantitative immune-detection assay is an enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, or bioluminescent assay.
  • the quantitative immune-detection assay is an ELISA.
  • the subject has dementia.
  • the dementia is vascular dementia.
  • the subject has an autoimmune disease.
  • the autoimmune disease is multiple sclerosis (MS).
  • the GSNO and/or GSNO reductase inhibitor is administered orally, intravenously, intraperitoneally, intratracheally, intratumor ally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.
  • the GSNO and/or GSNO reductase inhibitor is administered orally.
  • the GSNO reductase inhibitor is N6022.
  • the GSNO reductase inhibitor is N91115.
  • the subject has increased Thl and Thl7 immune response in the spleen, increased peripheral mononuclear cell infiltration, and/or demyelination in the spinal cord.
  • the GSNO and/or GSNO reductase inhibitor decreases microvessel pathology, endothelial ONOO synthesis.
  • the GSNO and/or GSNO reductase inhibitor decreases Ab40 and/or Ab42 levels.
  • the subject has MS. In some aspects, the subject has been determined to have an increased ratio of L-Arg to ADMA and/or increased L-homocysteine. In certain aspect, the subject has been determined to have increased IFNy and IL-17 and/or decreased IL-10.
  • the GSNO reductase inhibitor protects against myelin loss in the central nervous system and/or selectively modulates CD4+ T cells subsets.
  • the method further comprises administering at least a second therapy.
  • the second therapy is an anti-inflammatory, immunosuppressive agent, or immunomodulatory agent.
  • the second therapy is interferon-b, glatiramer acetate, teriflunomide, dimethyl fumarate, natalizumab, fingolimod, alemtuzumab, mitoxantrone, and/or simvastatin.
  • FIGS. 1A-1B Tg-SwDI mice have age dependent elevation of serum ADMA levels and ADMA vs. L-Arg ratio.
  • NS (not significant: P > .05); *P ⁇ .05, **P ⁇ .01, ***P ⁇ .001 vs 2 months old WT mice and #P ⁇ .05, and ###P ⁇ .001 vs age-matched WT mice.
  • B. Seram levels of ADMA, L-Arg (L- arginine), and ratio of ADMA vs. L-Arg were analyzed in WT and Tg-SwDI mice at age of 8 months. ** p ⁇ 0.01, *** p ⁇ 0.001 vs. WT. Both groups include equal numbers of females and males and there was no statistical difference between female and male groups. Studies were performed in double-blinded fashion.
  • FIGS. 2A-2B ADMA treatment increases blood ADMA levels and decreases spatial learning and memory performance of Tg-SwDI mice.
  • WT wild type
  • Tg-SwDI mice were treated with vehicle (saline; 100 u L) or ADMA (50 mg/kg/i.p.) for 10 days.
  • MAP mean arterial blood pressure
  • DDAH2 enzyme DDAH2 enzyme
  • FIGS. 3A-3B ADMA treatment increases A b accumulation in the brain of Tg-SwDI mice.
  • A Brain levels of human Ab40 and Ab42 levels were analyzed in saline- treated (-) or ADMA-treated (+) Tg-SwDI mice. For this, 6 months old Tg-SwDI mice were administered with daily doses of saline or ADMA (50 mg/kg/i.p./day) for 8 weeks.
  • For Ab4o measurement three of untreated Tg-SwDI mice and four of ADMA-treated Tg-SwDI mice were used.
  • For Ab42 measurement seven of untreated Tg-SwDI mice and 8 of ADMA-treated Tg- SwDI mice were used.
  • FIGS. 4A-4B ADMA treatment increases inflammation in the brain of Tg- SwDI mice.
  • Six months old WT and Tg-SwDI mice were treated with a daily dose of ADMA (50 mg/kg/i.p./day) or saline for 8 weeks.
  • A brain levels of glial acidic fibrillary protein (GFAP: a marker for activated glial cells) and Iba-1 (a marker for activated microglia) were analyzed in saline-treated (-) or ADMA-treated (+) WT and Tg-SwDl mice by Western analysis (i) and presented densitometric bar graph (ii).
  • GFAP glial acidic fibrillary protein
  • Iba-1 a marker for activated microglia
  • the bar graphs represent the average data of three mice per group. Data are expressed as mean ⁇ standard deviation (SD). ***P ⁇ .001 vs untreated WT mice and ++P .01(or ###P .001) vs as indicated.
  • B Coronal brain sections of WT and Tg-SwDI mice treated with saline or ADMA were double- stained for GFAP and Iba-1 and DAPI for nuclei and visualized by fluorescence microscope
  • FIGS. 5A-5B ADMA treatment increases BBB disruption in the brain of Tg-SwDI mice.
  • B. Brain levels of tight junctional proteins (ZO-1 and cluaidin-1) were analyzed by Western analysis (i) and quantified by densitometer (ii and iii). Data are expressed as mean ⁇ standard deviation (SD).
  • FIGS. 6A-6C ADMA treatment increases MLC phosphorylation and decreases microvessel density in the brain of Tg-SwDI mice.
  • A Protein expression of phospho and total MLC (p-MLC and t-MLC) and CD31 (a marker for endothelial cells) were analyzed by Western analyses (i) and quantified by densitometer (ii) in brains of saline (-) or ADMA (+) treated WT or Tg-SwDI mice b -actin was used for internal loading control.
  • ADMA (or saline) treatment was administered daily to 6 months old mice for 8 weeks (50 mg/kg/i.p./day).
  • B Colocalization of p-MLC and CD31 was visualized by double immunostaining of coronal sections of ADMA-treated Tg-SwDI mice and Z-stack confocal microscopy.
  • FIGS.7A-7C ADMA increases MLC phosphorylation via inducing ONOO - mediated RhoA activation in cultured human brain microvessel endothelial cells (hBMVECs).
  • hBMVECs human brain microvessel endothelial cells
  • hBMVECs were treated (+)/untreated (-) with L-NIO (NOS inhibitor) or FeTPPS (ONOO- scavenger) 30 min prior to ADMA + VEGFa treatments and cellular levels of N-Tyr were analyzed (iv).
  • the bar graphs represent the average data of three independent experiments.
  • C Human hBMVECs were treated with various concentrations of ADMA in the presence (+) or absence (-) of VEGFa and cellular levels of active- and total-RhoA, phosphorylated and total levels of MLC were analyzed by Western analysis (i).
  • the active-RhoA was analyzed by measuring the levels of agarose-GST-RBD (Rhotekin-Rho binding domain)-bound RhoA.
  • the bar graphs represent the average data of three independent experiments.
  • ***P ⁇ .001 vs untreated cells and ++P ⁇ .01 and ++-I-P ⁇ .001 vs VEGFa treated cells (i).
  • the cells were treated (+)/untreated (-) with L-NIO or FeTPPS 30 min prior to ADMA and VEGFa treatments and activity and cellular levels of RhoA, phosphorylated and total levels of MLC were analyzed (ii).
  • the bar graphs represent the average data of three independent experiments.
  • FIGS. 8A-8B ADMA increases F-actin stress fiber formation and barrier disruption in cultured human microvessel endothelial cells (MVECs).
  • A. Cultured human MVEC monolayers were treated with ADMA or VEGFa or ADMA and VEGFa combination and cellular phosphorylated MLC and F-actin bundles were visualized by fluorescence microscopy following the staining with anti-p-MLC and phalloidin. DAPI was used for staining of nuclei of MVECs.
  • B Following the treatment of human MVECs with ADMA, VEGFa, and ADMA and VEGFa combination, the endothelial barrier integrity was analyzed by transendothelial electrical resistance (TEER). Data are expressed as mean ⁇ standard deviation (SD). * p ⁇ 0.05, *** p ⁇ 0.001.
  • FIGS. 9A-9D ADMA is elevated in the blood of EAE animals: Female C57BL/6 mice were treated with complete Freund's adjuvant (CFA), myelin oligodendrocyte glycoprotein peptide (MOG), and pertussis toxin (PTX) for induction of EAE, then blood (serum) levels ADMA (A-i) and VEGF (A-ii) were analyzed in control (0-day post immunization) and EAE mice before the onset of the disease (day 7 post-immunization), on the day of disease onset (11 day of post-immunization), at the peak of disease (day 19 post immunization), and at the chronic disease phase (44 day of post-immunization) (A-iii).
  • CFA complete Freund's adjuvant
  • MOG myelin oligodendrocyte glycoprotein peptide
  • PTX pertussis toxin
  • FIGS. 10A-10B ADMA treatment exacerbates EAE disease: Female C57BL/6 mice were treated with complete Freund's adjuvant (CFA), myelin oligodendrocyte glycoprotein (MOG), and pertussis toxin (PTX) for induction of EAE and daily clinical scores of control, EAE, and ADMA-treated EAE mice (A) and the areas under the curves as overall disease severity (B) were analyzed. Data are expressed as mean ⁇ standard deviation (SD). * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 vs. EAE mice.
  • CFA complete Freund's adjuvant
  • MOG myelin oligodendrocyte glycoprotein
  • PTX pertussis toxin
  • FIGS. 11A-11E ADMA treatment increases myelin and axonal pathologies in EAE mice: Female C57BL/6 mice were treated with complete Freund's adjuvant (CFA), myelin oligodendrocyte glycoprotein (MOG), and pertussis toxin (PTX) for induction of EAE.
  • CFA complete Freund's adjuvant
  • MOG myelin oligodendrocyte glycoprotein
  • PTX pertussis toxin
  • LLB Luxol-fast-blue
  • H&E hismatoxylin and eosin
  • A electron microscope
  • C axon-myelination ultrastructure
  • D Bielchowsky silver staining for visualization of axonal pathology
  • Demyelination and axonal loss were also analyzed by Western analysis for myelin basic protein (MBP) (B) and b-tubulin (E) using the spinal cord tissue lysates b-actin was used for the internal loading standard. Data are expressed as mean ⁇ standard deviation (SD). * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 vs. as indicated. AMF (anterior median fissure, WM (white matter), GM (grey matter).
  • FIGS. 12A-12E ADMA treatment increases CNS infiltration of mononuclear cells and induces BBB dysfunction in EAE mice: Female C57BL/6 mice were treated with complete Freund's adjuvant (CFA), myelin oligodendrocyte glycoprotein (MOG), and pertussis toxin (PTX) for induction of EAE with/without exogenous ADMA treatment.
  • CFA complete Freund's adjuvant
  • MOG myelin oligodendrocyte glycoprotein
  • PTX pertussis toxin
  • FIGS. 13A-13C ADMA treatment induces EAE disease in MOG- immunized mice in the absence of PTX:
  • Female C57BL/6 mice were immunized with complete Freund's adjuvant (CFA) and myelin oligodendrocyte glycoprotein (MOG) with or without pertussis toxin (PTX) or ADMA treatment.
  • CFA complete Freund's adjuvant
  • MOG myelin oligodendrocyte glycoprotein
  • PTX pertussis toxin
  • ADMA 50mg/kg was treated daily starting day 0 and ending day 10 post immunization.
  • FIGS. 14A-14C ADMA treatment increased myelin and axonal pathologies in EAE mice: Female C57BL/6 mice were treated with complete Freund's adjuvant (CFA), myelin oligodendrocyte glycoprotein (MOG), and pertussis toxin (PTX) for induction of EAE with or without ADMA treatment.
  • CFA complete Freund's adjuvant
  • MOG myelin oligodendrocyte glycoprotein
  • PTX pertussis toxin
  • CD4 + IFN-y + T H I cells A-i
  • CD4 + IL-17 + T H 17 cells A-ii
  • CD4 + CD25 + FOXP3 + Treg cells A-iii
  • CD4 + CD25 + FOXP3 Treg cells A-iv
  • naive splenic CD4 + T cells were activated by IL-2 and anti-CD3 and anti-CD28 mAbs under unpolarized conditions in the presence or absence of ADMA (400 mM) and the numbers of CD4 + IFN-Y + T H I cells (C-i), CD4 + IL-17 + T H 17 cells (C-ii), CD4 + CD25 + FOXP3 + Treg cells (C-iii), and CD4 + CD25 + FOXP3 Treg cells (C-iv) were analyzed by fluorescence flow cytometry. Data are expressed as mean ⁇ standard deviation (SD). N.S. (not significant: P > 0.05); * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 vs. as indicated.
  • SD standard deviation
  • the present studies investigated the role of ADMA in VCID and AD associated microvascular pathology and neuropathology in transgenic mice expressing human b-amyloid precursor protein Swedish, Dutch, Iowa mutant ( Tg-SwDl ). It was found that VCID and AD associated brain pathologies of Tg-SwDI mice correlates with the elevation of blood ADMA levels. ADMA overburden in Tg-SwDI mice by daily treatment accelerates VCID and AD associated brain pathologies by increasing blood-brain barrier (BBB) disruption, loss of microvessel density, deposition of Ab4o/Ab42, and activation of astrocytes and microglia.
  • BBB blood-brain barrier
  • Vascular pathology is an important factor causing neuronal dysfunction/degeneration in multiple sclerosis (MS), yet the role of ADMA in MS remains elusive.
  • EAE experimental autoimmune encephalomyelitis
  • BBB blood-brain barrier
  • ADMA treatment promoted Thl and Thl7 immune responses in the spleen, increased peripheral mononuclear cell infiltration and demyelination in the spinal cord, and exacerbated the clinical EAE disease.
  • ADMA treatment also increased BBB disruption and induced EAE disease even without pertussis toxin treatment for BBB disruption in MOG immunized mice.
  • the present disclosure provides methods for detecting ADMA levels in subjects to determine the risk of vascular dysfunction in MS, the initiation and progression of vascular inflammatory disease including diabetes associated dementia and vascular associated dementia, Alzheimer’s associated dementia, stroke, traumatic brain injuries, neurological disorders, sickle cell disease and other vascular pathologies. Further embodiments concern the treatment of subjects identified to be at risk for these vascular pathologies.
  • essentially free in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts.
  • the total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%.
  • Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
  • “a” or“an” may mean one or more.
  • the words“a” or “an” when used in conjunction with the word“comprising,” the words“a” or “an” may mean one or more than one.
  • “Treatment” or“ treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.
  • inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease e.g., arresting further development of the pathology and/or symptomatology
  • ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease e.g., reversing the pathology and/or symptomatology
  • the term“subject” refers to a human or non-human mammal or animal.
  • Non-human mammals include livestock animals, companion animals, laboratory animals, and non-human primates.
  • Non-human subjects also specifically include, without limitation, chickens, horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, mink, and rabbits.
  • a subject is a patient.
  • a“patient” refers to a subject who is under the care of a physician or other health care worker, including someone who has consulted with, received advice from or received a prescription or other recommendation from a physician or other health care worker.
  • antibody herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g. , bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
  • determining an expression level means the application of a gene specific reagent such as a probe, primer or antibody and/or a method to a sample, for example a sample of the subject and/or a control sample, for ascertaining or measuring quantitatively, semi-quantitatively or qualitatively the amount of a gene or genes, for example the amount of mRNA.
  • a level of a gene can be determined by a number of methods including for example immunoassays including for example immunohistochemistry, ELISA, Western blot, immunoprecipitation and the like, where a biomarker detection agent such as an antibody for example, a labeled antibody, specifically binds the biomarker and permits for example relative or absolute ascertaining of the amount of polypeptide biomarker, hybridization and PCR protocols where a probe or primer or primer set are used to ascertain the amount of nucleic acid biomarker, including for example probe based and amplification based methods including for example microarray analysis, RT-PCR such as quantitative RT-PCR, serial analysis of gene expression (SAGE), Northern Blot, digital molecular barcoding technology, for example NanostringmCounterTM Analysis, and TaqMan quantitative PCR assays.
  • immunoassays including for example immunohistochemistry, ELISA, Western blot, immunoprecipitation and the like
  • a biomarker detection agent such as an antibody for example
  • mRNA in situ hybridization in formalin-fixed, paraffin-embedded (FFPE) tissue samples or cells can be applied, such as mRNA in situ hybridization in formalin-fixed, paraffin-embedded (FFPE) tissue samples or cells.
  • FFPE paraffin-embedded
  • QuantiGene®ViewRNA Affymetrix
  • This system for example can detect and measure transcript levels in heterogeneous samples; for example, if a sample has normal and tumor cells present in the same tissue section.
  • TaqMan probe-based gene expression analysis can also be used for measuring gene expression levels in tissue samples, and for example for measuring mRNA levels in FFPE samples.
  • TaqMan probe-based assays utilize a probe that hybridizes specifically to the mRNA target. This probe contains a quencher dye and a reporter dye (fluorescent molecule) attached to each end, and fluorescence is emitted only when specific hybridization to the mRNA target occurs.
  • the exonuclease activity of the polymerase enzyme causes the quencher and the reporter dyes to be detached from the probe, and fluorescence emission can occur. This fluorescence emission is recorded and signals are measured by a detection system; these signal intensities are used to calculate the abundance of a given transcript (gene expression) in a sample.
  • sample includes any biological specimen obtained from a patient. Samples include, without limitation, whole blood, plasma, serum, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), ductal lavage fluid, nipple aspirate, lymph (e.g., disseminated tumor cells of the lymph node), bone marrow aspirate, saliva, urine, stool (/. ⁇ ?
  • a“fixed” sample refers to a sample which has undergone preservation. The fixation can terminate any biochemical reactions and increase the tissue’s stability. Chemical fixation methods can include subjecting the sample to aldehydes, such as formaldehyde or glutaraldehyde, or alcohols, such as methanol or ethanol.
  • the terms “increased”, “elevated”, “overexpress”, “overexpression”, “overexpressed”,“up-regulate”, or“up-regulated” interchangeably refer to a biomarker that is present at a detectably greater level in a biological sample, e.g. plasma, from a patient with cancer, in comparison to a biological sample from a patient without cancer.
  • the term includes overexpression in a sample from a patient with cancer due to transcription, post-transcriptional processing, translation, post-translational processing, cellular localization (e.g, organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a sample from a patient without cancer. Overexpression can be detected using conventional techniques for detecting mRNA (/.
  • Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a sample from a patient without cancer. In certain instances, overexpression is 1-fold, 2-fold, 3-fold, 4-fold 5, 6, 7, 8, 9, 10, or 15-fold or more higher levels of transcription or translation in comparison to a sample from a patient without cancer.
  • the term“detecting” refers to observing a signal from a label moiety to indicate the presence of a biomarker in the sample. Any method known in the art for detecting a particular detectable moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical methods.
  • Certain embodiments of the present disclosure concern immunodetection methods for binding, purifying, removing, quantifying or otherwise generally detecting ADMA.
  • Any antibody-based method of detection is contemplated for use with the present methods.
  • the present methods could be used for the detection of immune checkpoint molecules such as by immunoblotting, quantitative ELISA, immunofluorescence (IF) imaging, and IHC staining.
  • IF immunofluorescence
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al. (1987), incorporated herein by reference.
  • sample may refer to a whole organism or a subset of its tissues, cells or component parts.
  • a sample may also refer to a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof.
  • samples include urine, blood, cerebrospinal fluid (CSF), pleural fluid, sputum, and peritoneal fluid, bladder washings, secretions, oral washings, tissue samples, touch preps, or fine-needle aspirates.
  • a sample may be a cell line, cell culture or cell suspension.
  • a sample corresponds to the amount and type of expression products present in a parent cell from which the sample was derived.
  • a sample can be from a human or non-human subject.
  • the sample used for performing antibody-based detection is a formalin fixed paraffin embedded (FFPE) specimen.
  • the sample may comprise body fluids and tissue samples that include but are not limited to blood, tissue biopsies, spinal fluid, meningeal fluid, urine, alveolar fluid.
  • tissue samples that include but are not limited to blood, tissue biopsies, spinal fluid, meningeal fluid, urine, alveolar fluid.
  • the cells can be dissociated by standard techniques known to those skilled in the art. These techniques include but are not limited to trypsin, collagenase or dispase treatment of the tissue.
  • the sample is a blood sample, such as a serum sample.
  • cells are harvested from a sample using standard techniques.
  • cells can be harvested by centrifuging a biological sample such as urine, and resuspending the pelleted cells.
  • the cells are resuspended in phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the cells can be fixed, for example, in acid alcohol solutions, acid acetone solutions, or aldehydes such as formaldehyde, paraformaldehyde, and glutaraldehyde.
  • a fixative containing methanol and glacial acetic acid in a 3:1 ratio, respectively can be used as a fixative.
  • a neutral buffered formalin solution also can be used, and includes approximately 1% to 10% of 37-40% formaldehyde in an aqueous solution of sodium phosphate. Slides containing the cells can be prepared by removing a majority of the fixative, leaving the concentrated cells suspended in only a portion of the solution.
  • the ADMA level (e.g., asymmetric dimethylarginine concentrations in plasma and serum) may be measured by mass spectrometry coupled with gas chromatography (i.e., gas chromatography-mass spectrometry and gas chromatography-mass spectrometry /mass spectrometry) or liquid chromatography (i.e., liquid chromatography-mass spectrometry and liquid chromatography-mass spectrometry /mass spectrometry).
  • gas chromatography i.e., gas chromatography-mass spectrometry and gas chromatography-mass spectrometry /mass spectrometry
  • liquid chromatography i.e., liquid chromatography-mass spectrometry and liquid chromatography-mass spectrometry /mass spectrometry
  • the detection methods comprises gas chromatography coupled with mass spectrometry (GC-MS), high- performance liquid chromatography (HPLC) with fluorescence detection after derivatization, HPLC with mass spectrometric detection (LC-MS and LC-MS/MS) underivatized or after derivatization, and capillary electrophoresis (CE) coupled with ultraviolet or CE coupled with mass-spectrometric detection.
  • GC-MS gas chromatography coupled with mass spectrometry
  • HPLC high- performance liquid chromatography
  • LC-MS and LC-MS/MS HPLC with mass spectrometric detection
  • CE capillary electrophoresis
  • the level of expression of ADMA may be measured by ELISA, western blotting, mass spectrometry, a capillary immune-detection method, isoelectric focusing, an immune precipitation method or immunohistochemistry.
  • Other methods include of detection include antibody-based optical imaging, ultrasound imaging, MRI imaging, PET imaging, and phototherapy.
  • the antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non- specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • An enzyme-linked immunosorbent assay may be used to measure the differential expression of a plurality of biomarkers.
  • ELISA tests may be formatted for direct, indirect, competitive, or sandwich detection of the analyte. All are based on the immobilization of an antigen or antibody on a solid surface, generally a microtiter plate.
  • the original ELISA method comprises preparing a sample containing the biomarker proteins of interest, coating the wells of a microtiter plate with the sample, incubating each well with a primary antibody that recognizes a specific antigen, washing away the unbound antibody, and then detecting the antibody- antigen complexes.
  • the antibody-antibody complexes may be detected directly.
  • the primary antibodies are conjugated to a detection system, such as an enzyme that produces a detectable product.
  • the antibody- antibody complexes may be detected indirectly.
  • the primary antibody is detected by a secondary antibody that is conjugated to a detection system, as described above.
  • the microtiter plate is then scanned and the raw intensity data may be converted into expression values using means known in the art.
  • Single- and Multi-probe kits are available from commercial suppliers, e.g., Meso Scale Discovery (MSD).
  • a first, or capture, binding agent such as an antibody that specifically binds the biomarker of interest
  • a suitable solid phase substrate or carrier is immobilized on a suitable solid phase substrate or carrier.
  • the test biological sample is then contacted with the capture antibody and incubated for a desired period of time.
  • a second, detection, antibody that binds to a different, non-overlapping, epitope on the biomarker is then used to detect binding of the polypeptide biomarker to the capture antibody.
  • the detection antibody is preferably conjugated, either directly or indirectly, to a detectable moiety.
  • detectable moieties examples include, but are not limited to, cheminescent and luminescent agents; fluorophores such as fluorescein, rhodamine and eosin; radioisotopes; colorimetric agents; and enzyme- substrate labels, such as biotin.
  • the ELISA is a competitive binding assay, wherein labeled biomarker is used in place of the labeled detection antibody, and the labeled biomarker and any unlabeled biomarker present in the test sample compete for binding to the capture antibody.
  • the amount of biomarker bound to the capture antibody can be determined based on the proportion of labeled biomarker detected.
  • the biomarker or antibody bound to the biomarker is directly or indirectly labeled with a detectable moiety.
  • the role of a detectable agent is to facilitate the detection step of the diagnostic method by allowing visualization of the complex formed by binding of the binding agent to the protein marker (or fragment thereof).
  • the detectable agent can be selected such that it generates a signal that can be measured and whose intensity is related (preferably proportional) to the amount of protein marker present in the sample being analyzed.
  • Methods for labeling biological molecules such as polypeptides and antibodies are well-known in the art. Any of a wide variety of detectable agents can be used in the practice of the present disclosure.
  • Suitable detectable agents include, but are not limited to: various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (such as, for example, quantum dots, nanocrystals, phosphors and the like), photosensitizers, enzymes (such as, those used in an ELISA, /. ⁇ ? ., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and biotin, digoxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.
  • the antibodies may be attached to imaging agents of use for imaging and diagnosis of various diseased organs, tissues or cell types.
  • the antibody may be labeled or conjugated with a fluorophore or radiotracer for use as an imaging agent.
  • a fluorophore or radiotracer for use as an imaging agent.
  • Many appropriate imaging agents are known in the art, as are methods for their attachment to proteins or peptides using metal chelate complexes, radioisotopes, fluorescent markers, or enzymes whose presence can be detected using a colorimetric markers (such as, but not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase).
  • the imaging conjugate will also be dual labeled with a radio-isotope in order to combine imaging through nuclear approaches and be made into a unique cyclic structure and optimized for binding affinity and pharmacokinetics.
  • agents can be administered by any number of methods known to those of ordinary skill in the art including, but not limited to, oral administration, inhalation, subcutaneous (sub-q), intravenous (I.V.), intraperitoneal (I.P.), intramuscular (I.M.), or intrathecal injection, or as described in greater detail below.
  • the imaging agent is a chromophore, such as a fluorophore.
  • fluorophores suitable for use with the present disclosure includes rhodamine, rhodol, fluorescein, thiofluorescein, aminofiuorescein, carboxyfiuorescein, chlorofluorescein, methylfluorescein, sulfofiuorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulforhodol; aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, and thiorhodamine; cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine
  • the detectable moiety may include, but is not limited to fluorodeoxyglucose (FDG); 2'-fluoro-2'deoxy-lbeta-D-arabionofuranosyl-5-ethyl-uracil (FEAU); 5-[ 123 I]- 2'- riuoro-5-iodo- 1 b-D-arabinoluranosyl-uracil; 5-[ 124 I]- 2'-lluoro-5-iodo- 1 b-D-arabinofuranosyl- uracil; 5-
  • the imaging agent is a radionuclide.
  • Suitable radionuclide labels are Tc, In, Ga, Cu, F, Lu, Y, Bi, Ac, and other radionuclide isotopes.
  • the radionuclide is selected from the group comprising i n In, 99m Tc, 94m Tc, 67 Ga, 66 Ga, 68 Ga, 52 Fe, 69 Er, 72 As, 97 RU, 203 Pb, 62 Cu, 64 Cu, 67 Cu, 186 Re, 188 Re, 86 Y, 90 Y, 51 Cr, 52m Mn, 157 Gd, 177 Lu, 161 Tb, 169 Yb, 175 Yb, 105 Rh, 166 Dy, 166 Ho, 153 Sm, 149 Pm, 151 Pm, 172 Tm, 121 Sn, 177m Sn, 213 Bi, 142 Pr, 143 Pr, 198 AU, 199 AU, 18
  • Methods of detecting and/or for quantifying a detectable label or signal generating material depend on the nature of the label.
  • the products of reactions catalyzed by appropriate enzymes can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light.
  • detectors suitable for detecting such detectable labels include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers. Any of the methods for detection can be performed in any format that allows for any suitable preparation, processing, and analysis of the reactions.
  • This can be, for example, in multi-well assay plates (e.g., 96 wells or 386 wells) or using any suitable array or microarray.
  • Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting a detectable label.
  • Imaging may be by optical imaging, ultrasound, PET, SPECT, MRI, or phototherapy.
  • the antigen- specific antibodies may be immobilized on a carrier or support (e.g., a bead, a magnetic particle, a latex particle, a microtiter plate well, a cuvette, or other reaction vessel).
  • suitable carrier or support materials include agarose, cellulose, nitrocellulose, dextran, Sephadex®, Sepharose®, liposomes, carboxymethyl cellulose, polyacrylamides, polystyrene, gabbros, filter paper, magnetite, ion- exchange resin, plastic film, plastic tube, glass, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, and the like.
  • Binding agents may be indirectly immobilized using second binding agents specific for the first binding agents (e.g., mouse antibodies specific for the protein markers may be immobilized using sheep anti-mouse IgG Fc fragment specific antibody coated on the carrier or support).
  • the protein may be detected by a multiplex ELISA to detect two or three proteina simultaneously.
  • the multiplex ELISA may comprise an antibody array with capture antibodies spotted in subarrays on which the sample is incubated, non-specific proteins are washed off, and the array is incubated with a cocktail of biotinylated detection antibodies followed by a streptavidin-conjugated fluorophore which is visualized by a fluorescence laser scanner (e.g., Quantibody Multiplex ELISA Array, RayBiotech).
  • a multiplex assay such as a multiplex ELISA.
  • Multiplex assays offer the advantages of high throughput, a small volume of sample being required, and the ability to detect different proteins across a board dynamic range of concentrations.
  • such methods employ an array, wherein multiple binding agents (for example, capture antibodies) specific for multiple proteins are immobilized on a substrate, such as a membrane, with each capture antibody being positioned at a specific, pre-determined, location on the substrate.
  • Methods for performing assays employing such arrays include those described, for example, in US Patent Publication Nos. US2010/0093557A1 and US2010/0190656A1, the disclosures of which are hereby specifically incorporated by reference.
  • Flow cytometric multiplex arrays also known as bead-based multiplex arrays, include the Cytometric Bead Array (CBA) system from BD Biosciences (Bedford, Mass.) and multi-analyte profiling (xMAP®) technology from Luminex Corp. (Austin, Tex.), both of which employ bead sets which are distinguishable by flow cytometry. Each bead set is coated with a specific capture antibody.
  • CBA Cytometric Bead Array
  • xMAP® multi-analyte profiling
  • Fluorescence or streptavidin-labeled detection antibodies bind to specific capture antibody-protein complexes formed on the bead set. Multiple proteins can be recognized and measured by differences in the bead sets, with chromogenic or fluorogenic emissions being detected using flow cytometric analysis.
  • a multiplex ELISA from Quansys Biosciences coats multiple specific capture antibodies at multiple spots (one antibody at one spot) in the same well on a 96-well microtiter plate. Chemiluminescence technology is then used to detect multiple proteins at the corresponding spots on the plate.
  • An antibody microarray may also be used to measure the differential expression of a plurality of proteins.
  • a plurality of antibodies is arrayed and covalently attached to the surface of the microarray or biochip.
  • a protein extract containing the proteins of interest is generally labeled with a fluorescent dye or biotin.
  • the labeled proteins are incubated with the antibody microarray. After washes to remove the unbound proteins, the microarray is scanned.
  • the raw fluorescent intensity data may be converted into expression values using means known in the art.
  • the present methods may be used in conjunction with both fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al, 1990; Abbondanzo el al, 1990; Allred el al, 1990).
  • frozen-sections may be prepared by rehydrating 50 ng of frozen "pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections.
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • Permanent- sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.
  • the slides may be dried at 40-45 °C in an oven overnight and then incubated at 58-65 C for 1-3 hours.
  • the slides can then be deparaffinized with xylene and ethanol and hydrated in distilled H2O.
  • Antigen retrieval can be performed in 10 mM citric acid (pH 6.0) in a microwave for 10 min (2 min 1000W, 8 min 200W), cooled down at room temperature for 60 min, and washed with PBS twice.
  • the slides can then be blocked in 3% H 2 0 2 /methanol for 10 min at room temperature and washed with PBS three times.
  • Normal horse serum or goat serum (10% normal serum in PBS) is applied for 30 min in a humid chamber at room temperature and normal serum is wiped off.
  • the primary antibody in applied in a humid chamber at 4°C overnight and then washed with PBS three times.
  • the secondary antibody is applied in a humid chamber for 1 hour at room temperature and then washed with PBS three times.
  • Peroxidase conjugated avidin biotin complex is applied in a humid chamber for 1 hour at room temperature and then washed with PBS three times.
  • AEC chromogen substrate is applied for 5-10 min and washed with distilled H2O three times.
  • the sample is then counterstained with Mayer’s hematoxylin for 30 seconds and washed with distilled H2O three times. Finally, the slides are mounted with aqua-mount (Lerner Laboratories Inc).
  • Embodiments of the present disclosure concern methods of treating diseases by administering an effective amount of a therapy, such as a GSNO inhibitor, to a subject determined to have an increased expression of ADMA by the methods provided herein.
  • a therapy such as a GSNO inhibitor
  • S-Nitrosoglutathione is an endogenous S-nitrosothiol (SNO) that plays a critical role in nitric oxide (NO) signaling and is a source of bioavailable NO.
  • GSNO reductase reduces S-nitrosoglutathione (GSNO) to an unstable intermediate, S-hydroxylaminoglutathione, which then rearranges to form glutathione sulfonamide, or in the presence of GSH, forms oxidized glutathione (GSSG) and hydroxylamine.
  • GSNOR regulates the cellular concentrations of GSNO and plays a central role in regulating the levels of endogenous S-nitrosothiols and controlling protein S- nitrosylation-based signaling.
  • GSNOR 5-Nitrosoglutathione reductase regulates S- nitrosothiols (SNOs) and nitric oxide (NO) in vivo through catabolism of S-nitrosoglutathione (GSNO).
  • SNOs S- nitrosothiols
  • NO nitric oxide
  • GSNOR and the anti-inflammatory and smooth muscle relaxant activities of SNOs, GSNO, and NO play significant roles in pulmonary, cardiovascular, and gastrointestinal function.
  • a subject is administered an inhibitor of GSNO reductase (GSNOR).
  • GSNOR GSNO reductase
  • N6022 is a potent and reversible GSNO reductase inhibitor that may be used in the methods of the present disclosure (Sun et al, 2011; Green et al, 2012; both incorporated herein by reference).
  • Further GSNO reductase inhibitors that may be used in the present disclosure include, but are not limited to, substituted pyrrole analogs (e.g., described in U.S. Patent No.
  • chromone inhibitors of GSNOR such as 4-(2-(difluoromethyl)-7-hydroxy-4-oxo-4H-chromen-3-yl)benzoic acid, as disclosed in U.S. Patent No. 8,669,381; incorporated herein by reference.
  • the GSNO and/or one or more GSNO reductase inhibitors are used to treat neurological deficits, neurological inflammation, brain edema, damaged ultrastructure of microvessels, and/or cognition.
  • the neurological deficits may be the result of increased permeability of the blood brain barrier, such as resulting from hyperglycemia associated with diabetes.
  • the neurological inflammation may be associated with immune-related disorders, such as autoimmune disorders including multiple sclerosis and rheumatoid arthritis.
  • the disorders can include pulmonary disorders associated with hypoxemia and/or smooth muscle constriction in the lungs and/or lung infection and/or lung injury (e.g., pulmonary hypertension, ARDS, asthma, pneumonia, pulmonary fibrosis/interstitial lung diseases, cystic fibrosis COPD) cardiovascular disease and heart disease, including conditions such as hypertension, ischemic coronary syndromes, atherosclerosis, heart failure, glaucoma, diseases characterized by angiogenesis (e.g., coronary artery disease), disorders where there is risk of thrombosis occurring, disorders where there is risk of restenosis occurring, chronic inflammatory diseases (e.g., AID dementia and psoriasis), diseases where there is risk of apoptosis occurring (e.g., heart failure, atherosclerosis, degenerative neurologic disorders, arthritis and liver injury (ischemic or alcoholic)), impotence, obesity caused by eating in response to craving for food, stroke, reperfusion injury (e.g., traumatic muscle injury), e
  • autoimmune diseases include: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac mandate-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulone
  • an autoimmune disease that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosis, type 1 diabetes mellitus, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis.
  • the subject can also have an allergic disorder such as Asthma.
  • the GSNO and/or at least one GSNO reductase inhibitor may be administered in combination with one or more additional therapies.
  • the additional therapies may comprise anti-inflammatories, immune-modulating agents, and/or immunosuppressive therapies.
  • the additional therapy may be a therapy known in the art for the treatment of diabetes or an autoimmune disease, such as multiple sclerosis.
  • “treating” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition or disorder. More specifically, “treating” includes reversing, attenuating, alleviating, minimizing, suppressing or halting at least one deleterious symptom or effect of a disease (disorder) state, disease progression, disease causative agent (e.g., bacteria or viruses), or other abnormal condition. Treatment is continued as long as symptoms and/or pathology ameliorate.
  • a disease disorder
  • disease causative agent e.g., bacteria or viruses
  • the patient can be any animal, domestic, livestock or wild, including, but not limited to cats, dogs, horses, pigs and cattle, and preferably human patients.
  • the terms patient and subject may be used interchangeably.
  • the GSNO or GSNO reductase inhibitors can be utilized in any pharmaceutically acceptable dosage form, including but not limited to injectable dosage forms, liquid dispersions, gels, aerosols, ointments, creams, lyophilized formulations, dry powders, tablets, capsules, controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, mixed immediate release and controlled release formulations, etc.
  • the GSNO reductase inhibitors described herein can be formulated: (a) for administration selected from the group consisting of oral, pulmonary, intravenous, intra-arterial, intrathecal, intra-articular, rectal, ophthalmic, colonic, parenteral, intracistemal, intravaginal, intraperitoneal, local, buccal, nasal, and topical administration; (b) into a dosage form selected from the group consisting of liquid dispersions, gels, aerosols, ointments, creams, tablets, sachets and capsules; (c) into a dosage form selected from the group consisting of lyophilized formulations, dry powders, fast melt formulations, controlled release formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations; or (d) any combination thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed, for example, in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the GSNOR inhibitor can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of GSNO or GSNOR inhibitor calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the GSNO or GSNOR inhibitor and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active agent for the treatment of individuals.
  • compositions according to the present disclosure comprising GSNO and/or at least one GSNOR inhibitor can comprise one or more pharmaceutical excipients.
  • excipients include, but are not limited to binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art.
  • Exemplary excipients include: (1) binding agents which include various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, silicified microcrystalline cellulose (ProSolv SMCCTM), gum tragacanth and gelatin; (2) filling agents such as various starches, lactose, lactose monohydrate, and lactose anhydrous; (3) disintegrating agents such as alginic acid, Primogel, com starch, lightly crosslinked polyvinyl pyrrolidone, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof; (4) lubricants, including agents that act on the flowability of a powder to be compressed, include magnesium stearate, colloidal silicon dioxide, such as Aerosil® 200, talc, stearic acid, calcium stearate, and silica gel; (5) gli
  • Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts.
  • Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate.
  • phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate.
  • the preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure.
  • animal (e.g., human) administration it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.
  • “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art.
  • aqueous solvents e.g.
  • therapeutic benefit or“therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
  • Tg-SwDI mice also showed decreased serum L-arginine levels (FIG. lB-ii) and thus increased serum [ADMA]/[L-arginine] ratio (FIG. lB-iii).
  • the serum ADMA levels in Tg-SwDI mice was 3 times higher than WT mice (FIG. 1A).
  • ADMA treatment of Tg-SwDI mice aggravates VCID/AD associated neuro-pathology: To investigate the role of ADMA in development of VCID and AD associated neuro and vascular pathologies, 6-month-old young mice were treated with daily dose of ADMA (40mg/kg/i.p.) for 6 weeks and spatial learning and memory performances were analyzed by Morris water maze test.
  • FIG. 2A shows that Tg-SwDI mice compared to WT mice had increased latency in finding the hidden target platform and the latency was further increased by ADMA treatment, indicating that ADMA aggravates spatial learning performance of Tg-SwDI mice. At the final day of test, the mice were subjected to spatial memory performance by removing the target platform.
  • Tg-SwDI mice compared to WT mice had decreased traveling time (FIG. 2B-i) and distance (FIG. 2B-ii) in the target quadrant and number of passing the target quadrant (FIG. 2B-iii), and ADMA treatment of Tg-SwDI mice further decreased the time, distance, and number of passing the target quadrant, indicating that ADMA aggravates spatial memory performance of Tg-SwDI mice.
  • ADMA treatment of WT mice did not induce any significant change in spatial learning and memory performances.
  • AD brains commonly involve inflammation in pathologically vulnerable regions although whether the inflammation causes brain damage or merely to remove the detritus from other is not fully understood at present (Akiyama et al, 2000).
  • GFAP and Iba-1 two cellular markers of astrocytic and microglial activations.
  • FIG. 4A shows that Tg-SwDI mice compared to WT mice had increased GFAP and Iba- 1 expression and ADMA treatment of Tg-SwDI mice further increased the expressions of these proteins.
  • ADMA an endogenous NOS inhibitor
  • BBB integrity and function a major structural protein of Tg-SwDI mice
  • ADMA treated Tg-SwDI mice compared to untreated Tg-SwDI mice significantly increased the BBB permeability (FIG. 5 A). Accordingly, it was observed that ADMA treatment of Tg-SwDI mice also decreased the levels of tight junction proteins, such as ZO-1 and claudin- 1 (FIG. 5B), which play important roles in maintenance of BBB.
  • Tg-SwDI mice compared to WT mice had increased MLC phosphorylation (Ser 19 ) and which was further increased by ADMA treatment (FIG. 6A).
  • Confocal microscopy of cortical sections from ADMA treated Tg-SwDI mice document the location of phosphorylated MLC in endothelial cells (FIG. 6B).
  • Tg-SwDI mice also showed decreased expression of CD31 (marker for endothelial cells) and decreased microvessel density in cortex as shown by decreased fluorescence of collagen type IV (FIG.
  • Tg-SwDI mice treated with ADMA compared to untreated Tg- SwDI mice showed significant reduction in micro vessel density (FIG. 6C), indicating that ADMA treatment increases microvessel pathology in Tg-SwDI mice.
  • ADMA induces eNOS/ONOO -mediated RhoA activation leading to MLC phosphorylation in human brain microvessel endothelial cells: eNOS produced ONOCT has a role in thrombin-induced RhoA/Ca 2+ mediated MLC phosphorylation and barrier disruption in in vitro cultured human BMVECs (Choi et ai, 2018).
  • ADMA treatment had no effect on cellular synthesis of ONOCT, as observed by cellular levels of protein associated nitro-tyrosine (N-Tyr) levels (FIG. 7A-i).
  • ADMA treatment increased ONOO- (N-Tyr) synthesis when VEGFa activated eNOS (phosphorylation at Ser 1177 ) (FIGS. 7A-ii and -iii).
  • ADMA treatment increased endothelial ONOO synthesis in the presence of VEGFa
  • ADMA treatment also increased RhoA activation and MLC phosphorylation in the presence of VEGFa
  • the increased RhoA activation and MLC phosphorylation were inhibited by NOS inhibitor (L-NIO) and ONOO scavenger (FeTPPS) and correlated with decreased cellular N-Tyr (ONOO-) levels (FIG. 7A-iii). It was previously reported that human BMVECs exclusively express eNOS (34), thus indicating that ADMA induces RhoA-dependent MLC phosphorylation via inducing eNOS dependent ONOO- production.
  • F-actin stress fiber formation is mediated by increased F-actin polymerization and prolonged MLC phosphorylation (Stevenson, 2005).
  • actin cytoskeleton changes were examined by Phalloidin (F-actin) and p-MLC staining after VEGFa and ADMA treatments.
  • F-actin Phalloidin
  • p-MLC p-MLC staining after VEGFa and ADMA treatments.
  • ADMA treatment along with VEGFa treatment significantly reduced the trans-endothelial electric resistance (TEER; FIG. 8B), thus indicating the role of ADMA in activation of endothelial cell signaling for barrier disruption via inducing eNOS/ONOO- mediated RhoA activation.
  • Tg-SwDI mice the most common model for study of neurovascular-pathology associated with AD and VCID, have age dependent serum ADMA elevation and overburden of ADMA promotes AD and VCID associated neurovascular pathologies.
  • ADMA induces endothelial cell signaling for BBB disruption (e.g. RhoA activation and MLC phosphorylation) via inducing eNOS/ONOO- mediated mechanisms. Therefore, the data suggest that increase in blood ADMA levels, which can be caused by a variety of metabolic and vascular risk factors, can promote neurovascular disease associated with AD and VCID.
  • ADMA as a potential intermediator that links metabolic and cardiovascular risk factors to neurovascular diseases associated with AD and VCID.
  • the observed effect of ADMA in induction of endothelial nitrosative stress and cell signaling for endothelial actin cytoskeleton rearrangement suggests potential role of ADMA for microvascular dysfunction and BBB disruption and thus ADMA as a target for AD and VCID therapeutics.
  • ADMA and Evans blue dye were purchased from Sigma-
  • L-NIO N 5 -(l- Iminoethyl)-L-ornithine dihydrochloride
  • FeTPPS 5,10,15,20-Tetrakis(4-sulfonatophenyl) porphyrinato Iron (III), Cl
  • EMD Millipore- Temecula, CA, USA, Cat#: 341492
  • Recombinant Human VEGFa 165 Protein, CF (VEGFa) was purchased from R&D System (Minneapolis, MN, USA, Cat#: 293-VE-OlO/CF).
  • ELISA Serum levels of ADMA and L-arginine were determined with commercial enzyme linked immunosorbent assay (ELISA) kits according to the manufacturers’ instructions (MyBioSource, San Diego, CA, USA, Cat# mbs705936 for ADMA and mbs726317 for L-arginine). Quantification of brain levels of Ab4o and Ab42, was performed as described previously (Won et al, 2013). Briefly, the isolated mice brain cortices were homogenized in 70% formic acid.
  • Morris water maze test Morris water maze was employed to assess spatial learning and memory according to previously published methods with modification (Won et al, 2013). The test was performed in a circular pool (124 cm in diameter/60 cm in depth) filled with water clouded by nontoxic white paint. The circular pool consisted of four equal virtual quadrants. A circular area (radius 20 cm from the center of the platform) was defined as the target zone, equivalent to 4.9% of the total water maze area. All other experimental conditions are identical with our previous report (Won et al, 2013).
  • hBMVECs Primary human brain microvascular endothelial cells
  • Angio-Proteomic Cat#: cAP-0002, Atlanta, GA, USA.
  • the cells were cultured in cell culture flasks or plates precoated with Quick Coating Solution (Angio-Proteomie; Cat#: cAP-01) and maintained in Endothelial Growth Medium (Angio- Proteomie; Cat#: cAP-02) at 37°C under 5% C0 2 /95% air.
  • Immuno-fluorescent staining Cryosections (40 pm thick) obtained from 4% paraformaldehyde-fixed brain tissues were used for immunofluorescent staining for Ab42 (BC05; Wako, Osaka, Japan, Cat# 010-26903), GFAP (Cell Signaling Technology, Beverly, MA, USA, Cat# 12389), Iba-1 (Fuji Film Wako Pure Chemical Inc., Saitama , Japan, Cat# 012-26723), CD31 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, Cat# scl506), p-MLC (Ser 19 ) (Abeam, Cat#: ab2480, and collagen type IV (EMD Millipore, Cat# AB769).
  • Ab42 BC05; Wako, Osaka, Japan, Cat# 010-26903
  • GFAP Cell Signaling Technology, Beverly, MA, USA, Cat# 12389
  • Iba-1 Feuji Film Wako Pure Chemical Inc
  • hBMVECs For immunofluorescent staining of cultured hBMVECs, the cells were cultured on fibronectin- coated 24 well plates (BD Bioscience). Following drug treatments, the cells were fixed with 4% (wt/vol) paraformaldehyde, permeabilized by the addition of 0.25% Triton X-100, and blocked by 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS).
  • BSA bovine serum albumin
  • the slides were immunostained for p-MLC as well as stained with Phalloidin for F-actin (F-actin Visualization kit, Cytoskeleton Inc., Denver, CO, USA, Cat#: BK005) and DAPI for nucleus (4',6-diamidino-2-phenylindole; ThermoFisher Scientific, Houston, TX, USA).
  • Phalloidin for F-actin F-actin Visualization kit, Cytoskeleton Inc., Denver, CO, USA, Cat#: BK005
  • DAPI nucleus (4',6-diamidino-2-phenylindole; ThermoFisher Scientific, Houston, TX, USA).
  • the tissue sections and cells were imaged by BX60 Olympus fluorescent/light microscope equipped with DP-70 digital camera (Olympus, Tokyo, Japan) or LSM 880 NLO confocal microscope (Zeiss, NY, USA).
  • the acquired images were analyzed by ImageJ (NIH
  • TEER trans-endothelial electrical resistance
  • hBMVECs were plated on fibronectin-coated polycarbonate filters (Transwell system, Coming, Midland, NC) containing Endothelial Growth Medium (Angio-Proteomie Cat#: cAP-02). The medium was renewed every other day. Five days after seeding, the medium was replaced with Endothelial Basal Medium (Angio- Proteomie Cat#: cAP-03) containing 0.5% FBS and incubated for 2 days. Following drug treatments, transendothelial electrical resistance (TEER) was measured by EVOM2 (Word Precision Instruments) as described previously (Choi et al, 2018).
  • RhoA activity assay RhoA activity in hBMVECs was analyzed as described previously (Choi et al, 2018) by RhoA Activation Assay Kit (Abeam Cat#: ab211164). Briefly, following drug treatments, the cells were lysed with lXAssay buffer provided in the kit. Lysates were centrifuged (14,000 x g for 10 sec), and supernatants were incubated with agarose beads coupled to GST- Rhotekin-Rho binding domain (RBD) for 2 h at 4 °C. Beads were then washed with lXAssay buffer and GTP-bound RhoA was eluted with 2X SDS-PAGE sample buffer. Amounts of active (GTP-bound) RhoA were determined by Western blot analysis using antibody specific to RhoA (Abeam).
  • Western blot analysis Western immunoblot analysis was performed by standard method using 50 pg of cell lysates. Following the SDS-PAGE electrophoresis, proteins were transferred from the gel onto the Polyvinylidene fluoride membrane (GE Healthcare Life Sciences, Marlborough, MA, USA).
  • Membranes were blocked with non-fat dry milk (Santa Cruz Biotechnology) or I-BlockTM (ThermoFisher Scientific, Waltham, MA, USA) for detection of phospho-proteins and incubated with primary antibodies, such as ZO-1 (Invitrogen- Thermo Fisher Inc., Cat# 33-9100), Claudin-1 (Invitrogen- Thermo Fisher Inc., Cat# 37-4900), p-myosin light chain (MLC) 2 (Ser 19 ) (Abeam, Cat#: ab2480), total (t)-MLC (Abeam, Cat#: at>79935), CD31, phospho(p)-eNOS (Ser 1177 ) (Cell Signaling, Cat#: 9571), total(t)-eNOS (Cell Signaling, Cat#: 32027), and RhoA (Santa Cruz Biotechnology; Cat#: sc418).
  • ZO-1 Invitrogen- Thermo Fisher Inc., Cat# 33
  • the membranes were incubated with horseradish peroxidase conjugated secondary antibody (Jackson Immunoresearch Lab, West Grove, PA), washed and then incubated with ECL reagent (Amersham Life Science, Pittsbrugh, PA), and exposed to Amersham Hyperfilm ECL film.
  • mice received 100 m ⁇ of a 5% solution of EB in saline administered intravenously.
  • cardiac perfusion was performed under deep anesthesia with 200 ml of saline to clear the brain circulation of EB.
  • the brain was removed and sliced.
  • the brain tissues were homogenized in 750 m ⁇ of N, N- dimethylformamide (DMF) and centrifuged at 10,000 x g for 25 minutes, and EB content in supernatant was fluorimetrically analyzed (l ec 620 nm, L em 680 nm).
  • DMF N, N- dimethylformamide
  • EAE increases the blood ratio of ADMA vs. L-Arg with reduction of DDAH activity: Blood levels of ADMA and VEGF were analyzed in EAE mice. For induction of EAE, female C57BL/6 mice were treated with complete Freund's adjuvant (CFA) and myelin oligodendrocyte glycoprotein (MOG) with/without pertussis toxin (PTX) treatment.
  • CFA complete Freund's adjuvant
  • MOG myelin oligodendrocyte glycoprotein
  • PTX pertussis toxin
  • FIG. 9A-i and -iii shows that ADMA levels were significantly increased on the day of disease onset (day 11 post immunization) and stayed at high levels till the day 44 post-immunization.
  • the EAE mice also had increased blood VEGF levels even before the onset of disease (day 7 post-immunization) and which was decreased during the chronic phase of EAE disease (day 44 post-immunization) (FIG. 9A-ii and -iii).
  • ADMA Alzheimer's disease
  • DDAH a redox-sensitive enzyme whose activity is affected by reactive thiols, such as HCy (Stuhlinger et al, 2001). Similar to the MS patients, it was observed that the blood levels of HCy were greatly increased in EAE animals (CFA+MOG+PTX) (FIG. 9C). Accordingly, significant reductions in DDAH activities were observed in the kidney and liver (FIG. 9D). Taken together, these data indicate the potential role of hyperhomocysteinemia in the inhibition of DDAH-mediated ADMA catabolism and blood accumulation in EAE.
  • ADMA treatment exacerbates clinical EAE disease: Next, the effect of ADMA overburden on clinical EAE disease was investigated. EAE mice were treated with a daily dose of ADMA starting on the day of disease onset (day 11 post-immunization). FIG. 10A shows that the treatment of EAE mice with ADMA had no significant effect till day 19 post-immunization. However, it significantly increased the disease starting day 20 post immunization. The areas under the curves (AUC) of the daily clinical scores (FIG. 10B) show that ADMA significantly increased the overall EAE disease symptom.
  • ADMA treatment increases myelin and axonal pathologies in the spinal cord of EAE animals: Next, the effect of ADMA overburden in EAE-induced demyelination and axonal degeneration was investigated in the spinal cord. LFB staining in FIG. 11A shows that EAE mice had decreased myelin content as compared to the control mice and which was further decreased with ADMA treatment. Accordingly, Western analysis of myelin basic protein (MBP) in FIG. 11B shows that ADMA treatment significantly enhanced the EAE-induced reduction in MBP levels in the spinal cord. Electron microscope study of the spinal cord for ultrastructural conformation of myelin and axonal loss (FIG.
  • ADMA treatment also enhanced the loss of myelin structure as shown by the loss of doughnut like structure of myelin sheet.
  • ADMA treatment also increased the loss of axons in the spinal cord of EAE mice as shown by Bielchowsky silver staining (FIG. 11D) and b-tubulin (axonal marker) western analysis (FIG. 11E).
  • FIG. 11D Bielchowsky silver staining
  • ADMA treatment increased the extravasation of mononuclear cells and BBB disruption in EAE animals:
  • H&E Hematoxylin and Eosin staining in FIGS. 12A and 12B show that daily ADMA treatment increased the extravasation of mononuclear cells in the lumbar area of the spinal cord in EAE mice.
  • H&E Hematoxylin and Eosin staining in FIGS. 12A and 12B show that daily ADMA treatment increased the extravasation of mononuclear cells in the lumbar area of the spinal cord in EAE mice.
  • BBB disruption shows by increased Evan’s blue extravasation into the CNS (brain and spinal cord) (FIG. 12C).
  • ADMA by itself had relatively little effect on Evan’s blue extravasation in control mice (FIG.
  • EAE mice had increased expression of MMP-9 and ADMA treatment further increased the MMP-9 expression (FIG. 12D).
  • Claudin-1 is one of the substrates of MMP-9 and an important constituent of tight junction in the endothelium. Accordingly, it was observed that the claudin-1 level is decreased in the spinal cord of EAE mice and ADMA treatment further reduced it (FIG. 12E).
  • ADMA treatment induces blood-brain barrier disruption and CNS infiltration of immune cells to induce EAE disease:
  • Current EAE models are based on peripheral immunization of animals with myelin-antigen (e.g. MOG) and CFA.
  • myelin-antigen e.g. MOG
  • CFA myelin-antigen
  • the myelin immunization by itself is not enough for developing clinical EAE disease due to a lack of CNS infiltration of immunocytes (Linthicum et al, 1982). Accordingly, a lack of induction of EAE disease was observed in CFA+MOG treated group (FIG. 13 A: solid inverted triangle) as well as a lack of extravasation of mononuclear cells into the spinal cord (FIG.
  • ADMA treatment of MOG immunized mice induced the CNS infiltration of immunocytes (FIG. 5B) and Evan’s blue dye (FIG. 5C) without PTX treatment and accordingly induced the clinical disease of EAE (FIG. 5A: circle).
  • ADMA treatment induces autoreactive THI and TH17 immune responses under EAE conditions:
  • CD4 + T cells purified from spleens of naive, EAE, and ADMA treated EAE mice on 30 day of post-immunization, were cultured ex vivo and the numbers of CD4 + IFN-y + (T H 1), CD4 + IL-17 + (T H 1), CD4 + CD25 + FOXP3 + (n/iTreg), and CD4 + CD25 + FOXP3 (Trl or NO-Treg) were analyzed (Niedbala et al, 2007).
  • EAE animals had increased numbers of T H I and T H 17 cells and which were further increased by treatment with ADMA. Accordingly, splenic CD4 + T cells isolated from EAE animals produced increased levels of IFN-g and IL-17a and ADMA treatment further increased the release of these pro-inflammatory cytokines (FIGS. 14B-i and ii). EAE animals also had decreased numbers of FOXP3 + and FOXP3 Treg cells compared to naive mice but ADMA treatment had no effect on these decreases (FIGS. 14A-iii and iv). Accordingly, ADMA had no effect on EAE-induced reduction in IL-10 production in splenic CD4 + T cells (FIG. 14B-iii).
  • ADMA treatment had no obvious effect on the differentiation of CD4 + T cells under unstimulatory conditions but it enhanced the T H I and T H 17 differentiation under stimulatory conditions with anti-CD3/CD28 mAh and IL-2 treatments.
  • CD4 + T cells from EAE animals in vitro stimulation of naive CD4 + T cells increased the numbers of FOXP3 + and FOXP3 Treg cells (FIG. 14C-iv).
  • ADMA treatment had no effect on these changes.
  • ADMA was also able to induce EAE disease in MOG-immunized mice by substituting PTX for BBB disruption and CNS infiltration of mononuclear cells (FIG. 13).
  • ADMA treatment also promoted T H I and T H 17 mediated immune responses in the spleen but without affecting Treg-mediated immune response under EAE conditions (FIG. 14).
  • mice were purchased from Jackson Laboratory (Stock no: 000664, Bar Harbor, ME). Mice were supplied with food and water ad libitum and kept in ventilated cages in specific pathogen-free animal care facility maintained by the Medical University of South Carolina throughout the entire study. They were housed at controlled temperature (22 °C), humidity (45-55%), and 12 h light/dark cycle. All animal studies were reviewed and approved by the Medical University of South Carolina’s Institutional Animal Care and Use Committee (IACUC) (AR # 2019-00761).
  • IACUC Institutional Animal Care and Use Committee
  • EAE was induced by the subcutaneous injection of female C57BL/6 mice (8-12 weeks old) with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide emulsified in the complete Freund’s adjuvant (CFA) as described in the kit instruction (Hook Laboratories, Lawrence, MA). Two hundred ng of pertussis toxin was injected intraperitoneally on days 0 and 1. Mice were weighed and assessed for clinical signs every day starting from day 0 through the day of experiment termination. Alternatively, MOG-immunized mice received daily ADMA (50 mg/kg body weight /day/ip; Sigma- Aldrich, St.
  • ADMA 50 mg/kg body weight /day/ip; Sigma- Aldrich, St.
  • a vehicle 100 pL of 10% DMSO in saline
  • ADMA 50 mg/Kg body weight/day/ip
  • ELISA for serum ADMA, L-Arg, VEGF, and HCy Seram levels of ADMA, L-arginine, VEGF, and HCy were determined with commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers’ instructions (MyBioSource, San Diego, CA, USA, Cat# mbs705936 for ADMA, mbs726317 for L- arginine, mbsl65329 for VEGF, and mbs7252797 for HCy).
  • DDAH activity was measured as reported previously (Tain and Baylis, 2007). Briefly, kidneys and livers were homogenized in sodium phosphate buffer containing 0.1% Triton X-100, preincubated with urease (100 U/ml) for elimination urea. The samples were mixed with 1 mM ADMA, incubated at 37°C, and treated with the sample volume of 4% sulfosalicylic acid to stop the reaction and followed by centrifugation at 3,000 g for 10 min.
  • the supernatant was mixed with an equal amount of color mixture (1 part of oxime reagent:2 parts of antipyrine/H 2 S0 4 ) and incubated for 110 min at 60°C in the dark. The reaction was stopped by cooling samples at 20°C for 10 min, and the levels of citrulline were measured at 466 nm.
  • Electron microscopy was performed as we described previously (Singh et al, 2018). Animals were anesthetized and perfused with 10 ml of normal saline containing 0.1% sodium nitrite followed by 15 ml of a mixture of 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Then the spinal cords were fixed in the same fixative (above) and post- fixed with 1% osmium tetroxide-1.5% ferrocyanide for 2 hours in the dark, then dehydrated and embedded in Epon LX 112 resin. Semi-thin sections, approximately 1 pm thick, were cut and stained with toluidine blue.
  • Membranes were blocked with non-fat dry milk (Santa Cruz Biotechnology) and incubated with a primary antibody specific to myelin basic protein (MBP; Santa Cruz Biotech, Delaware Avenue, CA), Claudin-1 (Invitrogen- Thermo Fisher Inc., Cat# 37-4900), b- actin (Cell Signaling Technology, Danvers, MA), b- tubulin (Cell Signaling Technology), or MMP-9 (Cell Signaling Technology).
  • MBP myelin basic protein
  • Claudin-1 Invitrogen- Thermo Fisher Inc., Cat# 37-4900
  • b- actin Cell Signaling Technology, Danvers, MA
  • b- tubulin Cell Signaling Technology
  • MMP-9 Cell Signaling Technology
  • mice received 100 m ⁇ of a 5% solution of EB in saline administered intravenously.
  • cardiac perfusion was performed under deep anesthesia with 200 ml of saline to clear the brain/spinal cord circulation of EB.
  • the brain and spinal cord were removed and sliced.
  • the tissues were homogenized in 750 m ⁇ of N, N-dimethylformamide (DMF) and centrifuged at 10,000 x g for 25 minutes, and EB content in the supernatant was fluorometrically analyzed (l ec 620 nm, L em 680 nm).
  • DMF N, N-dimethylformamide
  • CD4 + T cells were purified with CD4 microbeads (Miltenyi Biotech) then re-suspended with complete RPMI-media in 12-well plates (5 x 10 6 cells/ 2 ml per well) containing MOG peptide (25 pg/ml) for 48 hrs. Following the centrifugation, the resulting supernatants were collected for ELISA for CD4 + subset specific cytokines.
  • the cell pellets were washed with cell staining solution (ebioscience, Waltham, MA, USA) and stained with a fluorescence-labeled antibody specific to IFN-g for T H U IL-17 for TH17, CD25 and FOXP3 + for FOXP3 + Treg, and CD25 and FOXP3 for FOXP3 Tregs (ebioscience, Waltham, MA, USA).
  • the cells were counted and analyzed using a Beckman Coulter instrument (Beckman Coulter, Inc., Brea, CA, USA).
  • ELISA assay was performed for analysis of CD4 + T cell subset specific cytokines (IFN-g, IL-17, and IL-10) released from cultured lymphocytes.
  • the ELISA assay kits for IEN-g, IL-17, and IL-10 were purchased from R&D Systems (Minneapolis, MN, USA).
  • naive CD4 + T cells were isolated from naive C57BL/6 mice as described above.
  • the CD4 + T cells (2xl0 5 cells/well)suspended in RPMI-complete media were pretreated with ADMA (200 mM) and then stimulated with plate-bound anti-CD3s antibody and soluble anti-CD28 antibody and IL-2 (20 U/ml) as described previously (Nath et al, 2010).
  • the cells were collected at 36 to 96 h and the numbers of THI, TH17, and FOXP3 + Treg, and FOXP3 Treg cells were analyzed by fluorescence cytometric analysis as described above.

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Abstract

La présente invention concerne des méthodes qui permettent de détecter des niveaux d'ADMA pour déterminer le risque d'une pathologie vasculaire ou neurologique.
PCT/US2020/039613 2019-06-25 2020-06-25 Diméthylarginine asymétrique (adma) en tant que marqueur de pathologies vasculaires WO2020264156A1 (fr)

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